Roots are in capitals, and are not words in use at all, but serve as an elucidation of the words grouped together and a connection between them.

J.R.R. Tolkien's note in the Qenya Lexicon[1]

Sauron new

knew that he had been wrong - not everyone would want to use the ring for their own power and Glory

yes Frodo succumbed at the very very end but - he and Sam made it that far and - fate or Providence or the intervention of Uru? himself did the rest

some people are capable of selfless and purely good acts

it wasn't just just Sauron who fell - it was his entire worldview

hope and love and care and friendship - can triumph over evil - however powerful it may seem at the time

attention, pas d'espace avant la virgule. il y a une espace après tous les signes de ponctuation, mais l'espace avant n'est que pour les signes de ponctuation doubles (; : ! ?)

La Prévention des Conflits d'Intérêts : Collectivités et Associations

Synthèse

Ce document de synthèse analyse les enjeux juridiques et pratiques liés à la prévention des conflits d'intérêts dans les relations entre les collectivités territoriales et les associations.

Basé sur les interventions d'experts juridiques et de formateurs d'élus, il met en lumière les risques pénaux encourus et propose des préconisations concrètes.

Les points critiques à retenir sont les suivants :

• 1. Le conflit d'intérêts n'est pas une infraction, mais un signal d'alerte. La situation devient délictuelle lorsqu'un élu ou un agent public, conscient de ce conflit, ne se déporte pas et participe à une décision, tombant ainsi sous le coup de la prise illégale d'intérêt, une infraction pénale sévèrement sanctionnée (jusqu'à 5 ans d'emprisonnement et 500 000 € d'amende).

• 1. La notion d'intérêt est extrêmement large. Elle couvre les intérêts matériels, mais aussi moraux ou familiaux. Il n'est pas nécessaire que l'élu se soit enrichi personnellement ou que la collectivité ait subi un préjudice ; la simple apparence d'une impartialité compromise peut suffire à caractériser l'infraction.
• 1. La règle pour les élus impliqués dans une association est le "déport général". Qu'ils soient membres du bureau à titre personnel ou en tant que représentants de la commune, ils doivent s'abstenir de toute participation à une délibération concernant cette association.

Ce déport doit être total :

• ◦ Absence de participation à l'instruction du dossier.
• ◦ Absence de participation aux débats.
• ◦ Absence de participation au vote.

• ◦ Sortie physique de la salle du conseil durant les débats et le vote.

• 1. Les élus locaux sous-estiment massivement ce risque. Les formations de terrain révèlent que la préoccupation principale des élus concerne les aspects techniques des subventions, tandis que le risque de conflit d'intérêts est souvent ignoré, en particulier dans les petites communes où les interférences entre mandats électifs et vie associative sont pourtant maximales.
• 1. Des outils et des bonnes pratiques existent pour sécuriser les processus.

La responsabilité première incombe à chaque élu, qui doit s'auto-évaluer en permanence.

Pour sécuriser les décisions, il est préconisé de voter les subventions au cas par cas, de systématiser la déclaration des conflits en début de séance et de s'appuyer sur des ressources externes comme la Haute Autorité pour la Transparence de la Vie Publique (HATVP) et le référent déontologue, désormais obligatoire pour toutes les communes.

1. Le Cadre Juridique et les Risques Pénaux

L'analyse juridique, menée par Luc Brunet de l'Observatoire SMAC, souligne la nécessité de distinguer deux notions fondamentales qui sont souvent confondues.

Définitions Fondamentales : Conflit d'Intérêts vs. Prise Illégale d'Intérêt

Le conflit d'intérêts est une situation, tandis que la prise illégale d'intérêt est une infraction pénale qui découle de la mauvaise gestion de cette situation. Caractéristique Conflit d'Intérêts Prise Illégale d'Intérêt Nature

Une situation d'interférence entre un intérêt public et des intérêts (publics ou privés) de nature à influencer ou paraître influencer l'exercice d'une fonction.

Une infraction pénale. Le fait de prendre, recevoir ou conserver, directement ou indirectement, un intérêt de nature à compromettre son impartialité.

Source Légale Loi du 11 octobre 2013

Article 432-12 du Code pénal

Sanction

Aucune (ce n'est pas une infraction). La situation doit être prévenue ou résolue.

Jusqu'à 5 ans d'emprisonnement et 500 000 € d'amende.

"Le conflit d'intérêts, c'est la vie. Nous avons tous des conflits d'intérêts. [...] Là où c'est pas normal [...] c'est quand on va se dire 'je vais surtout pas le dire que je suis en situation de conflit d'intérêt'. Et c'est là qu'on franchit la ligne jaune et qu'on passe [...] du côté du code pénal avec le délit de prise illégale d'intérêt." - Luc Brunet

Le Champ d'Application Vaste de la Prise Illégale d'Intérêt

Le délit de prise illégale d'intérêt est l'infraction numéro un pour laquelle les élus locaux sont poursuivis. Son champ d'application est particulièrement étendu :

• Tous les domaines : Contrairement au délit de favoritisme (limité à la commande publique), il s'applique à toutes les décisions d'une collectivité : urbanisme, recrutement, vente de biens, et notamment les subventions aux associations.

• Intérêt moral ou familial : L'intérêt n'est pas nécessairement matériel ou financier.

• Absence de préjudice requis : L'infraction est constituée même si la collectivité n'a subi aucun préjudice, voire si elle a bénéficié de l'opération.

• Intérêts indirects : Le délit couvre les intérêts pris par personne interposée (conjoint, ascendants, descendants, mais aussi amis proches).

La jurisprudence retient une vision très large : "l'infraction s'arrête où le soupçon s'arrête".

• La notion d'apparence : Il ne faut pas seulement ne pas être en conflit d'intérêts, mais aussi ne pas donner l'apparence de l'être.

La Doctrine de la Haute Autorité pour la Transparence de la Vie Publique (HATVP)

La HATVP a établi une doctrine pour clarifier les niveaux de risque. Pour les relations avec les associations, le risque est considéré comme large.

• Zone Rouge (Risque Large) : Concerne la participation d'un élu au sein d'un organisme de droit privé, comme une association, que ce soit à titre personnel ou comme représentant de la commune.

• Règle Appliquée : Le déport général. L'élu concerné doit s'abstenir de participer à toute délibération relative à cet organisme, y compris en l'absence d'enjeu financier direct. Adhérent ou Dirigeant : Une Distinction Cruciale ?

La question se pose de savoir si un simple adhérent est soumis aux mêmes règles qu'un membre du bureau (président, trésorier, etc.).

• Position de la HATVP (Avis du 3 mai 2022) : Le simple fait d'être adhérent ne justifie pas un déport systématique.

Cependant, une analyse au cas par cas doit être menée en fonction de la nature de l'association, de son nombre d'adhérents et de l'objet de la délibération.

• Conseil de Prudence : Face à l'incertitude de l'analyse au cas par cas, il est recommandé aux simples adhérents, par mesure de sécurité, de se déporter systématiquement lors du vote d'une subvention.

2. Règles Pratiques et Préconisations La prévention repose sur une démarche rigoureuse et transparente.

Les Quatre Étapes de la Prévention

• 1. Identifier les situations à risque : L'élu doit se poser les bonnes questions sur ses liens personnels, familiaux ou associatifs en rapport avec les dossiers de la collectivité.

• 2. Déclarer le conflit d'intérêts : Conformément à la Charte de l'élu local, l'élu doit faire connaître ses intérêts personnels avant le débat et le vote.

• 3. Se déporter complètement : Le déport ne se limite pas au non-vote. L'élu ne doit participer ni à l'instruction du dossier, ni aux débats qui précèdent le vote.

• 4. Ne pas influencer : L'élu doit s'abstenir de toute intervention, même informelle ("tirer les ficelles par derrière").

Jurisprudence : Des Exemples Concrets et Marquants Deux cas illustrent la sévérité avec laquelle la justice appréhende ce délit :

• Le maire de Plougastel-Daoulas : Des élus membres du bureau d'une association ad hoc n'ont pas participé au vote de la subvention, mais sont restés dans la salle.

Ce simple fait a été jugé suffisant pour caractériser une influence et a conduit à leur condamnation pour prise illégale d'intérêt.

• Une commune rurale de 250 habitants : Des élus, membres du bureau d'une association organisant la fête du village, ont participé au vote d'une subvention de 250 €.

Ils ont été condamnés pour prise illégale d'intérêt suite à la plainte d'un opposant politique.

Ces exemples démontrent que ni la bonne foi, ni la poursuite de l'intérêt général, ni le faible montant de la subvention ne constituent des protections contre une condamnation.

Préconisations pour Sécuriser les Délibérations

• Pas de vote global : Les subventions aux associations doivent être votées une par une, jamais en bloc.

• Sortir de la salle : L'élu concerné doit physiquement quitter la salle du conseil avant le début des débats et ne revenir qu'une fois le point de l'ordre du jour traité. Cette sortie doit être consignée au procès-verbal.

• Instaurer un "tour de table" déontologique : En début de chaque conseil, le maire peut demander à chaque élu de signaler d'éventuels conflits d'intérêts au regard de l'ordre du jour.

3. Le Témoignage du Terrain : Entre Méconnaissance et Difficultés d'Application

Le témoignage de Sophie Van migom, directrice d'un centre de formation pour élus, révèle un décalage important entre les exigences légales et la perception des élus sur le terrain.

Une Prise de Conscience Limitée chez les Élus

Lors des formations, les préoccupations des élus portent majoritairement sur des questions techniques (conventionnement, prêt de matériel, contrôle financier).

Le risque de conflit d'intérêts est très rarement abordé spontanément, en particulier par les élus des petites communes.

"Sur 90 participants, je n'ai que deux élus qui m'ont parlé de conflit d'intérêt. [...] Les élus des petites communes ne se posent pas la question, alors qu'il y a forcément des interférences entre leur mandat électif, leur vie familiale, leur vie associative." - Sophie Van migom

Les Conséquences Pratiques et les Défis Opérationnels

L'application stricte des règles de déport peut engendrer des difficultés de fonctionnement :

• Problèmes de quorum : Dans une commune de 620 habitants, la mise en place de règles de déport strictes a conduit à ce que la moitié du conseil municipal sorte de la salle, empêchant le quorum d'être atteint. La seule solution est de reconvoquer le conseil, ce qui retarde la décision.

• Paralysie de l'action des élus : Un élu engagé pour son expertise associative (ex: président de l'association des parents d'élèves devenu adjoint aux écoles) peut se retrouver dans l'incapacité d'agir sur les dossiers pour lesquels il a été élu.

Les Doubles Sanctions : Pénale et Administrative Le non-respect des règles de déport expose l'élu et la collectivité à un double risque :

1. Le risque pénal : L'élu est poursuivi pour prise illégale d'intérêt et le maire pour complicité.

2. Le risque administratif : La délibération elle-même est illégale.

Elle peut être annulée par le juge administratif suite à un recours d'un opposant, d'un contribuable ou du préfet. L'association pourrait alors être contrainte de rembourser la subvention perçue.

4. Outils et Bonnes Pratiques

La Responsabilité Personnelle de l'Élu

C'est à chaque élu d'évaluer sa propre situation, d'informer le maire et le conseil, et de prendre la décision de se déporter.

Cette réflexion doit être menée dès le début du mandat pour clarifier les limites de ses fonctions.

Les Aides à la Décision

Les élus ne sont pas seuls face à ces questionnements complexes. Ils peuvent solliciter :

• La Haute Autorité pour la Transparence de la Vie Publique (HATVP) : Il est possible de saisir la HATVP pour obtenir un avis confidentiel et rapide sur une situation personnelle.

• Le référent déontologue : Sa désignation est une obligation pour toutes les collectivités. Il offre un avis qui va au-delà du strict droit, en abordant les questions de probité et d'exemplarité.

Cas Spécifiques Abordés

• Agents de la collectivité : Ils sont également concernés par le délit.

S'ils sont en situation de conflit d'intérêts sur un dossier (ex: instruction d'un marché public pour l'entreprise d'un proche), ils doivent le signaler à leur hiérarchie pour que le dossier leur soit retiré.

• Subventions en nature : La mise à disposition de locaux, de matériel ou d'agents est considérée comme un avantage et suit exactement les mêmes règles de déport que les subventions financières.

• Associations "transparentes" : Une association qui n'est en réalité que le prolongement de la collectivité (ex: toutes les décisions sont prises par la commune) pose des problèmes juridiques majeurs.

Toutes les règles de la collectivité (comptabilité publique, marchés publics) s'appliquent alors à elle, créant un risque juridique élevé.

Medición de cohesión social longitudinal en Chile

Y en la presentación mencionar que es con ELSOC ... y qué es ELSOC, qué es COES, sentido del documento, etc etc. Es decir, falta redactar bien la presentación.

la conceptualizacion y medicion de cohesion social para AL del OCS

Relevaría aquí que, dada la relevancia de la cohesión social para mantener la estabilidad de la sociedad, el lazo etc, poder medirlo empiricamete es clave y ello es algo fragmentado a nivel internacional y no existe un esfuerzo como tal en Chile antes del OCS

/hyperpost/🌐/🎭/gyuri/do/web/snarf/📅/2025/10/2/Metaphysics.of.Adjacency.html

self

view

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

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

all driven from future orientated awareness of possibilities

or even impossibiities that desrve to be rendered inevitable and present

How ridiculous LLMs look from this perspective?

not to mention bogus notions like the manyverse?

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

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

Gyuri Web Snarf 2025-10-2

exp - lore - eriment - perience

the info-morphic-communication flow

Let the spice of Life flow

Reviewer #1 (Public review):

Summary:

The authors present MerQuaCo, a computational tool that fills a critical gap in the field of spatial transcriptomics: the absence of standardized quality control (QC) tools for image-based datasets. Spatial transcriptomics is an emerging field where datasets are often imperfect, and current practices lack systematic methods to quantify and address these imperfections. MerQuaCo offers an objective and reproducible framework to evaluate issues like data loss, transcript detection variability, and efficiency differences across imaging planes.

Strengths:

(1) The study draws on an impressive dataset comprising 641 mouse brain sections collected on the Vizgen MERSCOPE platform over two years. This scale ensures that the documented imperfections are not isolated or anecdotal but represent systemic challenges in spatial transcriptomics. The variability observed across this large dataset underscores the importance of using sufficiently large sample sizes when benchmarking different image-based spatial technologies. Smaller datasets risk producing misleading results by over-representing unusually successful or unsuccessful experiments. This comprehensive dataset not only highlights systemic challenges in spatial transcriptomics but also provides a robust foundation for evaluating MerQuaCo's metrics. The study sets a valuable precedent for future quality assessment and benchmarking efforts as the field continues to evolve.

(2) MerQuaCo introduces thoughtful metrics and filters that address a wide range of quality control needs. These include pixel classification, transcript density, and detection efficiency across both x-y axes (periodicity) and z-planes (p6/p0 ratio). The tool also effectively quantifies data loss due to dropped images, providing tangible metrics for researchers to evaluate and standardize their data. Additionally, the authors' decision to include examples of imperfections detectable by visual inspection but not flagged by MerQuaCo reflects a transparent and balanced assessment of the tool's current capabilities.

Weaknesses:

(1) The study focuses on cell-type label changes as the main downstream impact of imperfections. Broadening the scope to explore expression response changes of downstream analyses would offer a more complete picture of the biological consequences of these imperfections and enhance the utility of the tool.

(2) While the manuscript identifies and quantifies imperfections effectively, it does not propose post-imaging data processing solutions to correct these issues, aside from the exclusion of problematic sections or transcript species. While this is understandable given the study is aimed at the highest quality atlas effort, many researchers don't need that level of quality to compare groups. It would be important to include discussion points as to how those cut-offs should be decided for a specific study.

(3) Although the authors demonstrate the applicability of MerQuaCo on a large MERFISH dataset, and the limited number of sections from other platforms, it would be helpful to describe its limitations in its generalizability.

Reviewer #1 (Public review):

The authors present MerQuaCo, a computational tool that fills a critical gap in the field of spatial transcriptomics: the absence of standardized quality control (QC) tools for image-based datasets. Spatial transcriptomics is an emerging field where datasets are often imperfect, and current practices lack systematic methods to quantify and address these imperfections. MerQuaCo offers an objective and reproducible framework to evaluate issues like data loss, transcript detection variability, and efficiency differences across imaging planes.

Strengths

(1) The study draws on an impressive dataset comprising 641 mouse brain sections collected on the Vizgen MERSCOPE platform over two years. This scale ensures that the documented imperfections are not isolated or anecdotal but represent systemic challenges in spatial transcriptomics. The variability observed across this large dataset underscores the importance of using sufficiently large sample sizes when benchmarking different image-based spatial technologies. Smaller datasets risk producing misleading results by over-representing unusually successful or unsuccessful experiments. This comprehensive dataset not only highlights systemic challenges in spatial transcriptomics but also provides a robust foundation for evaluating MerQuaCo's metrics. The study sets a valuable precedent for future quality assessment and benchmarking efforts as the field continues to evolve.

(2) MerQuaCo introduces thoughtful metrics and filters that address a wide range of quality control needs. These include pixel classification, transcript density, and detection efficiency across both x-y axes (periodicity) and z-planes (p6/p0 ratio). The tool also effectively quantifies data loss due to dropped images, providing tangible metrics for researchers to evaluate and standardize their data. Additionally, the authors' decision to include examples of imperfections detectable by visual inspection but not flagged by MerQuaCo reflects a transparent and balanced assessment of the tool's current capabilities.

Comments on revisions:

All previous concerns have been fully addressed. The revised manuscript presents a robust, well-documented, and user-friendly tool for quality control in image-based spatial transcriptomics, a rapidly advancing area where objective assessment tools are urgently needed.

Author response:

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

Reviewer #1 (Public review):

The authors present MerQuaCo, a computational tool that fills a critical gap in the field of spatial transcriptomics: the absence of standardized quality control (QC) tools for image-based datasets. Spatial transcriptomics is an emerging field where datasets are often imperfect, and current practices lack systematic methods to quantify and address these imperfections. MerQuaCo offers an objective and reproducible framework to evaluate issues like data loss, transcript detection variability, and efficiency differences across imaging planes.

Strengths:

(1) The study draws on an impressive dataset comprising 641 mouse brain sections collected on the Vizgen MERSCOPE platform over two years. This scale ensures that the documented imperfections are not isolated or anecdotal but represent systemic challenges in spatial transcriptomics. The variability observed across this large dataset underscores the importance of using sufficiently large sample sizes when benchmarking different image-based spatial technologies. Smaller datasets risk producing misleading results by over-representing unusually successful or unsuccessful experiments. This comprehensive dataset not only highlights systemic challenges in spatial transcriptomics but also provides a robust foundation for evaluating MerQuaCo's metrics. The study sets a valuable precedent for future quality assessment and benchmarking efforts as the field continues to evolve.

(2) MerQuaCo introduces thoughtful metrics and filters that address a wide range of quality control needs. These include pixel classification, transcript density, and detection efficiency across both x-y axes (periodicity) and z-planes (p6/p0 ratio). The tool also effectively quantifies data loss due to dropped images, providing tangible metrics for researchers to evaluate and standardize their data. Additionally, the authors' decision to include examples of imperfections detectable by visual inspection but not flagged by MerQuaCo reflects a transparent and balanced assessment of the tool's current capabilities.

Weaknesses:

(1) The study focuses on cell-type label changes as the main downstream impact of imperfections. Broadening the scope to explore expression response changes of downstream analyses would offer a more complete picture of the biological consequences of these imperfections and enhance the utility of the tool.

Here, we focused on the consequences of imperfections on cell-type labels, one common use for spatial transcriptomics datasets. Spatial datasets are used for so many other purposes that there are almost endless ways in which imperfections could impact downstream analyses. It is difficult to see how we might broaden the scope to include more downstream effects, while providing enough analysis to derive meaningful conclusions, all within the scope of a single paper. Existing studies bring some insight into the impact of imperfections and we expect future studies will extend our understanding of consequences in other biological contexts.

(2) While the manuscript identifies and quantifies imperfections effectively, it does not propose post-imaging data processing solutions to correct these issues, aside from the exclusion of problematic sections or transcript species. While this is understandable given the study is aimed at the highest quality atlas effort, many researchers don't need that level of quality to compare groups. It would be important to include discussion points as to how those cut-offs should be decided for a specific study.

Studies differ greatly in their aims and, as a result, the impact of imperfections in the underlying data will differ also, preventing us from offering meaningful guidance on how cut-offs might best be identified. Rather, our aim with MerQuaCo was to provide researchers with tools to generate information on their spatial datasets, to facilitate downstream decisions on data inclusion and cut-offs.

(3) Although the authors demonstrate the applicability of MerQuaCo on a large MERFISH dataset, and the limited number of sections from other platforms, it would be helpful to describe its limitations in its generalizability.

In figure 9, we addressed the limitations and generalizability of MerQuaCo as best we could with the available datasets. Gaining deep insight into the limitations and generalizability of MerQuaCo would require application to multiple large datasets and, to the best of our knowledge, these datasets are not available.

Reviewer #2 (Public review):

The authors present MerQuaCo, a computational tool for quality control in image-based spatial transcriptomic, especially MERSCOPE. They assessed MerQuaCo on 641 slides that are produced in their institute in terms of the ratio of imperfection, transcript density, and variations of quality by different planes (x-axis).

Strengths:

This looks to be a valuable work that can be a good guideline of quality control in future spatial transcriptomics. A well-controlled spatial transcriptomics dataset is also important for the downstream analysis.

Weaknesses:

The results section needs to be more structured.

We have split the ‘Transcript density’ subsection of the results into 3 new subsections.

Reviewer #3 (Public review):

MerQuaCo is an open-source computational tool developed for quality control in imagebased spatial transcriptomics data, with a primary focus on data generated by the Vizgen MERSCOPE platform. The authors analyzed a substantial dataset of 641 freshfrozen adult mouse brain sections to identify and quantify common imperfections, aiming to replace manual quality assessment with an automated, objective approach, providing standardized data integrity measures for spatial transcriptomics experiments.

Strengths:

The manuscript's strengths lie in its timely utility, rigorous empirical validation, and practical contributions to methodology and biological discovery in spatial transcriptomics.

Weaknesses:

While MerQuaCo demonstrates utility in large datasets and cross-platform potential, its generalizability and validation require expansion, particularly for non-MERSCOPE platforms and real-world biological impact.

We agree that there is value in expanding our analyses to non-Merscope platforms, to tissues other than brain, and to analyses other than cell typing. The limiting factor in all these directions is the availability of large enough datasets to probe the limits of MerQuaCo. We look forward to a future in which more datasets are available and it’s possible to extend our analyses

Reviewer #1(Recommendation for the Author):

(1) To better capture the downstream impacts of imperfections, consider extending the analysis to additional metrics, such as specificity variation across cell types, gene coexpression, or spatial gene patterning. This would deepen insights into how these imperfections shape biological interpretations and further demonstrate the versatility of MerQuaCo.

These are compelling ideas, but we are unable to study so many possible downstream impacts in sufficient depth in a single study. Insights into these topics will likely come from future studies.

(2) In Figure 7 legend, panel label (D) is repeated thus panels E-F are mislabelled. 

We have corrected this error.

(3) Ensure that the image quality is high for the figures. 

We will upload Illustrator files, ensuring that images are at full resolution.

Reviewer #2 (Recommendation for the Author):

(1) A result subsection "Transcript density" looks too long. Please provide a subsection heading for each figure. 

We have split this section into 3 with new subheadings.

(2) The result subsection title "Transcript density" sounds ambiguous. Please provide a detailed title describing what information this subsection contains. 

We have renamed this section ‘Differences in transcript density between MERSCOPE experiments’.

Minor: 

(1) There is no explanation of the black and grey bars in Figure 2A.

We have added information to the figure legend, identifying the datasets underlying the grey and black bars.

(2) In the abstract, the phrase "High-dimension" should be "High-dimensional". 

We have changed ‘high-dimension’ to ‘high-dimensional’.

(3) In the abstract, "Spatial results" is an unclear expression. What does it stand for? 

We have replaced the term ‘spatial results’ with ‘the outputs of spatial transcriptomics platforms’.

Reviewer #3 (Recommendation for the Author):

(1) While the tool claims broad applicability, validation is heavily centered on MERSCOPE data, with limited testing on other platforms. The authors should expand validation to include more diverse platforms and add a small analysis of non-brain tissue. If broader validation isn't feasible, modify the title and abstract to reflect the focus on the mouse brain explicitly.

We agree that expansion to other platforms is desirable, but to the best of our knowledge sufficient datasets from other platforms are not available. In the abstract, we state that ‘… we describe imperfections in a dataset of 641 fresh-frozen adult mouse brain sections collected using the Vizgen MERSCOPE.’

(2) The impact of data imperfections on downstream analysis needs a more comprehensive evaluation. The authors should expand beyond cluster label changes to include a) differential expression analysis with simulated imperfections, b) impact on spatial statistics and pattern detection, and c) effects on cell-cell interactions. 

Each of these ideas could support a substantial study. We are unable to do them justice in the limited space available as an addition to the current study.

(3) The pixel classification workflow and validation process need more detailed documentation. 

The methods and results together describe the workflow and validation in depth. We are unclear what details are missing.

(4) The manuscript lacks comparison to existing. QC pipelines such as Squidpy and Giotto. The authors should benchmark MerQuaCo against them and provide integration options with popular spatial analysis tools with clear documentation.

To the best of our knowledge, Squidpy and Giotto lack QC benchmarks, certainly of the parameters characterized by MerQuaCo. Direct comparison isn’t possible.

Puisque la thèse est désormais soutenue, y a-t-il un lien où il serait possible de renvoyer le lectorat ? Elle pourrait minimalement être intégrée à la bibliographie.

Un peu de contexte supplémentaire serait nécessaire ici. Dans le passage précédent, on croit comprendre que la demande de stage de Phan Vô Kỵ à la bibliothèque nationale est refusée ; il est pourtant ici question de « retour à Sài Gòn » (depuis la France, croit-on comprendre). Vous citez également le dossier personnel d'employé de Phan Vô Kỵ à la BnF : y a-t-il donc finalement occupé un rôle ? Si c'est le cas, il serait bon de préciser lequel.

Y a-t-il une raison pour laquelle « Sud Viêtnam » est orthographié de deux manières différentes ?

Synthèse sur le rôle de l'alcool dans la société

Résumé

Ce document de synthèse analyse le rôle complexe et paradoxal de l'alcool dans la société, en se basant sur des perspectives historiques, socioculturelles, scientifiques et politiques.

L'alcool est présenté comme une substance à double tranchant : d'une part, un puissant lubrifiant social et un pilier de rituels culturels et de moments de convivialité, profondément ancré dans l'histoire de l'humanité depuis des millénaires.

D'autre part, il est une force destructrice majeure, responsable de 2 200 décès par jour en Europe selon l'OMS, lié à plus de 200 maladies, et engendrant des coûts sociétaux colossaux, estimés à 57 milliards d'euros par an rien qu'en Allemagne.

Le document met en lumière l'ambivalence fondamentale de la société face à l'alcool, oscillant entre sa célébration dans les rituels et la stigmatisation de la dépendance individuelle.

Les tentatives historiques et modernes de régulation se sont souvent heurtées à une forte résistance populaire, illustrant la difficulté de gérer une substance si intimement liée au plaisir, à l'identité et à la cohésion sociale.

En définitive, les politiques les plus efficaces pour réduire les méfaits de l'alcool, à savoir l'augmentation des prix et la limitation de l'accès, se heurtent à cette acceptation culturelle profondément enracinée.

1. Le Paradoxe Fondamental de l'Alcool : Plaisir et Destruction

L'alcool occupe une place centrale et ambivalente dans la société, incarnant à la fois le plaisir et le danger.

Cette dualité est au cœur de notre rapport à cette substance.

• Le Côté Positif : L'alcool est associé à des sensations agréables, comme une "douce sensation de chaleur dans le ventre", et à des contextes plaisants.

Il est perçu comme un facilitateur de convivialité, pouvant donner lieu à des "conversations intéressantes" et favoriser le sentiment d'appartenance.

Une citation résume bien ce paradoxe :

"je dis toujours que j'ai passé certaines des meilleures nuits de ma vie avec de l'alcool et aussi certaines des pires."

• Le Côté Sombre : Son pouvoir destructeur est immense.

◦ Mortalité : L'OMS estime qu'environ 2 200 personnes meurent chaque jour en Europe à cause de l'alcool.      ◦ Maladies : Des études récentes lient une consommation régulière d'alcool à plus de 200 maladies.   

◦ Dépendance : L'alcool est la troisième substance la plus addictive en Allemagne, après le tabac et les médicaments.

En France, une personne sur dix a un problème avec l'alcool.   

◦ Conséquences Sociales : Il mène à la solitude, l'anxiété, la dépression et la dépendance.

Bien que la consommation globale soit en baisse en Europe, elle reste significative.

En Allemagne, elle est passée de 141 L à 115 L de boisson alcoolisée par an et par habitant depuis 2008, ce qui équivaut encore à "une bière par jour".

2. Une Perspective Historique : Un Compagnon de l'Humanité

La relation de l'humanité avec l'alcool est millénaire, suggérant qu'il a pu jouer un rôle dans notre évolution et le développement de nos civilisations.

• Origines Ancestrales : Des indices suggèrent que l'alcool est "aussi vieux que l'humanité".

◦ Des archéologues ont découvert en Chine des récipients contenant des restes de vin vieux de 9 000 ans.   

◦ En Géorgie, la consommation d'alcool remonte à au moins 8 000 ans.  

◦ La découverte est probablement fortuite, issue de fruits fermentés naturellement.

• Avantages Historiques :

◦ Source d'Énergie : 1 gramme d'alcool contient 7 calories, soit presque le double des protéines ou des glucides.  

◦ Sécurité Sanitaire : L'alcool dissout la membrane des germes, rendant les boissons fermentées (bière, vin) plus sûres à consommer que l'eau potentiellement contaminée.  

◦ Moyen de Paiement : La bière était utilisée comme une quasi-monnaie.

Un bulletin de paie en argile de Mésopotamie, vieux de 5 000 ans, indique des unités de bière.

En Égypte, les ouvriers des pyramides étaient rémunérés en bière.

• Consommation Massive : Au Moyen Âge en Europe, des chercheurs estiment la consommation à 3 litres de boisson alcoolisée par jour et par habitant, y compris pour les enfants.

3. Le Rôle Socioculturel : Ciment des Relations Humaines

L'alcool est omniprésent dans les structures sociales, agissant comme un "lubrifiant social" et un marqueur des moments importants.

• Cohésion Sociale :

◦ Il favorise le "sentiment d'appartenance" en créant une expérience collective.   

◦ Une expérience a montré qu'un groupe consommant un peu de vodka "interagissait davantage, riait beaucoup plus et passait globalement un moment plus agréable".  

◦ Des études indiquent que les personnes qui fréquentent régulièrement les bars avec modération sont mieux intégrées socialement.

• Rituels et Célébrations : L'alcool sert à marquer la frontière entre le "quotidien et la normalité de l'exceptionnel".

◦ Il est présent à chaque étape de la vie : naissance ("mouiller la tête"), mariages (champagne), enterrements.   

◦ Même dans un contexte religieux, le vin est utilisé pour représenter le sang du Christ.  

◦ Utiliser une boisson plus chère et exceptionnelle comme le champagne pour un anniversaire est une façon de "marquer un moment solennel".

• Influence sur le Développement Sociétal :

◦ Sédentarisation : Une théorie postule que la production de bière sur des sites comme Göbekli Tepe (il y a 12 000 ans) a pu renforcer la cohésion sociale et inciter les groupes humains à se sédentariser.    

◦ Infrastructures : La production d'alcool a influencé le développement des moyens de transport (fûts), des espaces de stockage et des bâtiments (brasseries).

• Variations Culturelles : Les coutumes de consommation varient :

◦ Norvège : Sobriété la semaine, forte consommation le week-end.  

◦ France/Italie : Un verre de vin au déjeuner.

4. Impacts sur la Santé et Mécanismes d'Action

D'un point de vue chimique et biologique, les effets de l'alcool sur le corps expliquent à la fois son attrait et sa dangerosité.

• La Molécule d'Éthanol : Petite molécule (deux atomes de carbone, six d'hydrogène, un d'oxygène), elle traverse facilement la barrière hémato-encéphalique pour agir sur le cerveau.

• Action sur les Neurotransmetteurs : L'alcool influence trois systèmes principaux : | Système | Effet Principal | Conséquence | | :--- | :--- | :--- | | GABA | Anxiolytique | Sensation de détente, réduction de l'anxiété | | Glutamate | Augmente la vigilance | Stimulation de la présence et de l'attention | | Dopamine | Rend heureux | Sensation de plaisir, voire d'euphorie |

• Toxicité Métabolique :

◦ Le foie transforme l'alcool en acétaldéhïde, qui est un "poison".   

◦ Cette substance circule dans le sang et atteint tous les organes (cerveau, peau, etc.).  

◦ Dommages Spécifiques : L'alcool peut provoquer des gastrites (attaque des muqueuses de l'estomac), endommager le foie, entraîner une atrophie du cervelet et être toxique pour le pancréas.  

◦ Risque de Cancer : La consommation régulière d'alcool augmente le risque de tumeurs et de cancer.

5. Dépendance, Coûts et Ambivalence Sociétale

La société entretient une relation contradictoire avec l'alcool, le célébrant tout en laissant les individus gérer seuls ses conséquences les plus graves.

• La Dépendance :

◦ La plus grande difficulté est le déni : "plus les gens sont dépendants, moins ils se rendent compte qu'ils le sont."   

◦ La dépendance isole l'individu, produisant l'effet inverse du sentiment d'appartenance initialement recherché.

• Coûts Économiques :

◦ Selon l'annuaire des addictions, l'alcool coûte 57 milliards d'euros par an en Allemagne.  

◦ Ces coûts incluent les délits, la violence, la conduite en état d'ivresse, les arrêts maladie et les traitements.

• L'Hypocrisie Sociale :

◦ La société vend l'alcool comme "quelque chose de positif associé à des fêtes", mais "ceux qui ne savent pas gérer leur consommation sont livrés à eux-mêmes".

La responsabilité est individualisée.   

◦ Cette ambivalence se reflète dans les politiques publiques : en 2024, la Société allemande de nutrition a recommandé "zéro alcool", tandis que 30 % du budget de prévention des addictions était supprimé.  

◦ La publicité pour l'alcool reste peu réglementée et la "consommation accompagnée" (dès 14 ans) est autorisée en Allemagne.

6. Les Tentatives de Régulation et la Résistance Populaire

L'histoire montre que les tentatives de contrôle de la consommation d'alcool par les autorités se sont souvent soldées par des échecs face à la pression sociale.

• Le Cas de la Bavière (1844) : Le roi Louis Ier a tenté d'augmenter le prix de la bière.

La mesure a provoqué de tels "remous au sein de la population" qu'elle a été annulée après seulement quatre jours.

L'alcool est perçu comme un "dernier bastion qui nous permet de nous distinguer en tant qu'être humain".

• La Campagne de Gorbatchev (années 1980) : Mikhaïl Gorbatchev a lancé une campagne anti-alcool en URSS pour améliorer la santé publique.

◦ Résultats sanitaires : La mortalité a considérablement diminué durant cette période.   

◦ Échec politique : La campagne a été un "désastre" pour Gorbatchev, contribuant à sa chute. L'ironie veut qu'il ait cédé le pouvoir à Boris Eltsine, "notoirement alcoolique".

• La Prohibition aux États-Unis : Bien qu'elle ait généré un marché noir, la prohibition a entraîné une baisse considérable de la consommation d'alcool et des maladies et décès qui y sont liés.

• L'Ambivalence de l'Église : L'Église chrétienne a prêché la modération ("l'idéal chrétien de la juste mesure") tout en intégrant le vin dans ses rites les plus sacrés (la Cène, les noces de Cana), illustrant une "hypocrisie généralisée vis-à-vis de l'alcool".

7. Vers des Politiques Efficaces ?

Le document suggère que les campagnes de sensibilisation actuelles sont largement inefficaces et que des mesures plus structurelles sont nécessaires pour réduire les méfaits de l'alcool.

• Inefficacité des Campagnes : Les campagnes de sensibilisation sont jugées peu efficaces ; elles servent surtout à "donner bonne conscience".

• Les Deux Leviersefficaces : Pour réduire la consommation, deux mesures sont jugées primordiales :

1. Limiter l'accès à l'alcool.    2. Augmenter son prix.

• L'Exemple du Tabac : Le Royaume-Uni est cité en exemple.

Avec un paquet de cigarettes à 16 €, le taux de fumeurs est de 11,9 %, contre 24,5 % en France et 20,1 % en Allemagne, où les prix sont plus bas.

• La Question de la Fiscalité : Il est noté que l'alcool est "très bon marché" dans de nombreuses régions d'Europe. Par exemple, la taxe minimale sur le vin fixée au sein de l'UE est de 0 €.

8. Conclusion : Accepter une Réalité Humaine et Complexe

L'attrait pour l'alcool, malgré ses dangers connus, semble être une caractéristique profondément humaine, liée à une "dimension autodestructrice" ou à un "désir d'échapper à la réalité de la vie".

Les individus réagissent souvent avec colère aux avertissements, les percevant comme une forme d'infantilisation.

La conclusion suggère qu'il est peut-être impossible d'apprécier l'alcool "sans la double morale qui l'accompagne".

La première étape serait de reconnaître pleinement le paradoxe de l'alcool, ses avantages et ses inconvénients, afin d'apprendre à vivre avec cette substance complexe qui ne semble pas prête de disparaître de nos sociétés.

L'Éducation comme Instrument de Pouvoir : Une Analyse Historique

https://www.youtube.com/watch?v=JCKbqhfFKy8

Résumé

Ce document de synthèse analyse le rôle historique de l'éducation, démontrant qu'au-delà de son idéal d'épanouissement personnel et de service du bien commun, elle a principalement été un instrument stratégique utilisé par les élites pour asseoir et maintenir leur pouvoir.

L'analyse, qui s'étend de la Sparte antique à l'époque contemporaine, révèle un schéma récurrent :

la mise en place de systèmes d'instruction publique est souvent une réponse directe aux troubles sociaux et vise à former des citoyens obéissants, à consolider des empires et à imposer des normes culturelles.

Des cas d'étude allant de la Prusse, pionnière de l'école obligatoire pour mater les révoltes paysannes, à la Chine impériale, utilisant des examens méritocratiques pour briser le pouvoir de la noblesse, illustrent cette thèse.

L'exemple tragique des pensionnats autochtones au Canada expose la forme la plus extrême de cette instrumentalisation, où l'éducation devient une arme de domination culturelle et d'éradication.

En conclusion, l'histoire révèle une tension fondamentale entre une éducation visant l'autonomie et la pensée critique, et une formation axée sur la performance, l'obéissance et la consolidation du statu quo.

1. Introduction : Le Droit à l'Éducation et ses Desseins Cachés

L'idéal moderne de l'éducation, tel que conçu par Platon comme une sortie de "la caverne de notre propre ignorance" et consacré par l'article 26 de la Déclaration universelle des droits de l'homme de 1948, postule l'instruction comme un droit fondamental au service de l'intérêt général.

Des études américaines corrèlent même un diplôme universitaire à une espérance de vie accrue de près de neuf ans.

Cependant, un examen historique approfondi soulève une question essentielle : l'éducation a-t-elle toujours poursuivi cet objectif d'émancipation ?

L'histoire suggère que l'instruction publique a souvent été un outil au service d'intérêts politiques et de stratégies de pouvoir bien définies.

2. Les Origines du Contrôle Social par l'Instruction

Loin d'être une invention des démocraties modernes, l'instruction publique obligatoire trouve ses racines dans des régimes autocratiques qui y ont vu un moyen efficace de garantir l'ordre social et la stabilité de leur pouvoir.

Sparte : Former le Guerrier-Citoyen Obéissant

Le premier exemple d'un système éducatif public structuré ne se trouve pas dans l'Athènes démocratique, mais dans la dictature militaire et esclavagiste de Sparte.

• Contexte de Domination : La société spartiate était composée d'une minorité de citoyens libres (les Spartiates) dominant une très large population d'hilotes, des serfs autochtones.

Le rapport était estimé à sept hilotes pour un Spartiate.

• L'Agogé, un Outil de Contrôle : Pour maintenir le contrôle sur cette population asservie et supérieure en nombre, Sparte a mis en place l'agogé.

Il s'agissait d'un système éducatif public et obligatoire pour les garçons spartiates dès l'âge de 7 ans, conçu comme un camp d'entraînement militaire visant à former des "guerriers surhumains".

• Objectifs Pédagogiques : L'accent était mis sur l'endurance, l'obéissance et la suppression de toute faiblesse, comme en témoigne le "concours de flagellation".

• Une Alphabétisation Stratégique : Bien que les hilotes en soient exclus, le programme incluait l'alphabétisation.

L'objectif n'était pas l'épanouissement intellectuel, mais une compétence militaire :

"Si un spartiate est envoyé en mission d'espionnage et qu'il intercepte un message écrit, il doit être capable de le lire."

• Conclusion : L'éducation spartiate n'avait pas pour but le développement personnel mais la formation de citoyens-soldats obéissants, un instrument essentiel à la survie du pouvoir en place.

L'Empire Carolingien : Unifier pour Mieux Régner

Après la chute de l'Empire romain d'Occident, Charlemagne initia la première grande expansion de l'éducation en Europe.

Son projet, loin d'être purement altruiste, était une manœuvre calculée pour consolider son vaste empire.

• Besoin Administratif : Pour contrôler son territoire, Charlemagne avait besoin d'une administration solide et unifiée.

L'école de la cour servait de "vivier de futur haut fonctionnaire".

• Unification Religieuse et Culturelle : Le pouvoir de l'empereur reposant sur Dieu, il était crucial de diffuser un christianisme uniformisé.

La réforme éducative visait à améliorer le niveau des ecclésiastiques et à standardiser la liturgie dans tout l'empire.

• Harmonisation de l'Écriture : Pour une administration efficace, une écriture commune était nécessaire.

La "minuscule Caroline" fut développée à cette fin, unifiant la communication écrite.

Cette police est l'ancêtre directe de la police de caractères Times New Roman.

• Conclusion : Pour Charlemagne, l'éducation n'était pas une fin en soi, mais un "instrument nécessaire pour maintenir la cohésion de l'empire".

La Prusse : L'École Obligatoire comme Rempart contre les Révoltes

C'est en Prusse, en 1763, que Frédéric II promulgua la loi créant le premier système d'enseignement primaire obligatoire au monde.

L'analyse de la politologue Agustina Paglayan révèle que cette initiative, loin d'être un progrès démocratique, était une stratégie de contrôle social.

• Le Paradoxe des Autocraties : Paglayan souligne que "ce ne sont pas les démocraties qui ont conduit à la création d'un enseignement primaire dans le monde occidental.

Celui-ci s'est surtout développé et étendu avant que les pays ne deviennent démocratiques."

• L'Éducation en Réponse aux Crises : Un schéma récurrent a été identifié :

la plupart des lois sur la scolarité obligatoire ont été adoptées juste après des révoltes populaires.

◦ Prusse (milieu du 18e siècle) : La loi est promulguée suite à des rebellions paysannes.    ◦ Massachusetts (années 1780) :

La première loi américaine sur la scolarité obligatoire répond à la révolte de Shays.  

◦ France (1833) : La loi suit la révolution de Juillet.   

◦ Pérou (2000) : La scolarité est imposée dans les anciennes zones rebelles après une guerre civile de 20 ans.

• Objectif : l'Endoctrinement : Face à la peur des masses, les élites politiques ont utilisé l'école primaire pour "enseigner aux enfants que le statut quo est acceptable et qu'il n'y a aucune raison de se rebeller".

L'enfance est ciblée car c'est la période où "les valeurs morales et les comportements politiques se façonnaient le mieux".

• L'École comme "Prison de Jour" : Reprenant les idées de Michel Foucault, le document décrit l'école comme une institution disciplinaire.

Les enseignants agissent comme des gardiens, inculquant la ponctualité, l'immobilité, la sagesse et la soumission.

Le but est de "créer une machine sociale bien huilée".

• Le Modèle Humboldtien : Une vision alternative fut proposée par le Prussien Wilhelm von Humboldt, pour qui l'éducation devait viser l'épanouissement personnel de chaque individu, "quel que soit leur origine sociale".

Cependant, après la défaite de Napoléon, ses idées jugées "dangereuses" furent écartées au profit d'un retour à l'"obéissance aveugle".

3. L'Éducation comme Outil de Sélection et de Pouvoir

Au-delà de l'inculcation de l'obéissance, l'éducation a aussi servi à structurer les hiérarchies du pouvoir, comme le montre l'exemple de la Chine impériale.

La Chine Impériale et le Système des Examens (Keju)

Pendant plus de 1000 ans, la Chine a utilisé un système d'examens (le Keju, institué au 7e siècle) pour attribuer les postes de la fonction publique.

• Une Méritocratie de Façade : En apparence, le système était basé sur le mérite.

Les candidats, parfois près d'un million pour environ 400 postes de finalistes, devaient mémoriser des classiques confucéens comptant jusqu'à 400 000 caractères.

• Un Objectif Politique : L'objectif réel de l'empereur était de limiter l'emprise des familles nobles qui contrôlaient traditionnellement l'administration.

En instituant un système basé sur des examens, il étendait son propre pouvoir en créant une bureaucratie qui lui était directement redevable.

• Influence Globale : Ce modèle, basé sur le mérite pour contrer le népotisme, a inspiré des réformes similaires jusqu'en Angleterre au milieu du 19e siècle.

4. L'Éducation comme Arme de Domination Culturelle

Le cas des pensionnats pour autochtones au Canada représente l'utilisation la plus sinistre de l'éducation, où elle est détournée pour devenir un outil d'éradication culturelle.

Le Témoignage de Gary Godfriitson (Peuple Sir Weepom)

Gary Godfriitson, gardien du savoir de la communauté Sir Weepom, décrit le système éducatif autochtone traditionnel comme étant basé sur "une étude attentive des enfants" pour découvrir leurs talents individuels et leur assigner des mentors experts.

Ce système, jugé "rétrograde" par les colons européens, fut systématiquement démantelé.

• Les Pensionnats : Des écoles spéciales, ou pensionnats, furent créées avec pour objectif de "détruire les cultures autochtones du Canada".

Gary Godfriitson, entré à 5 ans, se souvient : "Nous avons appris à nous taire. Nous avons appris que nous n'avions pas de voix dans ces pensionnats."

• Un Système d'Abus : Les enfants étaient soumis à un régime de discipline stricte, de prières constantes et de travail forcé ("un camp de travail pour enfants").

Ils subissaient "toutes sortes de violences (...) sexuel, physique, émotionnel".

• Bilan Tragique : Environ 150 000 enfants autochtones sont passés par ces établissements.

Un institut de recherche canadien estime qu'au moins 4 100 d'entre eux y sont morts de maladie, de négligence, de mauvais traitements ou en tentant de fuir.

• Un Projet Colonial Global : Ces pensionnats n'étaient pas une exception mais "l'un des outils majeurs pour la domination culturelle et soumettre l'autre".

5. Conclusion : Quelle Finalité pour l'Éducation de Demain ?

L'histoire démontre que l'éducation a trop rarement été "vouée au seul bien commun".

Elle a plus souvent servi à "garantir le pouvoir, à orienter les carrières et à imposer des normes".

Aujourd'hui, une tension persiste entre deux modèles :

1. L'Éducation comme Formation : Un modèle axé sur la performance, la fonctionnalisation et la monétisation des connaissances, qui forme des individus adaptés à une "machine sociale bien huilée".

2. L'Éducation comme Épanouissement : Le modèle de Humboldt, qui privilégie le développement personnel, la recherche de la connaissance et du sens, et qui promeut la pensée critique et la créativité comme compétences fondamentales.

La question finale demeure : "Quelle formule souhaitons-nous pour l'avenir ?

Une éducation qui nous dicte ce que nous devons savoir ou une éducation qui nous aide à découvrir qui nous voulons vraiment être ?"

Esa parte me llamó mucho la atención porque siento que tiene razón. Hoy todo el mundo habla de la inteligencia artificial como si fuera algo que va a solucionarlo todo, pero en realidad todavía no sabemos si nos está ayudando tanto como creemos o si más bien nos está volviendo cómodos. A veces parece que dependemos demasiado de ella y se nos olvida pensar o crear por nuestra cuenta. Yo creo que, como con todo, lo importante es usarla con medida, sin dejar que nos quite lo que nos hace humanos.

Point

DOI: 10.1242/dev.177022

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

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What is this?

DOI: 10.1242/dev.177022

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1242/dev.177022

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1242/dev.177022

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1093/genetics/iyae181

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

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DOI: 10.1091/mbc.e19-12-0673

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

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DOI: 10.1002/dneu.22731

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

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DOI: 10.1016/j.ccell.2025.09.007

Resource: (BioLegend Cat# 304822, RRID:AB_830801)

Curator: @scibot

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Synthèse sur les Impacts de la Séparation Parentale sur les Enfants

Résumé (11 sources)

La séparation parentale est un phénomène sociétal majeur qui a des répercussions profondes et multidimensionnelles sur les enfants.

Les impacts varient considérablement en fonction de l'âge de l'enfant au moment de la rupture, du niveau de conflit entre les parents, du contexte socio-économique et du mode de garde adopté.

Cette synthèse, basée sur une analyse de plusieurs études et rapports, met en lumière les conséquences psychologiques, scolaires, professionnelles et économiques de la séparation sur les enfants et les jeunes adultes.

Les impacts les plus significatifs sont :

1. Conséquences Économiques et Matérielles : La séparation entraîne une baisse de niveau de vie marquée et durable pour les enfants, estimée à 19 % en moyenne l'année de la rupture et persistant à 12 % cinq ans après. Le taux de pauvreté des enfants concernés double, passant à 29 % l'année de la séparation. Cette précarité est particulièrement notable pour les enfants résidant principalement avec leur mère et ceux issus de ménages au niveau de vie intermédiaire avant la rupture. La séparation provoque également des déménagements fréquents (six enfants sur dix dans les trois ans) et une diminution de l'accès à la propriété pour le parent gardien.

2. Répercussions sur la Réussite Scolaire et Professionnelle : Les études convergent pour montrer que la séparation parentale avant 18 ans est associée à une réussite scolaire plus faible. Cela se traduit par une durée d'études réduite, une probabilité moindre d'obtenir un diplôme et des performances académiques inférieures. Les garçons semblent particulièrement affectés en matière de rendement scolaire. De plus, les jeunes issus de familles recomposées manifestent un désir d'indépendance plus précoce, les poussant vers des "petits boulots" ou des formations courtes au détriment d'études longues, souvent pour éviter de peser financièrement sur une structure familiale perçue comme fragile.

3. Impacts Psychologiques et Comportementaux : L'âge de l'enfant est un facteur déterminant de sa compréhension, de ses émotions et de ses réactions. Les plus jeunes (moins de 5 ans) peuvent subir des retards de développement et développer un fort sentiment d'insécurité. Les enfants d'âge scolaire (6-12 ans) sont confrontés à des conflits de loyauté et peuvent développer des stratégies d'adaptation complexes. Les adolescents, en pleine construction identitaire, peuvent voir leur estime de soi diminuer et remettre en question leur capacité à nouer des relations futures. Le conflit parental est un facteur aggravant majeur, augmentant les risques d'anxiété et de dépression.

4. La Question de la Résidence Alternée : Bien que la loi privilégie souvent l'hébergement égalitaire, son application et ses bénéfices font l'objet de débats. Des craintes subsistent quant à son adéquation pour les très jeunes enfants (moins de 3 ans) en raison de la théorie de l'attachement principal. Cependant, un large consensus scientifique international, s'appuyant sur des décennies de recherche, affirme que la résidence alternée est bénéfique pour les enfants de tous âges, y compris les plus jeunes, car elle favorise le maintien de liens d'attachement multiples et solides avec les deux parents, ce qui est crucial pour leur bien-être psychologique et leur développement, même en cas de conflit parental.

En conclusion, si la séparation est un choc indéniable, ses effets négatifs peuvent être atténués par des facteurs de protection clés : le maintien d'une coparentalité de qualité, une communication ouverte et adaptée à l'enfant, la réduction du conflit parental, la stabilité des routines et un soutien socio-économique adéquat, incluant le versement régulier des pensions alimentaires et des politiques publiques efficaces.

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1. Contexte et Ampleur du Phénomène

La séparation parentale est devenue une réalité sociétale courante. Les statistiques confirment l'ampleur du phénomène :

• En Belgique, 23 059 divorces ont été prononcés en 2017.

• En France, en 2020, près de quatre millions d'enfants mineurs avaient des parents séparés. Chaque année, environ 380 000 enfants sont concernés par la séparation de leurs parents.

La part des individus dont les parents se sont séparés a considérablement augmenté au fil des générations, passant de 3 % pour la génération née en 1946 à 15 % pour celle née en 1988. Ce changement structurel a des impacts à court, moyen et long terme sur les enfants, qui se répercutent sur l'ensemble du corps social.

2. Impacts Psychologiques, Émotionnels et Comportementaux par Âge

L'âge de l'enfant au moment de la séparation est un facteur déterminant dans la manière dont il vit, comprend et réagit à l'événement. L'analyse de l'UFAPEC, corroborée par d'autres études, permet de dresser un tableau détaillé des impacts selon les tranches d'âge.

2.1. La Compréhension de la Séparation

La capacité de l'enfant à comprendre la situation évolue avec son développement cognitif.

Tranche d'Âge

Niveau de Compréhension

Moins de 2 ans

Ne comprend pas le concept de divorce mais perçoit le changement, l'état émotionnel des parents et leur absence, ce qui peut se traduire par un sentiment d'abandon et d'insécurité.

2 à 5 ans

Commence à comprendre que quelque chose a changé, mais la situation reste complexe et confuse. Pose beaucoup de questions pour se rassurer.

6 à 12 ans

Comprend le divorce, ses raisons et le point de vue de chaque parent. Fait preuve d'empathie, mais nourrit souvent l'espoir d'une réconciliation.

Plus de 12 ans

Saisit la complexité des relations et comprend le divorce comme une incompatibilité du couple.

Témoignage de Clotilde, 37 ans : « Mes parents ont divorcé quand j’avais 2 ans et se sont fait une guerre sans merci pendant vingt ans, à coup de procès. Je garde un souvenir d’incompréhension totale, d’abandon. De honte, aussi, vis-à-vis des autres enfants. [...] J’en veux à mes parents de ne m’avoir jamais rien expliqué. »

2.2. Les Émotions de l'Enfant

Diverses émotions peuvent être ressenties, avec des dominantes selon l'âge.

Tranche d'Âge

Émotions et Sentiments Prédominants

Moins de 5 ans

Insécurité, peur de l'abandon, possessivité envers la figure d'attachement. Les mensonges ou le manque de clarté accentuent le sentiment que le monde est devenu un endroit peu sûr.

6 à 12 ans

Tristesse, deuil de la famille unie, conflit de loyauté. Peut se sentir personnellement rejeté ou, à l'inverse, développer une forte empathie et chercher à consoler ses parents.

Plus de 12 ans

Colère, tristesse, repli sur soi. Peut ressentir une diminution de l'estime de soi et remettre en question sa propre capacité future à établir des relations durables.

2.3. Les Réactions Comportementales

Les réactions observables varient également, allant de la régression à l'indépendance précoce.

Tranche d'Âge

Réactions Typiques

Âge préscolaire (<5 ans)

Comportements régressifs (ex: propreté), troubles du langage, anxiété, tristesse, sentiment de culpabilité. Peut manifester un retard dans l'acquisition des facultés psychomotrices.

Âge scolaire (6-12 ans)

Insécurité, peur de l'abandon, conflits de loyauté. Difficultés scolaires ou relationnelles. Peut développer des "stratégies d'affrontement" comme le refoulement des émotions.

Préadolescence

Colère envers les parents, sentiment de honte, troubles psychosomatiques.

Adolescence (>12 ans)

Comportements "parentifiés" (prise de responsabilité excessive), tendance à l'indépendance précoce, fugues, comportements déviants (délinquance, addictions), activités sexuelles précoces. Peut surinvestir ou désinvestir la sphère scolaire.

3. Impacts sur la Réussite Scolaire et Professionnelle

La séparation parentale est statistiquement corrélée à une performance scolaire et à un parcours éducatif moins favorables.

3.1. Baisse de la Réussite Scolaire

Plusieurs études quantitatives françaises démontrent un effet négatif de la séparation sur le parcours scolaire :

• Réussite scolaire plus faible : Les individus ayant vécu une séparation parentale avant leur majorité ont une réussite scolaire globalement plus faible.

• Durée des études : La durée moyenne des études est raccourcie de six mois à plus d'un an.

• Obtention de diplômes : La probabilité d'obtenir un diplôme, notamment le baccalauréat, est plus faible. L'avantage d'être issu d'un milieu social favorisé est fortement amoindri par la séparation.

• Facteur de genre : Les garçons semblent plus affectés que les filles, notamment en matière de rendement scolaire, lorsque la séparation intervient à l'aube de l'adolescence.

• Âge à la séparation : L'effet négatif est particulièrement prononcé pour les enfants jeunes (0-6 ans) et à des âges charnières comme l'entrée au CP ou en sixième.

3.2. Désir d'Indépendance et Stratégies d'Orientation

Une étude qualitative de Sylvie Cadolle met en lumière comment la situation familiale post-séparation influence les choix d'orientation des jeunes adultes :

• Conscience du coût : Les jeunes de familles recomposées sont très conscients du "coût" qu'ils représentent, ce qui peut générer des tensions.

• Recherche d'autonomie financière : Pour ne plus être un "enjeu" financier et échapper aux conflits, beaucoup cherchent l'autonomie le plus tôt possible en occupant des "petits boulots" pendant leurs études.

• Impact sur les études : Ce désir d'indépendance peut les pousser à choisir des formations plus courtes et rémunérées (comme les BTS en alternance) au détriment d'études longues et potentiellement plus qualifiantes.

• Conflits avec les beaux-parents : Des relations difficiles avec un beau-parent sont un facteur majeur poussant à une décohabitation précoce. Le soutien financier du côté paternel est souvent perçu comme amoindri, notamment en cas de réticence de la belle-mère.

4. Impacts Économiques et sur les Conditions de Vie

L'étude de France Stratégie (2024) détaille les conséquences économiques sévères de la séparation pour les enfants.

4.1. Baisse du Niveau de Vie et Augmentation de la Pauvreté

• Chute du niveau de vie : L'année de la séparation, le niveau de vie des enfants chute de 19 % en moyenne. Cette baisse reste significative cinq ans après, à -12 %.

◦ La baisse initiale est plus forte pour les enfants résidant principalement avec leur mère (-25 %) qu'avec leur père (-11 %).    ◦ Les enfants en résidence alternée connaissent une baisse de 12 %.

• Explosion de la pauvreté : Le taux de pauvreté des enfants de parents séparés passe de 13,5 % avant la rupture à 29 % l'année de celle-ci, et se maintient à 21 % cinq ans plus tard.

• Facteurs d'amortissement : Cette baisse est partiellement amortie par :

◦ Les transferts sociaux et fiscaux, qui jouent un rôle crucial pour les ménages les plus modestes.    ◦ Les pensions alimentaires, qui sont plus significatives pour les ménages aisés. Cependant, deux ans après le divorce, 20 % des pensions ne sont pas versées régulièrement.    ◦ La reprise d'activité des mères.

• Remise en couple : La remise en couple du parent gardien fait disparaître la baisse de niveau de vie, mais ne concerne que 30 % des enfants six ans après la séparation.

4.2. Impact sur le Logement

• Déménagements : Six enfants sur dix déménagent dans les trois ans suivant la séparation, dont 38 % l'année même de la rupture.

• Statut d'occupation : Après la séparation, la part d'enfants vivant dans un logement dont un parent est propriétaire chute de 59 % à 38 %.

• Logement social : La part des enfants vivant en logement social augmente considérablement, surtout pour ceux résidant avec leur mère (passant de 15 % à 34 % l'année de la rupture).

5. La Question de la Résidence Alternée

Le mode de garde est un enjeu central. Si la législation tend à favoriser la résidence alternée, sa mise en œuvre et ses effets sont débattus, notamment en France où, en cas de désaccord, les juges ne l'accordent que dans 12 % des cas.

5.1. Arguments et Controverse

• Vision Traditionnelle (crainte pour les tout-petits) : L'analyse de l'UFAPEC (2018) relaie une opinion selon laquelle la résidence alternée avant 3 ans serait assimilée à de la "maltraitance", car le bébé n'aurait pas intégré la permanence des personnes et aurait besoin d'une figure d'attachement principale stable.

• Consensus Scientifique International : De nombreuses études récentes et méta-analyses contredisent fortement ce point de vue. Un consensus international, validé par des centaines de spécialistes, démontre les bienfaits de la résidence alternée pour les enfants de tous âges.

5.2. Synthèse des Études Internationales en Faveur de la Résidence Alternée

Chercheur / Étude

Année

Pays

Conclusion Principale

Consensus de 70 spécialistes

2021

Monde

Les enfants développent plusieurs relations d'attachement. Prioriser un seul parent peut compromettre ce réseau bénéfique et altérer la confiance de l'enfant.

Michel Grangeat

2018

France

S'appuyant sur les travaux de Michael Lamb, il affirme que les enfants sont prédisposés à des liens d'attachement multiples. La qualité de la relation dépend du temps passé, argumentant en faveur de la résidence alternée même pour les bébés.

Malin Bergström

2018

Suède

Une étude sur 3 662 enfants (2-9 ans) montre que ceux en résidence alternée souffrent de moins de problèmes psychologiques que ceux en garde exclusive.

William Fabricius

2017

États-Unis

Les enfants de moins de 2 ans passant un temps équivalent avec chaque parent développent des relations plus saines et solides avec eux à l'adolescence et à l'âge adulte.

Linda Nielsen

2014

États-Unis

Une synthèse de 40 études conclut que les enfants en résidence alternée ont un meilleur cursus scolaire, sont moins déprimés et plus équilibrés psychologiquement, même en cas de conflit parental.

Richard Warshak

2014

États-Unis

Une méta-analyse de 40 ans de recherche, validée par 110 experts, recommande la garde alternée comme norme pour tous les âges, soulignant ses bénéfices même en cas d'opposition initiale d'un parent.

Ces études suggèrent que les arguments contre la résidence alternée pour les jeunes enfants ne sont pas soutenus par les données scientifiques les plus récentes et les plus robustes.

6. Facteurs de Protection et Recommandations

Si les risques sont réels, tous les enfants de parents séparés ne subissent pas des conséquences négatives à long terme. Plusieurs facteurs peuvent protéger l'enfant et favoriser son adaptation.

• Qualité de la Coparentalité : Le facteur le plus important est la capacité des parents à coopérer, à communiquer et à maintenir un faible niveau de conflit.

• Communication avec l'Enfant : Il est crucial de parler à l'enfant de la séparation de manière claire, honnête et adaptée à son âge, en le rassurant sur le fait qu'il n'est pas responsable et qu'il continue d'être aimé par ses deux parents.

• Maintien des Relations : Maintenir une relation de qualité avec les deux parents est un facteur de protection majeur.

• Stabilité : Assurer une continuité dans la vie de l'enfant (maison, école, amis, activités) aide à son adaptation.

• Soutien Externe : L'école, les amis et la famille élargie peuvent jouer un rôle de soutien important. Les parents ne doivent pas hésiter à chercher de l'aide auprès de professionnels (médiateurs, psychologues).

• Soutien Public : Les politiques publiques doivent mieux accompagner les familles, notamment en assurant le versement effectif des pensions alimentaires et en fournissant des aides suffisantes pour amortir le choc économique de la séparation.

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Reply to the reviewers

_Below we address all the comments by the reviewers. However, the figures that were used in our response are unfortunately not displayed in this format. _

Reviewer #1

Evidence, reproducibility and clarity

Thanks to the development of Ribo-Seq, translational buffering has been reported in the database of published Ribo-Seq and matched RNA-Seq, Rao et al. attempt to understand the mechanism underlying translational buffering of mRNA variation across diverse materials. Although the authors' report provides a step forward in our understanding of translational buffering, this reviewer found a series of concerns in this paper. These points could be tackled to improve the reliability of their findings, the strength of their main message, and the global understandability of the paper.

Major comments: 1. This paper heavily relies on the reference 18. However, this paper was not properly stated (no page or journal number); the study in Bioinformatics is nowhere to be found on the website, despite being out in 2024 apparently. Either title is wrong (yet a biorxiv can be found). This reviewer guessed that the reference 18 may be accepted. However, without a proper reference, this paper could not be judged since nearly all the parts of this work have been based on the reference 18. Also, the Ribobase data used in this manuscript comes from this reference, so it had better be well defined, especially when another Ribobase data set seems to be available online: http://www.bioinf.uni-freiburg.de/~ribobase/index.html

We apologize for the citation issue. This citation by Liu et al , 2024 (18) was a preprint from BioRxiv. This manuscript is now published in Nature Biotechnology. The reference has been updated in the revised version of the manuscript. The reference number in revised manuscript is Liu et al, 2025 (23).

In the Discussion, the authors mentioned "TE is based on a compositional regression model (18) rather than the commonly applied approach of using a logarithmic ratio of ribosome occupancy to mRNA abundance." This important information should be mentioned in the early section of the manuscript. Related to this, there are other published methods for exploring change in translation efficiency (e.g., 10.1093/bioinformatics/btw585; 10.1093/nar/gkz223) that could also be suitable in this context. It is not entirely clear if their approach is better than before. Again, the improper reference to 18 made our assessment of this work difficult.

We apologize and acknowledge the impact of the citation issue on this point. In Liu et al (2025), we have provided a comparison between our approach and the log-ratio strategy. We also agree that additional context was needed within the current study. Hence, we have now included more detailed information about the TE calculations in the initial results section (line 94).

As noted by the reviewer, several other methods have been developed previously for measuring changes in translation efficiency. These methods are designed to be used in cases of paired designs where there is a treatment or manipulation that is assayed along with controls. While these methods are highly valuable in assessing differential TE, they are unable to accommodate the type of meta-analyses described in our study. In particular, we do not report changes/differential TE with respect to a control sample but instead focus on the coordinated patterns of TE across experiments. We now note this important distinction in the manuscript in the discussion section (line 494).

The paper mainly relies on detecting a set of buffered genes using mRNA-TE correlation and MAD ratios (Ribo-Seq/RNA-Seq). While the concept seems sound, the authors should ensure that this method is reliable. Several controls could be used to confirm this. First, if any studies in humans or mice have described a set of genes as buffered, it would be worth checking for overlap between the authors' set of 'TB high' genes and the previously established list. Furthermore, the authors could use packages explicitly developed for translational buffering detection, such as annota2seq (https://academic.oup.com/nar/article/47/12/e70/5423604?login=true). Not all of the data used by the authors may be suitable for such packages, but the authors could at least partially use them on some of their datasets and see whether the buffered genes reported by these packages match their predictions.

We thank the reviewer for this constructive suggestion. To the best of our knowledge, no prior study in humans or mice has systematically analyzed translational buffering across a wide range of conditions. As a result, defining a gold-standard set for benchmarking is currently not feasible.

While packages such as anota2seq have proven highly valuable for identifying buffering effects in controlled experimental designs (e.g., comparing a treatment to a matched control), they are not readily applicable to the type of large-scale meta-analysis we present here.Our study integrates ribosome profiling and RNA-seq data across diverse datasets and conditions, which lies outside the design scope of such tools.

The most relevant point of comparison to our work is Wang et al. 2020 Nature, which examined a related but distinct form of translational buffering across species for a given tissue. We now present the overlap of genes identified as buffered in our study vs Wang et al. 2020. The details are presented in the reviewer's comment 5-2.

The threshold of 'TB high' or 'TB low' (top and bottom 250) is somewhat arbitrary. Why not top 100 or 500? The authors should provide a rationale for this choice. Also, they could include a numeric measure of buffering (the sum of the two rankings is probably suitable for this purpose). Several of the authors' explorations are suitable for numerical quantification (GO enrichment can be turned into GSEA, and the boxplot can be shown as correlations)

Thanks for these suggestions. We agree that the threshold used to define TB high and low are somewhat subjective. We ensure that changing this cutoff as suggested is easily achievable with the provided R script. These can be used to reproduce all of the reported analyses of translational buffering with different cutoffs.

To further assess whether our conclusions are robust to the selection of these thresholds, we tested several different values to define the TB high and TB low groups. As an example, we show here that the effect on protein variation and association of intrinsic features like the UTR lengths with the buffering potential of genes for different thresholds (i.e. if the TB high = top 100 or TB high = top 200) remain similar to the current cutoff of 250. However, if we increase the cutoff of TB high to 2000 and TB low to top 2000-4000 , the difference between the various features is diminished (Figure A& B). Further, protein variation (human cancer cell line and tissue) also becomes more similar across the three categories, possibly indicating a reduced regulatory potential of genes as their rank increases (Figure C& D).Our analyses reveal that highly ranked genes show associations with particular features, indicating an underlying hierarchy in translational buffering potential. This point is now discussed in the manuscript (line 177).

Legend: Effect of different thresholds on . A. Length features B. Median RNA expression C. Protein variation in human cancer cell line and D. on Primary human tissues

In response to the reviewer's suggestion of presenting data using numerical quantitation, we incorporated several additional inclusions in the manuscript.

1. We now report association of CDS / UTR length with translational buffering as a function of their translational buffering rank with highly ranked genes showing associations with particular features, indicating an underlying hierarchy in translational buffering potential (Sup Fig 3 A-B) Ii. We now include scatter plots which show that highly ranked genes have lower variation at the protein level in both cancer cell line and primary tissues (Sup Fig. 6 A-C).

Iii. We have now carried out modified GO enrichment analyses. Specifically, Gene Ontology enrichment analysis was performed for the TB high genes in humans and mouse using the clusterProfiler R package. Lists of TB high genes in human or mouse were analyzed against the Gene Ontology (GO) database using the enrichGO() function, with the organism-specific annotation database (org.Hs.eg.db for human or org.Mm.eg.db for mouse) as reference. Gene identifiers were supplied as gene symbols, and all genes in the current study were used as the background universe. Enrichment was carried out for the Biological Process (BP) ontology, with significance assessed by the hypergeometric test. P-values were adjusted for multiple testing using the Benjamini–Hochberg method, and terms with an adjusted p-value Legend: Gene Ontology (GO) enrichment analysis of the TB high gene set, performed with the clusterProfiler R package. Enriched GO Biological Process terms are shown after redundancy reduction using clusterProfiler::simplify. Each dot represents a GO term, with dot size indicating the number of genes associated with the term and color reflecting the adjusted p-value (Benjamini–Hochberg correction). Only the top non-redundant terms are displayed.

• *

Additionally, we performed Gene set enrichment analysis using the list of genes ordered according to their RNA-TE correlation. Hence lower ranks have lower RNA-TE correlations. The GSEA plots show significantly enriched Gene Ontology Biological Process (GO:BP) terms at the lower ranks of the ordered gene list. Together, these analyses further emphasize the observation that genes involved in macromolecular complexes are translationally buffered.

• *

Legend: Curves represent the enrichment score (ES) across the ranked gene list, with vertical bars indicating the positions of pathway-associated genes. The enrichment was identified using the gseGO() function from clusterProfiler.

Several of the statements of the authors in the Introduction or Discussion sections are not entirely true regarding the literature on the topics, or lack major papers on the topic, and therefore, they are a bit misleading. Among others, here are some:

We thank the reviewer for the suggestions and now have been incorporated in the revised manuscript, accordingly.

5-1 "In addition, genetic differences arising from aneuploidy, cell type differences or variability observed in the natural population can further determine the amplitude of variation (4-7). The effect of mRNA variation under these conditions is mostly reflected at the protein levels (2, 4-8).". Several recent or more ancient papers suggest that mRNA variation coming from aneuploidy, natural genetic variation, or CNV is buffered or not well reflected at the protein level: DOI: 10.1038/s41586-024-07442-9 DOI: 10.1073/pnas.2319211121 DOI: 10.1016/j.cels.2017.08.013 DOI: 10.15252/msb.20177548

We agree that mRNA variation coming from aneuploidy, natural genetic variation, or CNV is buffered or not well reflected at the protein level for some genes. This point has now been revised in the introduction. We have incorporated all the suggested literature into the revised manuscript (line 38).

5-2: The authors should also consider mentioning these studies and softening their initial statement. "Similarly, translational buffering of certain genes have been reported in mammalian cells, specifically under estrogen receptor alpha (ERα) depletion conditions (16).". Translational buffering has been deeply explored in mammalian tissues and even across several mammalian species in this study (DOI: 10.1038/s41586-020-2899-z). In this, the authors also provide a nice exploration of the gene characteristics that are associated with translational buffering. The authors should mention it and compare the study's findings to theirs ultimately.

We thank the reviewer for this suggestion. We have now cited the recommended study in the revised manuscript (line 65). Here, we provide a comparison of its findings with ours. While this related work offers important insights into translational buffering, its focus is on buffering across species within a given tissue, whereas our study emphasizes buffering across conditions, cell types, and treatments within a species. Despite this difference in focus, the comparison is highly informative, and we now highlight both the similarities and distinctions between the two studies in the relevant section of the revised manuscript.

Wang et al. calculate the variation at the transcriptome level vs at the translatome level and is represented as delta ∆ value for each gene. A lower value represents lower variation at the ribosome occupancy level than at the mRNA levels across various species. We classified the genes in the Wang et al study as TB high, TB low genes or others as identified in the current study while indicating the calculated delta ∆ from Wang et al. Many of the genes with a lower delta value (are delta ∆ Legend: A. Dot plot to highlight the delta value of all genes in the Wang et al study (also present in RiboBase) which are further grouped as TB high, low or others in (A) brain and (B) liver.

5-3: "Differences in species evaluated and statistical methods have resulted in conflicting interpretations (13, 28).". These conflicting results have been previously discussed in reviews on the topic that would be worth mentioning: DOI: 10.1016/j.cell.2016.03.014 DOI: 10.1038/s41576-020-0258-4

We have added these reviews at the appropriate location of the manuscript.

1. In addition to the p-values stated in the main text, the authors should annotate their plots when they find significant differences between groups to greatly facilitate the visual interpretation of the graphs.

We have now annotated many of the relevant graphs with p-values to facilitate visual interpretation, adding them where space and figure design allow.

Based on the data of Figure 4D, apparently, ribosome occupancy was not buffered even in high TB sets. The authors may argue that translational buffering may not cope with such a strong mRNA reduction. In that case, how big a difference in mRNA level does the buffering system adjust in protein synthesis? The authors should test gradual gene knockdown and/or overexpression and conduct Ribo-Seq/RNA-Seq to survey the buffering range.

We appreciate the reviewer’s suggestion regarding the experiment to determine the buffering range.To understand this for multiple genes, we attempted a series of knockdowns using CRISPR/gRNA approach using a MutiCas12a approach. We targeted 8 buffered and 2 non-buffered genes using a 10-plex crRNA along with 10-plex gRNA serving as a negative control (Figure below). The fold change at the mRNA level of the targeted gene was within the variation range observed in replicates for other non-targeted genes. The challenge in performing a gradual knockdown is the subtle changes in RNA expression falls within the margin of error of estimation, making it difficult to understand the clear implications of the mRNA levels on buffering. Hence, the precise experimental manipulation of mRNA expression levels that would be conducive to translational buffering remains highly technically challenging. As noted in our manuscript (Figure 4D), the conventional approaches for manipulation of transcript abundance lead to larger changes than typically observed as a result of natural variation.

*Legend: Validation of translational buffering by targeted knockdown of genes. A. The scatter plot shows the coefficient of variation of mRNA and ribosome occupancy between HEK293T cells targeted with sgRNA of different efficiencies. The genes indicated in blue are buffered and those in green are non buffered genes. B. The plot shows the fold change in mRNA abundance and ribosome occupancy as compared to cells that were infected with non-targeting crRNA array control (ratio of cpm in test vs control). Each color represents a gene and each point of a gene represents cells targeted by one of the four CRISPR arrays. *

"differential transcript accessibility model" could not be functional if mRNA is reduced beyond the accessible pool (i.e., less than the threshold, all the mRNAs are translated without buffering). The authors should carefully reconsider this model and the effective range of mRNAs.

We agree with the reviewer that according to the 'differential transcript accessibility model,' transcripts with abundances below a certain threshold should be completely accessible to the translational pool. Further, this could also be true for the other model, wherein initiation rate cannot increase beyond a particular threshold for transcripts of very low abundance. However, our observation from our haploinsufficiency analysis (Figure 4 B& C) and siRNA knockdown analysis from RiboBase (Figure 4 D) suggests that buffering might be possible within a given range of transcript abundance. Testing the buffering range by serial knockdowns might help in determining the threshold at which transcripts exhibit buffering. However, due to the challenges of serial knockdown as discussed above, makes this analysis difficult with Ribosome profiling and matched RNA-seq approach. An alternative approach could involve imaging translating and non-translating mRNA of buffered genes in different cells, which may help distinguish the two models. However, this falls outside the scope of the manuscript.

Minor comments:

1. Some figures are of poor quality as they seem to have points outside of the panel representations... Like Figure 3C, one point is out of the square, same for Figure 4E. Similarly, on figure 5F, some outliers seem to be clearly cut from the figure (maybe not, but then the author should put a larger space between the end of the figure and the max y points). Same for panel S2D and S6D, this does not sound so rigorous.

We agree and apologize for this issue. The axes of the figures have been annotated appropriately to indicate the presence of outliers in the figures.

1. There are several typos or weird sentences. Here are some (but maybe not all): 2-1: [...]with lower sums corresponding to higher final ranks. "two rankings". Based on these final ranks[...] 2-2: For each dataset, median absolute deviation (MAD) "i" protein abundance was calculated across samples 2-3: [...]neighbor method implemented in the MatchIT package (38) Differences in protein[...] a point is missing here. 2-4: Additionally a second dataset providing predictions of haploinsufficiency (pHaplo score) and triplosensitivity (pTriplo score) for all autosomal genes (25) was used to asses the distribution of these score"S" across buffered and non-buffered gene sets . There is a missing "s" at "score" and there is a space between the last word and the final point.

The necessary corrections have been incorporated in the revised version of the manuscript.

1. In the "Lymphoblastoid cell line data analysis:" section, this reviewer wonders why the authors used a different method to calculate buffering compared to before.

The main reason is the limited sample of the lymphoblastoid cell line data. In our larger analyses, we could use median absolute deviation as a robust metric of dispersion across heterogeneous samples. However, given the smaller dataset in that study we decided CV would be a better indicator of dispersion. To evaluate the potential for translational buffering of genes from RiboBase, we used two metrics. The first was the negative correlation between translation efficiency and RNA abundance across samples. The second metric relied on the ratio of variation in ribosome occupancy to variation in RNA levels. Given the limited sample size of the lymphoblastoid cell line dataset, we used the coefficient of variation (CV) instead of the median absolute deviation (MAD), as the data in this study were normalized using counts per million (CPM) rather than the centered log-ratio (clr) normalization used in RiboBase. This CV ratio allowed us to assess the effect of natural variation in RNA abundance on ribosome occupancy.

1. "Samples which had R2 less than 0.2 were removed as the residuals calculated for these samples could be unreliable". These samples for which the correspondence between RNA-Seq and Ribo-Seq is low wouldn't be the ones most impacted by translational buffering? Is it sure that the authors are not missing something here?

We agree with the reviewer that genes that show translational buffering may not conform to linear relationships between the two parameters. However, the proportion of genes exhibiting this buffering effect is not expected to significantly influence the overall regression fit. Instead, we hypothesized that low quality samples or truly different relationships between the two parameters can make this relationship nonlinear, rendering it unsuitable for linear regression analysis for calculation of TE.

To address these possibilities, we first analysed a commonly used proxy for data quality. Given the characteristic movement of ribosomes across mRNAs, periodicity of sequencing reads is a useful metric to assess whether reads are randomly fragmented, as in RNA-seq, or specifically represent ribosome-protected footprints. For this, we compared two groups: samples that were removed (~30) and those retained for analysis. We plotted the distribution of periodicity scores for all samples in both groups. For the calculation of periodicity scores, first the percentage of reads mapped to the dominant frame position across the dynamic ribosome footprint read length range was calculated for each sample. The periodicity score was calculated by taking the weighted sum of these dominant percentages, with weights based on the total read counts at each length.

The results indicate that the removed samples did not have lower periodicity scores, suggesting that their quality in terms of periodicity was comparable to the retained samples.

        To assess the second possibility, we checked if the study involved major perturbations, which may skew the relationship towards non linearity. The 30 samples that were removed came from 14 unique studies, 18 of which involved perturbation which possibly affected either of the two parameters. In addition to the genetic/pharmacological perturbations specific to the study, the overall conditions of the cells during an experiment could influence this relationship. Another point to note is that many of the filtered-out samples are HeLa and HEK293T cells, which show a normal relationship between ribosome occupancy and RNA abundance for the majority of cases.

        These considerations suggest that removing these samples is most appropriate, as their inclusion could bias the TE calculations.

For Figure 4B and 4C, the authors should provide statistical tests and p-values to confirm the observed trends.

The haploinsufficiency and triplosensitivity analyses are now supported by a chi-squared test. The details of the statistical test are now mentioned in the text and the p-values have been noted on the respective figures.

In Figure 2A, the "all genes" color doesn't correspond to the point color.

The color in the figure has been modified in the revised version of the manuscript.

1. "To understand if codon usage patterns are[...]". This comes slightly out of the blue. The authors could maybe explain why codon usage should be explored for translational buffering. The authors should cite recent key works in the fields: DOI: 10.1016/j.celrep.2023.113413 DOI: 10.1101/2023.11.27.568910

We would like to thank the reviewer for their suggestion. The references have been incorporated in the revised version of the manuscript. We have now explained why codon usage could be a contributor in determining the translational buffering potential (line 190).

"The change in each metric was calculated by subtracting the mean value in the control samples from that in the knockdown samples. This yielded the differential mRNA abundance and ribosome occupancy resulting from gene knockdown.". This looks statistically weak. The authors should consider using more robust methods like DESeq.

We thank the reviewer for the suggestion. We reanalyzed the selected studies using edgeR and the modified figure is included in the revised version of the manuscript (Figure 4D). The conclusion after this analysis remains essentially the same. In particular, translational buffering is ineffective when mRNA abundance is perturbed drastically. Additionally, the limited number of experiments with direct perturbation of buffered genes limit the generalizability of this observation. This limitation is included in the result section (line 342).

Legend: Scatter plot represents log2 fold change in RNA abundance and ribosome occupancy. Each point represents a gene and the fold change in its RNA and ribosome occupancy with respect to their controls. The line represents the line of equivalence. Buffered genes do not show less change in ribosome occupancy upon reduction in their RNA levels than other genes.

1. "Genes in the buffered gene set had a higher codon adaptation index than the non-buffered set, indicating that candidates in the buffered gene set are relatively well expressed due to the presence of a higher proportion of the codons observed in highly expressed genes". What do the authors mean by "relatively well expressed"? Abundantly expressed? This sentence and the causality under it is unclear and should be modified or better explained.

We thank the reviewer for pointing out the lack of clarity in the sentence. We have now quantitatively measured the CAI in the three categories and modified the sentence to better explain the rationale in the revised version (line 183). “To understand if codon usage patterns are associated with translational buffering, we next analyzed codon properties across buffered and non-buffered human gene sets. The codon adaptation index quantifies how closely a gene’s codon usage aligns with that of highly expressed genes. Genes in the buffered gene set had a higher codon adaptation index than the non-buffered set. Specifically, 28.4% of TB high genes, 14% of TB low genes and 9.3% of genes in the other category fall within the top decile (>90th percentile) of codon adaptation index.”

The panel 4D is unclear. Is one point associated with one gene? Or is it the average of several genes? If it's one point for one gene, it is important to clearly state it because the number of cases is therefore quite low, especially for the TB high and low.

Each point and line are associated with a single gene. This is now clarified in the legend of the figure (line 364). The number of genes in this analysis is limited to the available ribosome profiling data with gene knockdown experiments.

1. In Figure 2J, GGU (Gly), AAG (Lys), and ACU (Arg) provide negative effects on prediction, although these were enriched in the high TB set (Figure 2E). This contradiction should be explained.

While this appears to be a seeming contradiction, it is in line with what we expected. In particular, the objective of Figure 2J is to illustrate the features that predict the mRNA–TE correlation of genes, as identified using a LGBM model. The Spearman correlation shown reflects the relationship between each feature and the mRNA–TE correlation values. A negative correlation for codons such as GGU (Gly), AAG (Lys), and ACU (Thr) suggests that enrichment of these codons is associated with lower mRNA–TE correlation. This is in agreement with our observation in Figure 2E which suggests that high TB genes are enriched in these codons. In contrast, transcript size exhibits a positive correlation, indicating that shorter transcripts tend to have lower mRNA–TE correlation values.

Given that the choice of colors is a potential source of confusion, we have revised the text (line 230) and the figure (& legend) to try to clarify this relationship.

The subtitle of "Translationally buffered genes exhibit variable association kinetics with the translational machinery in response to mRNA variation" sounds unfair to this reviewer. Since the authors did not work on kinetics directly, the use of this word is misleading.

We agree and revised the subtitle to “The association of translationally buffered genes with the translational machinery varies in response to changes in mRNA abundance"

1. The explanation of Figure 5A "We next explored the potential mechanisms that may give rise to translational buffering. Specifically, we considered two non-mutually exclusive models by which mRNA abundance might be decoupled from ribosome occupancy. In the first, the "differential transcript accessibility model", mRNA abundance determines the fraction of transcripts that are accessible to the translational pool. In this scenario, an increase in mRNA abundance would be accompanied by a proportionally smaller increase in the fraction of transcripts entering the translating pool for buffered genes, compared to non-buffered genes. In the second, the "initiation rate model", the rate of translation initiation per transcript scales inversely with mRNA abundance. Under this model, the proportion of mRNA entering the translational pool would be comparable across buffered and non-buffered genes (Fig 5A)." is hard to understand. The authors should rewrite for a better understanding of the readers.

This section has been rewritten in the revised version of the manuscript. The text now reads as

“We next explored the potential mechanisms that may give rise to translational buffering. Specifically, we considered two non-mutually exclusive models by which mRNA abundance might be decoupled from ribosome occupancy. In the first, the “differential transcript accessibility model”, mRNA abundance determines the fraction of transcripts that are accessible to the translational pool. In this scenario, an increase in mRNA abundance would be accompanied by a proportionally smaller increase in the fraction of transcripts entering the translating pool for buffered genes, compared to non-buffered genes. In the second, the “initiation rate model”, the rate of translation initiation per transcript scales inversely with mRNA abundance. Under this model, as mRNA abundance increases, translation initiation on each transcript is reduced, thereby lowering the number of ribosomes per transcript. However, this mechanism allows a proportional increase in transcripts entering the translational pool for buffered genes, similar to non-buffered genes”

Significance

Thanks to the development of Ribo-Seq, translational buffering has been reported in various works. However, the systematic investigation has remained challenging. Employing the database of published Ribo-Seq and matched RNA-Seq, Rao et al. attempt to understand the mechanism underlying translational buffering of mRNA variation across diverse materials. A group of mRNAs whose expression variance is buffered at the translation level was comprehensively surveyed in humans and mice. The authors found a series of features in the translationally buffered genes, including high GC contents in the 5′ UTR, optimal codon usage, and mRNA length. The depletion or increase of one allele of the genes in the group may be particularly detrimental to cells. The authors' report provides a step forward in our understanding of translational buffering, appealing to the broad scientific community in basic and applied biology. However, this reviewer found a series of concerns in this paper, including clarity in the methods, experimental validation, referring the earlier works, etc. These points could be tackled to improve the reliability of their findings, the strength of their main message, and the global understandability of the paper.

We thank the reviewer for noting the significance of the work and for their constructive feedback.

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

In this manuscript, Rao and colleagues present a comprehensive analysis of translational buffering in human and mouse by mining 1515 matched ribosome profiling and RNAseq datasets from diverse tissues and cell lines. They define translational buffering as genes whose TE is negatively correlated with mRNA abundance across conditions, and further identify candidates by comparing median absolute deviations of ribosome occupancy versus mRNA levels. The authors find a conserved set of buffered genes enriched for components of multiprotein complexes, demonstrate that buffered genes exhibit lower protein variability and greater dosage sensitivity, and propose two non-mutually exclusive mechanistic models (differential accessibility and initiation rate modulation). Finally, they perform complementary fractionation experiments in HEK293T cells to support these models.

These findings propose a novel, conserved mechanism of translational buffering that tunes gene expression in mouse and human, showing how intrinsic sequence features and cellular context cooperate to stabilize protein output across diverse conditions. However, further evidence is required to fully support the authors conclusions, particularly direct validation of the proposed models of buffering.

We thank the reviewer for their positive assessment and thoughtful suggestions that we address below.

Below are my main concerns:

1. The choice of the top 250 genes by spearman correlation and MAD ratio as "TB high" seems arbitrary. The authors should justify these cut offs (via permutation analysis or FDR control) and show that conclusions are robust to different thresholds.

We agree that the threshold used to define TB high and TB low is somewhat subjective, and we now clearly acknowledge this in the discussion section (line 485). We now provide an R script that reproduces all analyses of translational buffering, where changing this cutoff to higher or lower values is straightforward.

To ensure the robustness of our conclusions, we evaluated several thresholds for defining TB high and TB low. We observed that the conclusions hold within a reasonable range of values (100-250). For example, the effects on protein variation and the association of intrinsic features such as UTR lengths with buffering potential remain consistent when TB high is defined as the top 100 or the top 200 genes, compared with the current cutoff of 250. In contrast, when we define TB high as the top 2000 and TB low as ranks 2000–4000, the difference between the various features is diminished (Figure A& B). Further, protein variation (human cancer cell line and tissue) also becomes more similar across the three categories, possibly indicating a reduced regulatory potential of genes as their rank increases (Figure C& D). Our results show that highly ranked genes consistently associate with specific features, suggesting an underlying hierarchy in translational buffering potential.

Legend: Effect of different thresholds on . A. Length features B. Median RNA expression C. Protein variation in human cancer cell line and D. on Primary human tissues

The modified compositional regression approach for TE and imputation of missing values are central to the study, but details are relegated to supplemental methods. The manuscript would benefit from a clear comparison of this method against standard log-ratio TE estimates, including sensitivity analyses to missing-data imputation strategies

We thank the reviewer for the feedback. We have now added further description of the modified compositional regression and the imputation strategy in the results section (line 94). Comparison to standard log-ratio TE estimates and their limitations has already been detailed in Liu et al. 2025, Nature Biotechnology. Therefore, in the current manuscript we specifically focus on the effect of the imputation strategy.

        Specifically, the modified imputation slightly improved concordance between the set of genes that are identified to be translationally buffered using the negative RNA-TE relationship or using RNA -Ribosome occupancy correlation (0.91 to 0.94). Further, we assessed the correlation between TE and protein abundance as measured by mass spectrometry from seven human cell lines (A549, HEK293, HeLa, HepG2, K562, MCF7 and U2OS). The protein measurements were obtained from PaxDb. The new imputation strategy slightly increased mean correlation between the TE and proteome abundance as compared to naive strategy. It specifically showed improved correlation for HepG2, A549 and HeLa cell lines. 3507 genes were used for this analysis that were common between PaxDb, Liu et al., 2005 and the current study.

Legend: Proteomics vs TE correlation of cell types without or with imputation strategy. Spearman correlation between compositional TE calculated as calculated by Liu et al., 2025 from 68 samples from 11 studies (HEK293), 86 samples from 10 studies (HeLa), 58 samples from four studies (U2OS), 29 samples from five studies (A549), five samples from two studies (MCF7), seven samples from two studies (K562) and 10 samples from two studies (HepG2) or from the current study. 57 samples from 10 studies (HEK293), 82 samples from 9 studies (HeLa), 58 samples from four studies (U2OS), 29 samples from five studies (A549), 5 samples from two studies (MCF7), one samples from one studies (K562) and 9 samples from two studies (HepG2) . 3507 genes were used for this analysis that were common between Paxdb, Liu et al., 2005 and the current study.

Human data are derived mainly from immortalized cell lines, whereas mouse data are from primary tissues. Pooling these heterogeneous sources may conflate cell type-specific regulation with intrinsic buffering. The authors should either stratify analyses by context or demonstrate buffering signatures remain consistent within more homogeneous subsets

We thank the reviewer for the suggestion and agree that heterogeneity could potentially mask cell type-specific buffering effects. The TB-high genes we report are those that show consistent and robust expression across diverse contexts. However, unlike RNA-seq datasets, the current number of ribosome profiling samples per cell type is still limited, and a more comprehensive assessment of context-specific buffering will require larger datasets that will accumulate over time.

Nonetheless, we have stratified the analysis by cellular context. Specifically, we grouped samples of the same cell-type and repeated the buffering analysis. We provide a new table listing TB-ranks of genes for the five cell types with the largest sample sizes as a table in github.

https://github.com/CenikLab/Translational-buffering/blob/Translational-Buffering/combined_tables.xlsx

As an additional control, we compared buffering patterns between related and unrelated cell lines. For example, the correlation of TB ranks between related cell lines HEK293T (n = 98) and HEK293 (n = 57) is higher (0.46) than between either and an unrelated cell line, HeLa (n = 82). Similarly, the correlation between two liver cell lines, Huh7 (n = 39) and HepG2 (n = 9), is higher (0.20) than between Huh7 and a similarly sampled but unrelated lymphoblastoid cell line (LCL, n = 9; correlation = 0.05). While these analyses suggest that cell type-specific patterns may exist, their exploration is currently limited by sample size, as detecting buffering requires substantial variability in mRNA expression. We now highlight this as a limitation in the Discussion section (line 573).

*Legend: Spearman correlation between TB ranks of different pairs of cell lines. The first set indicates comparison with HEK293T. The second set indicates comparison between liver cells (HepG2 and Huh 7). *

The HEK293T fractionation experiments offer preliminary support for both the "accessibility" and "initiation" models, but only slope analyses are shown. To validate these models, the authors should perform targeted reporter assays (dual luciferase constructs with 5′UTR swaps) or manipulations of initiation factors (eIF4E knockdown) to directly test how transcript abundance alters initiation rates versus pool entry

We thank the reviewer for suggesting experiments to validate the proposed models. In the luciferase reporter experiments, constructs bearing the endogenous UTRs from non-buffered genes would be expected to result in expression that is proportional to transcript abundance. In contrast, swapping a 5’ UTR from buffered genes would mitigate this effect of translation buffering via “initiation rate model” depending on the 5 UTR sequence of transcript. However, as outlined below, this experiment has important caveats:

1. Role of coding sequence: Such assays primarily test the contribution of the 5′UTR and do not address potential cooperative effects between the 5′UTR and the coding sequence (CDS). Thus, if 5′UTRs fails to recapitulate translational buffering, it would be unclear whether the buffering requires coordinated action of the 5′UTR and CDS or whether the gene in question simply does not conform to the initiation-rate model.
2. Sensitivity of measurements: Reporter-based measurements often rely on RT-qPCR to quantify expression changes. While suitable for large fold-changes, small shifts may fall within the assay’s technical margin of error, limiting the interpretability of the results. iii. Gene-to-gene variability: Buffered and non-buffered transcripts likely span a wide range of intrinsic initiation rates. Selecting only a few “representative” transcripts for 5′UTR swapping could yield results that are not broadly generalizable.

Similarly, knockdown of general initiation factors will likely impact on both buffered and non-buffered genes, which could limit the ability to distinguish the effect of transcript abundance on translational buffering via either of the proposed models. We envision an alternative future approach that would involve single molecule imaging translating and non-translating mRNAs of buffered and non-buffered genes under varying abundance conditions in a physiological context. Such experiments are likely the most suitable for disentangling the contributions of accessibility versus initiation. While we find this an exciting direction for future work, it lies beyond the scope of the present manuscript.

The conclusion that buffering reduces protein variability relies on mass-spec comparisons, but ribosome occupancy does not always reflect functional protein output (due to elongation stalling or co-translational degradation). Incorporating orthogonal measures, such as pulse-labeling or western blots for key buffered versus non-buffered genes, would strengthen the link between buffering and proteome stability

We agree with the reviewer’s concern and have been acknowledged as a limitation in the discussion section. To address this with orthogonal approaches, we carried out several additional experiments. Specifically, we identified a study from RiboBase (GSE132703) that exhibited significant variation in FUS transcript (a translationally buffered gene) abundance across conditions—namely HEK293T wild type, LARP1A single knockout (SKO), and LARP1A/B double knockout (DKO) using their RNA-seq data. We reached out to the authors of the study and obtained these knockout cell lines. We reanalyzed RNA abundance under the different conditions by RT-qPCR and assessed protein levels by Western blot. Despite observing differences in RNA abundance, FUS protein levels did not exhibit corresponding change at the protein level.

We also selected a non-buffered gene; DNAJC6, that also showed RNA-level differences. However, the change in RNA expression was not consistent at the protein level. Some caveats of Western blot is its limited sensitivity which may prevent detection of subtle changes and that the measurements are steady-state protein levels which cannot resolve whether differences arise from altered synthesis or degradation.

*Legend : Validation of buffering gene by western blot: A. Plot showing the RNA abundance and ribosome occupancy of buffered gene ; FUS and non buffered genes; DNAJC6 with variation in HEK293T-wild type, LARP1A single knockout and LARP1A/B double knockout. B. Validation of the RNA seq data by qPCR. C. Western Blot showing the FUS, DNAJC6 and Actin in wild type and different mutants. D. Bar plot showing the quantification of western blot. *

        In addition to this targeted analysis , we performed quantitative mass spectrometry to evaluate the effect of mRNA variation at the protein level at global scale.

LC MS/MS analysis was performed on the above samples in triplicates at the Proteomics facility of the University of Texas. A total of 4,048 proteins were identified using a peptide confidence threshold of 95% and a protein confidence threshold of 99%, with a minimum of two peptides required for identification. Total precursor intensities for all peptides of a protein was summed and was used for protein quantification using DEP (Differential Enrichment of Proteomics Analysis) Package, in Bioconductor, R (https://rdrr.io/bioc/DEP/man/DEP.html). DEP was used for variance normalization and statistical testing of differentially expressed proteins. As expected LARP1 protein was identified in the control cells but not in the single or double knockouts.

We then plotted the fold change in RNA as determined by edgeR analysis of RNA-seq from (Philippe et al. 2020) and the fold change in protein abundance from our mass spectrometry data. We observed that genes in the TB high group show reduced changes at the protein level compared to TB low or others as determined by the linear regression analysis in both single and double LARP1 KO mutants. This finding is consistent with our findings that buffered genes show lower variation in the protein abundance in response to change in mRNA expression.

Legend: Scatter plot showing the log2fold change in the RNA and protein levels as determined by RNA seq from (Philippe et al. 2020) or mass spectroscopy. Differential analysis of RNA was done using the edgeR package and the DEP (Differential Enrichment of Proteomics Analysis) Package *was used for mass spectrometry analysis. Only genes with an FDR We have not included this data in the manuscript given the deviation of the approach from our original analysis, but we are happy to reconsider the inclusion of this data to supplement our proteomic analysis.

While the LGBM modeling shows modest predictive power of sequence features alone, the manuscript stops short of exploring what cellular factors might drive context dependence. Integrating public datasets on RNA-binding protein expression or mTOR pathway activity across samples could illuminate trans-acting determinants of buffering and move beyond correlative sequence analyses,

We thank the reviewer for this suggestion. To investigate potential trans-acting determinants of buffering, we focused on 1,394 human RBPs as classified by Hentze et al. (2018), reasoning that some of these factors may facilitate translational buffering. Specifically, we examined correlations between the RNA expression of each RBP and the TE of all other genes across samples. p-values were corrected using the Bonferroni procedure. For each RBP, we then performed a Fisher’s exact test to assess whether the number of significant correlations was enriched among buffered versus non-buffered genes.

This analysis revealed that the expression levels of many RBPs are significantly enriched for either positive or negative correlations with the TE of buffered genes. In particular, we note that RNA expression of many buffered RBPs is enriched for negative correlations with the TE of other buffered transcripts. These results suggest that, rather than considering translational buffering in isolation for each transcript, buffering effects may be coordinated at the translational level and influenced by shared trans-acting factors such as RBPs. Network-based approaches have been valuable for RNA co-expression and are only now being applied to TE covariation. However, the correlative nature of these analyses limits causal inference. For example, although many ribosomal proteins appear to influence the buffering of other ribosomal proteins, they themselves may be regulated by a non-ribosomal RBP—so the apparent effects could reflect upstream regulatory influences. This analysis is now included as a supplementary figure (Sup. Fig. 5) of the revised manuscript.

Legend: A scatter plot of odds ratio log₂ of number of significant correlations (RNA abundance of RBPs ::TE of genes) and the p value from fisher test. The vertical dashed line represents the threshold odds ratio, above which RBPs exhibit a higher number of significant correlations with buffered genes. P values were corrected using Bonferroni procedure* and the horizontal dashed line represents the adjusted p value cutoff. *

Reviewer #2 (Significance (Required)):

Overall, this manuscript leverages an unprecedented compendium of matched ribosome profiling and RNAseq datasets across human cell lines and mouse tissues, combined with improved TE estimation, to robustly catalog genes exhibiting translational buffering, a clear methodological and conceptual strength. The main limitations stem from heterogeneous sample sources, largely correlative analyses, and a lack of targeted mechanistic validation. Compared to prior yeast focused studies, it fills a key gap by demonstrating conservation of buffering in mammals and linking it to dosage sensitivity and protein stability, representing a conceptual advance in understanding post-transcriptional homeostasis and a methodological step forward in TE analysis. This work will interest researchers in RNA biology, gene expression regulation, systems biology, and cancer proteomics, as well as those studying dosage-sensitive pathways and translational control. My expertise is on translational control in cancer.

We thank the reviewer for noting the broader significance of the work and for their constructive feedback.

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

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Referee #1

Evidence, reproducibility and clarity

Thanks to the development of Ribo-Seq, translational buffering has been reported in various works. However, the systematic investigation has remained challenging. Employing the database of published Ribo-Seq and matched RNA-Seq, Rao et al. attempt to understand the mechanism underlying translational buffering of mRNA variation across diverse materials. Although the authors' report provides a step forward in our understanding of translational buffering, this reviewer found a series of concerns in this paper. These points could be tackled to improve the reliability of their findings, the strength of their main message, and the global understandability of the paper.

Major comments:

1. This paper heavily relies on the reference 18. However, this paper was not properly stated (no page or journal number); the study in Bioinformatics is nowhere to be found on the website, despite being out in 2024 apparently. Either title is wrong (yet a biorxiv can be found). This reviewer guessed that the reference 18 may be accepted. However, without a proper reference, this paper could not be judged since nearly all the parts of this work have been based on the reference 18. Also, the Ribobase data used in this manuscript comes from this reference, so it had better be well defined, especially when another Ribobase data set seems to be available online: http://www.bioinf.uni-freiburg.de/~ribobase/index.html
2. In the Discussion, the authors mentioned "TE is based on a compositional regression model (18) rather than the commonly applied approach of using a logarithmic ratio of ribosome occupancy to mRNA abundance." This important information should be mentioned early section of the manuscript. Related to this, there are other published methods for exploring change in translation efficiency (e.g., 10.1093/bioinformatics/btw585; 10.1093/nar/gkz223) that could also be suitable in this context. It is not entirely clear if their approach is better than before. Again, the improper reference to 18 made our assessment of this work difficult.
3. The paper mainly relies on detecting a set of buffered genes using mRNA-TE correlation and MAD ratios (Ribo-Seq/RNA-Seq). While the concept seems sound, the authors should ensure that this method is reliable. Several controls could be used to confirm this. First, if any studies in humans or mice have described a set of genes as buffered, it would be worth checking for overlap between the authors' set of 'TB high' genes and the previously established list. Furthermore, the authors could use packages explicitly developed for translational buffering detection, such as annota2seq (https://academic.oup.com/nar/article/47/12/e70/5423604?login=true). Not all of the data used by the authors may be suitable for such packages, but the authors could at least partially use them on some of their datasets and see whether the buffered genes reported by these packages match their predictions.
4. The threshold of 'TB high' or 'TB low' (top and bottom 250) is somewhat arbitrary. Why not top 100 or 500? The authors should provide a rationale for this choice. Also, they could include a numeric measure of buffering (the sum of the two rankings is probably suitable for this purpose). Several of the authors' explorations are suitable for numerical quantification (GO enrichment can be turned into GSEA, and the boxplot can be shown as correlations)
5. Several of the statements of the authors in the Introduction or Discussion sections are not entirely true regarding the literature on the topics, or lack major papers on the topic, and therefore, they are a bit misleading. Among others, here are some:

5-1 "In addition, genetic differences arising from aneuploidy, cell type differences or variability observed in the natural population can further determine the amplitude of variation (4-7). The effect of mRNA variation under these conditions is mostly reflected at the protein levels (2, 4-8).". Several recent or more ancient papers suggest that mRNA variation coming from aneuploidy, natural genetic variation, or CNV is buffered or not well reflected at the protein level:

DOI: 10.1038/s41586-024-07442-9 DOI: 10.1073/pnas.2319211121 DOI: 10.1016/j.cels.2017.08.013 DOI: 10.15252/msb.20177548

5-2: The authors should also consider mentioning these studies and softening their initial statement. "Similarly, translational buffering of certain genes have been reported in mammalian cells, specifically under estrogen receptor alpha (ERα) depletion conditions (16).". Translational buffering has been deeply explored in mammalian tissues and even across several mammalian species in this study (DOI: 10.1038/s41586-020-2899-z). In this, the authors also provide a nice exploration of the gene characteristics that are associated with translational buffering. The authors should mention it and compare the study's findings to theirs ultimately.

5-3: "Differences in species evaluated and statistical methods have resulted in conflicting interpretations (13, 28).". These conflicting results have been previously discussed in reviews on the topic that would be worth mentioning: DOI: 10.1016/j.cell.2016.03.014 DOI: 10.1038/s41576-020-0258-4 6. In addition to the p-values stated in the main text, the authors should annotate their plots when they find significant differences between groups to greatly facilitate the visual interpretation of the graphs. 7. Based on the data of Figure 4D, apparently, ribosome occupancy was not buffered even in high TB sets. The authors may argue that translational buffering may not cope with such a strong mRNA reduction. In that case, how big a difference in mRNA level does the buffering system adjust in protein synthesis? The authors should test gradual gene knockdown and/or overexpression and conduct Ribo-Seq/RNA-Seq to survey the buffering range. 8. "differential transcript accessibility model" could not be functional if mRNA is reduced beyond the accessible pool (i.e., less than the threshold, all the mRNAs are translated without buffering). The authors should carefully reconsider this model and the effective range of mRNAs.

Minor comments:

1. Some figures are of poor quality as they seem to have points outside of the panel representations... Like Figure 3C, one point is out of the square, same for Figure 4E. Similarly, on figure 5F, some outliers seem to be clearly cut from the figure (maybe not, but then the author should put a larger space between the end of the figure and the max y points). Same for panel S2D and S6D, this does not sound so rigorous.
2. There are several typos or weird sentences. Here are some (but maybe not all):

2-1: [...]with lower sums corresponding to higher final ranks. "two rankings". Based on these final ranks[...]

2-2: For each dataset, median absolute deviation (MAD) "i" protein abundance was calculated across samples

2-3: [...]neighbor method implemented in the MatchIT package (38) Differences in protein[...] a point is missing here.

2-4: Additionally a second dataset providing predictions of haploinsufficiency (pHaplo score) and triplosensitivity (pTriplo score) for all autosomal genes (25) was used to asses the distribution of these score"S" across buffered and non-buffered gene sets . There is a missing "s" at "score" and there is a space between the last word and the final point. 3. In the "Lymphoblastoid cell line data analysis:" section, this reviewer wonders why the authors used a different method to calculate buffering compared to before. 4. "Samples which had R2 less than 0.2 were removed as the residuals calculated for these samples could be unreliable". These samples for which the correspondence between RNA-Seq and Ribo-Seq is low wouldn't be the ones most impacted by translational buffering? Is it sure that the authors are not missing something here? 5. For Figure 4B and 4C, the authors should provide statistical tests and p-values to confirm the observed trends. 6. In Figure 2A, the "all genes" color doesn't correspond to the point color. 7. "To understand if codon usage patterns are[...]". This comes slightly out of the blue. The authors could maybe explain why codon usage should be explored for translational buffering. The authors should cite recent key works in the fields: DOI: 10.1016/j.celrep.2023.113413 DOI: 10.1101/2023.11.27.568910 8. "The change in each metric was calculated by subtracting the mean value in the control samples from that in the knockdown samples. This yielded the differential mRNA abundance and ribosome occupancy resulting from gene knockdown.". This looks statistically weak. The authors should consider using more robust methods like DESeq. 9. "Genes in the buffered gene set had a higher codon adaptation index than the non-buffered set, indicating that candidates in the buffered gene set are relatively well expressed due to the presence of a higher proportion of the codons observed in highly expressed genes". What do the authors mean by "relatively well expressed"? Abundantly expressed? This sentence and the causality under it is unclear and should be modified or better explained. 10. The panel 4D is unclear. Is one point associated with one gene? Or is it the average of several genes? If it's one point for one gene, it is important to clearly state it because the number of cases is therefore quite low, especially for the TB high and low. 11. In Figure 2J, GGU (Gly), AAG (Lys), and ACU (Arg) provide negative effects on prediction, although these were enriched in the high TB set (Figure 2E). This contradiction should be explained. 12. The subtitle of "Translationally buffered genes exhibit variable association kinetics with the translational machinery in response to mRNA variation" sounds unfair to this reviewer. Since the authors did not work on kinetics directly, the use of this word is misleading. 13. The explanation of Figure 5A "We next explored the potential mechanisms that may give rise to translational buffering. Specifically, we considered two non-mutually exclusive models by which mRNA abundance might be decoupled from ribosome occupancy. In the first, the "differential transcript accessibility model", mRNA abundance determines the fraction of transcripts that are accessible to the translational pool. In this scenario, an increase in mRNA abundance would be accompanied by a proportionally smaller increase in the fraction of transcripts entering the translating pool for buffered genes, compared to non-buffered genes. In the second, the "initiation rate model", the rate of translation initiation per transcript scales inversely with mRNA abundance. Under this model, the proportion of mRNA entering the translational pool would be comparable across buffered and non-buffered genes (Fig 5A)." is hard to understand. The authors should rewrite for a better understanding of the readers.

Significance

Thanks to the development of Ribo-Seq, translational buffering has been reported in various works. However, the systematic investigation has remained challenging. Employing the database of published Ribo-Seq and matched RNA-Seq, Rao et al. attempt to understand the mechanism underlying translational buffering of mRNA variation across diverse materials. A group of mRNAs whose expression variance is buffered at the translation level was comprehensively surveyed in humans and mice. The authors found a series of features in the translationally buffered genes, including high GC contents in the 5′ UTR, optimal codon usage, and mRNA length. The depletion or increase of one allele of the genes in the group may be particularly detrimental to cells. The authors' report provides a step forward in our understanding of translational buffering, appealing to the broad scientific community in basic and applied biology. However, this reviewer found a series of concerns in this paper, including clarity in the methods, experimental validation, referring the earlier works, etc. These points could be tackled to improve the reliability of their findings, the strength of their main message, and the global understandability of the paper.

Document d'Information : La Santé Mentale en France

Synthèse

La santé mentale en France est au cœur d'un paradoxe critique : bien que décrétée "grande cause nationale" pour 2025, elle demeure la "grande cause oubliée" des politiques publiques, souffrant d'un sous-financement chronique et d'une crise structurelle profonde.

Le système de soins psychiatriques est au bord de la rupture, avec un taux de vacance de 47 % pour les postes de psychiatres hospitaliers, des fermetures de lits et des délais d'attente pour les consultations pouvant atteindre deux ans en pédopsychiatrie.

Cette situation a des conséquences dramatiques, notamment pour la jeunesse, population la plus vulnérable où un lycéen sur quatre a déjà eu des pensées suicidaires.

Tandis que la parole se libère progressivement grâce aux témoignages de personnalités publiques et à des œuvres culturelles qui contribuent à lever le tabou, les défis systémiques restent immenses.

L'investissement dans la prévention est quasi inexistant, entraînant des retards de diagnostic de près d'une décennie.

Les nouvelles initiatives, telles que les applications mobiles et la formation aux premiers secours en santé mentale, offrent des pistes complémentaires mais se heurtent à la réalité d'un manque criant de professionnels vers qui orienter les personnes en souffrance.

La crise est aggravée par des problématiques concrètes telles que les disparités territoriales d'accès aux soins et une pénurie inédite de médicaments psychotropes, soulignant l'urgence d'une politique ambitieuse et financée à la hauteur des enjeux.

1. La Crise Paradoxale de la Santé Mentale en France

"Grande Cause Nationale" : Une Déclaration Sans Moyens

La santé mentale a été officiellement désignée "grande cause nationale" pour l'année 2025. Cependant, cette annonce politique peine à se traduire par des actions concrètes et financées.

Selon le psychiatre Stéphane Oriette, cette déclaration s'est faite "sans financements associés", ce qui a été "la condition qui a été énoncée dès le début".

Les politiques publiques successives (Assises de la santé mentale, Conseil national de la refondation) ont identifié les difficultés mais n'ont pas déployé les moyens nécessaires, laissant les professionnels et les patients face à une pression croissante.

Angèle Malâtre-Lansac souligne le paradoxe : la santé mentale est le premier poste de dépense de l'Assurance Maladie, devant le cancer et les maladies cardiovasculaires, et pourtant, 50 % des personnes concernées ne sont pas prises en charge.

Une Filière en Souffrance : Pénuries et Manque d'Attractivité

Le secteur de la psychiatrie fait face à une grave crise de ressources humaines, symptomatique d'un manque de valorisation.

• Pénurie de personnel : Le taux de postes de psychiatres vacants en milieu hospitalier atteint 47 %, en augmentation par rapport aux 43 % enregistrés quelques années auparavant.

• Déficit d'attractivité : La psychiatrie est perçue négativement par une partie des futurs médecins. Une enquête révèle que 60 % des internes considèrent la psychiatrie comme une "sous-spécialité" et 30 % en ont peur. Pourtant, 90 % des psychiatres interrogés affirment qu'ils choisiraient à nouveau cette spécialité.

• Manque de moyens matériels : Stéphane Oriette insiste sur le besoin de "personnel", de "médicaments" et de "locaux adaptés" pour pouvoir soigner correctement.

Conséquences Directes sur les Soins aux Patients

Cette crise systémique impacte directement la qualité et l'accès aux soins pour les 13 millions de personnes concernées en France. Une infirmière de l'hôpital de Tours, où la suppression de 80 lits en psychiatrie est prévue, témoigne :

"Les patientes, ils ont à peu près en moyenne entre 1 et 2 mois pour qu'un patient schizophrène soit stabilisé. Là, il y aura pas de place.

Donc ça va être 15 jours d'hospitalisation et ces gens-là seront mis à la rue. Donc, on va les retrouver aux urgences psychiatriques. C'est pas possible."

2. La Jeunesse : Une Population Particulièrement Vulnérable

L'état de la santé mentale des jeunes en France est particulièrement alarmant, exacerbé par les crises récentes comme celle du Covid.

Le suicide est l'une des premières causes de mortalité chez les jeunes.

Statistiques Alarmantes

Indicateur

Donnée Clé

Pensées suicidaires (Lycéens)

1 jeune sur 4 a eu au moins une pensée suicidaire au cours de l'année.

Pensées suicidaires (18-24 ans)

1/3 des jeunes de cette tranche d'âge a déjà eu des idées suicidaires.

Perception de la santé mentale (Filles)

Seulement 49 % des jeunes filles estiment être dans une santé mentale convenable.

Ligne de prévention suicide

Le numéro national est le 3114.

L'actrice et réalisatrice Isabelle Carré a été motivée à réaliser son film "Les Rêveurs", inspiré de sa propre hospitalisation à 14 ans, en voyant "monter sur la désespérance, les fragilités psychologiques des jeunes".

Le Cas Spécifique des Jeunes Filles

Isabelle Carré souligne que les jeunes filles semblent souffrir davantage, une question qui, selon elle, n'est pas suffisamment débattue publiquement.

Le chiffre de près d'une fille sur deux ne se sentant pas en bonne santé mentale est qualifié de "dramatique".

Des Délais d'Attente Inacceptables en Pédopsychiatrie

L'accès aux soins pour les enfants et adolescents est un point noir majeur du système. Il faut parfois attendre jusqu'à deux ans pour obtenir un rendez-vous en pédopsychiatrie. Stéphane Oriette exprime le dilemme des soignants :

"Qu'est-ce que c'est aussi pour un soignant de prendre cette responsabilité là de dire ben rentre chez toi alors qu'il demande de l'aide ?".

Par ailleurs, les enfants de l'aide sociale à l'enfance (ASE) représentent les deux tiers des lits en pédopsychiatrie, soulignant la vulnérabilité de cette population.

3. Briser le Tabou : L'Émergence d'une Nouvelle Parole Publique

Malgré la crise, un changement culturel s'opère lentement, avec une libération de la parole qui contribue à déstigmatiser la maladie mentale.

Le Rôle des Témoignages et des Œuvres Culturelles

• Témoignages publics : Le journaliste Nicolas de Moran a publiquement parlé de sa bipolarité avec des mots forts : "Oui, je suis malade mental.

C'est cru, c'est violent à dire, peut-être à entendre aussi, mais je ne veux plus le cacher et je ne veux plus me cacher."

• Productions culturelles : Des séries, des émissions et des films, comme "Les Rêveurs" d'Isabelle Carré, abordent le sujet.

Le festival "Cinéma à la Folie", dont elle est la marraine, est également un vecteur de sensibilisation. L'objectif est de changer le regard sur la maladie psychiatrique, pour qu'elle ne soit plus vue "comme de la faiblesse, de la folie, de la violence".

Ce mouvement est comparé à celui qui a eu lieu dans les pays anglo-saxons, où un travail important a été fait sur la "déstigmatisation".

4. Prévention et Nouvelles Approches : Entre Espoirs et Limites

Face aux défaillances du système traditionnel, de nouvelles stratégies émergent, axées sur la prévention, le numérique et l'entraide.

L'Enjeu Crucial de la Prévention et de l'Intervention Précoce

Selon Angèle Malâtre-Lansac, la France investit "très très peu en prévention". Cette carence a des conséquences lourdes :

• 75 % des maladies mentales se développent avant l'âge de 25 ans.

• Les délais entre les premiers symptômes et un diagnostic peuvent atteindre 8 à 10 ans, comme l'illustre le cas de Nicolas de Moran. Certaines personnes ne sont diagnostiquées que vers 50 ans, voire jamais.

Les Outils Numériques : Complément ou Danger ?

Les jeunes se tournent massivement vers les réseaux sociaux et les applications pour s'informer et chercher de l'aide.

• Applications dédiées : L'application "Link", créée par l'influenceuse Miel (18 ans), a été téléchargée 300 000 fois.

Elle propose un calendrier des émotions, un journal intime et un "kit de secours".

D'autres applications comme "Jardin Mental" (gratuite et soutenue par l'État) existent également.

• Risques de désinformation : Une enquête du Guardian révèle que la moitié des vidéos les plus populaires sur TikTok concernant la santé mentale diffusent de fausses informations (ex: manger une orange contre l'anxiété).

En France, près de 90 % des contenus sur le sujet sont postés par des non-professionnels de santé.

• Intelligence Artificielle : Le recours à des IA comme ChatGPT pour se confier est perçu par Stéphane Oriette comme un signe que les jeunes "ne trouvent pas de réponses du côté de l'humain" et les cherchent ailleurs.

Les experts s'accordent à dire que ces outils peuvent être un complément utile pour l'information ou le suivi, mais ne remplaceront "jamais le facteur humain".

Les Premiers Secours en Santé Mentale et le Soutien par les Pairs

• Secourisme en santé mentale : Inspiré d'un modèle australien, ce programme vise à former des citoyens pour repérer les signes de détresse psychique et orienter vers des professionnels. L'ambition est de former 750 000 personnes en France.

• Pair-aidance : Des associations comme "La Maison Perchée" proposent des lieux de rencontre avec des "pairs-aidants", des personnes ayant traversé des expériences similaires et pouvant offrir un soutien.

La limite de ces dispositifs est soulignée par Stéphane Oriette : "La question c'est vers qui on oriente, vers quoi on oriente ?" si les structures de soin professionnelles sont saturées.

5. Enjeux Spécifiques et Systémiques

La Santé Mentale en Entreprise

C'est un "enjeu majeur, trop négligé" selon Angèle Malâtre-Lansac.

La santé mentale est la première cause d'arrêt de travail de longue durée.

Une charte d'engagement pour la santé mentale au travail a été créée pour inciter les entreprises à former leurs équipes, notamment aux premiers secours psychiques.

Disparités Territoriales

L'accès aux soins est extrêmement inégal sur le territoire. Isabelle Carré insiste sur ce point, mentionnant qu'il y a "des régions entières où il y a rien".

La Pénurie de Médicaments Psychotropes

Un phénomène qualifié d'"assez inédit" et particulièrement inquiétant est apparu depuis le printemps : une pénurie de médicaments psychotropes.

Due à des problèmes sur une chaîne de production en Grèce, cette situation empêche des patients d'accéder à leurs traitements, avec des conséquences potentiellement graves lors de l'arrêt brutal de ces médicaments.

Me pregunto si algún gobierno o agencia ya ha reconocido el Antropoceno y si ya ha tenido influencia en la política pública o si ha sido de forma más indirecta su influencia

Aqui se decribe qué es lo que tiene en mente hacer, me recuerda a Arjun Appadurai y la vida social de las cosas y a Latour con las traducciones, translations

Wikipedia:Reliable sources/Perennial sources

Author response:

The following is the authors’ response to the original reviews

General Statements:

In our manuscript, we demonstrate for the first time that RNA Polymerase I (Pol I) can prematurely release nascent transcripts at the 5' end of ribosomal DNA transcription units in vivo. This achievement was made possible by comparing wild-type Pol I with a mutant form of Pol I, hereafter called SuperPol previously isolated in our lab (Darrière at al., 2019). By combining in vivo analysis of rRNA synthesis (using pulse-labelling of nascent transcript and cross-linking of nascent transcript - CRAC) with in vitro analysis, we could show that Superpol reduced premature transcript release due to altered elongation dynamics and reduced RNA cleavage activity. Such premature release could reflect regulatory mechanisms controlling rRNA synthesis. Importantly, This increased processivity of SuperPol is correlated with resistance with BMH-21, a novel anticancer drugs inhibiting Pol I, showing the relevance of targeting Pol I during transcriptional pauses to kill cancer cells. This work offers critical insights into Pol I dynamics, rRNA transcription regulation, and implications for cancer therapeutics.

We sincerely thank the three reviewers for their insightful comments and recognition of the strengths and weaknesses of our study. Their acknowledgment of our rigorous methodology, the relevance of our findings on rRNA transcription regulation, and the significant enzymatic properties of the SuperPol mutant is highly appreciated. We are particularly grateful for their appreciation of the potential scientific impact of this work. Additionally, we value the reviewer’s suggestion that this article could address a broad scientific community, including in transcription biology and cancer therapy research. These encouraging remarks motivate us to refine and expand upon our findings further.

All three reviewers acknowledged the increased processivity of SuperPol compared to its wildtype counterpart. However, two out of three questions our claims that premature termination of transcription can regulate ribosomal RNA transcription. This conclusion is based on SuperPol mutant increasing rRNA production. Proving that modulation of early transcription termination is used to regulate rRNA production under physiological conditions is beyond the scope of this study. Therefore, we propose to change the title of this manuscript to focus on what we have unambiguously demonstrated:

“Ribosomal RNA synthesis by RNA polymerase I is subjected to premature termination of transcription”.

Reviewer 1 main criticisms centers on the use of the CRAC technique in our study. While we address this point in detail below, we would like to emphasize that, although we agree with the reviewer’s comments regarding its application to Pol II studies, by limiting contamination with mature rRNA, CRAC remains the only suitable method for studying Pol I elongation over the entire transcription units. All other methods are massively contaminated with fragments of mature RNA which prevents any quantitative analysis of read distribution within rDNA.  This perspective is widely accepted within the Pol I research community, as CRAC provides a robust approach to capturing transcriptional dynamics specific to Pol I activity. 

We hope that these findings will resonate with the readership of your journal and contribute significantly to advancing discussions in transcription biology and related fields.

Description of the planned revisions:

Despite numerous text modification (see below), we agree that one major point of discussion is the consequence of increased processivity in SuperPol mutant on the “quality” of produced rRNA. Reviewer 3 suggested comparisons with other processive alleles, such as the rpb1-E1103G mutant of the RNAPII subunit (Malagon et al., 2006). This comparison has already been addressed by the Schneider lab (Viktorovskaya OV, Cell Rep., 2013 - PMID: 23994471), which explored Pol II (rpb1-E1103G) and Pol I (rpa190-E1224G). The rpa190-E1224G mutant revealed enhanced pausing in vitro, highlighting key differences between Pol I and Pol II catalytic ratelimiting steps (see David Schneider's review on this topic for further details).

Reviewer 2 and 3 suggested that a decreased efficiency of cleavage upon backtracking might imply an increased error rate in SuperPol compared to the wild-type enzyme. Pol I mutant with decreased rRNA cleavage have been characterized previously, and resulted in increased errorrate. We already started to address this point. Preliminary results from in vitro experiments suggest that SuperPol mutants exhibit an elevated error rate during transcription. However, these findings remain preliminary and require further experimental validation to confirm their reproducibility and robustness. We propose to consolidate these data and incorporate into the manuscript to address this question comprehensively. This could provide valuable insights into the mechanistic differences between SuperPol and the wild-type enzyme. SuperPol is the first pol I mutant described with an increased processivity in vitro and in vivo, and we agree that this might be at the cost of a decreased fidelity.

Regulatory aspect of the process:

To address the reviewer’s remarks, we propose to test our model by performing experiments that would evaluate PTT levels in Pol I mutant’s or under different growth conditions. These experiments would provide crucial data to support our model, which suggests that PTT is a regulatory element of Pol I transcription. By demonstrating how PTT varies with environmental factors, we aim to strengthen the hypothesis that premature termination plays an important role in regulating Pol I activity.

We propose revising the title and conclusions of the manuscript. The updated version will better reflect the study's focus and temper claims regarding the regulatory aspects of termination events, while maintaining the value of our proposed model.

Description of the revisions that have already been incorporated in the transferred manuscript:

Some very important modifications have now been incorporated:

Statistical Analyses and CRAC Replicates:

Unlike reviewers 2 and 3, reviewer 1 suggests that we did not analyze the results statistically. In fact, the CRAC analyses were conducted in biological triplicate, ensuring robustness and reproducibility. The statistical analyses are presented in Figure 2C, which highlights significant findings supporting the fact WT Pol I and SuperPol distribution profiles are different. We CRAC replicates exhibit a high correlation and we confirmed significant effect in each region of interest (5’ETS, 18S.2, 25S.1 and 3’ ETS, Figure 1) to confirm consistency across experiments. We finally took care not to overinterpret the results, maintaining a rigorous and cautious approach in our analysis to ensure accurate conclusions.

CRAC vs. Net-seq:

Reviewer 1 ask to comment differences between CRAC and Net-seq. Both methods complement each other but serve different purposes depending on the biological question on the context of transcription analysis. Net-seq has originally been designed for Pol II analysis. It captures nascent RNAs but does not eliminate mature ribosomal RNAs (rRNAs), leading to high levels of contamination. While this is manageable for Pol II analysis (in silico elimination of reads corresponding to rRNAs), it poses a significant problem for Pol I due to the dominance of rRNAs (60% of total RNAs in yeast), which share sequences with nascent Pol I transcripts. As a result, large Net-seq peaks are observed at mature rRNA extremities (Clarke 2018, Jacobs 2022). This limits the interpretation of the results to the short lived pre-rRNA species. In contrast, CRAC has been specifically adapted by the laboratory of David Tollervey to map Pol I distribution while minimizing contamination from mature rRNAs (The CRAC protocol used exclusively recovers RNAs with 3′ hydroxyl groups that represent endogenous 3′ ends of nascent transcripts, thus removing RNAs with 3’-Phosphate, found in mature rRNAs). This makes CRAC more suitable for studying Pol I transcription, including polymerase pausing and distribution along rDNA, providing quantitative dataset for the entire rDNA gene.

CRAC vs. Other Methods:

Reviewer 1 suggests using GRO-seq or TT-seq, but the experiments in Figure 2 aim to assess the distribution profile of Pol I along the rDNA, which requires a method optimized for this specific purpose. While GRO-seq and TT-seq are excellent for measuring RNA synthesis and cotranscriptional processing, they rely on Sarkosyl treatment to permeabilize cellular and nuclear membranes. Sarkosyl is known to artificially induces polymerase pausing and inhibits RNase activities which are involved in the process. To avoid these artifacts, CRAC analysis is a direct and fully in vivo approach. In CRAC experiment, cells are grown exponentially in rich media and arrested via rapid cross-linking, providing precise and artifact-free data on Pol I activity and pausing.

Pol I ChIP Signal Comparison:

The ChIP experiments previously published in Darrière et al. lack the statistical depth and resolution offered by our CRAC analyses. The detailed results obtained through CRAC would have been impossible to detect using classical ChIP. The current study provides a more refined and precise understanding of Pol I distribution and dynamics, highlighting the advantages of CRAC over traditional methods in addressing these complex transcriptional processes.

BMH-21 Effects:

As highlighted by Reviewer 1, the effects of BMH-21 observed in our study differ slightly from those reported in earlier work (Ref Schneider 2022), likely due to variations in experimental conditions, such as methodologies (CRAC vs. Net-seq), as discussed earlier. We also identified variations in the response to BMH-21 treatment associated with differences in cell growth phases and/or cell density. These factors likely contribute to the observed discrepancies, offering a potential explanation for the variations between our findings and those reported in previous studies. In our approach, we prioritized reproducibility by carefully controlling BMH-21 experimental conditions to mitigate these factors. These variables can significantly influence results, potentially leading to subtle discrepancies. Nevertheless, the overall conclusions regarding BMH-21's effects on WT Pol I are largely consistent across studies, with differences primarily observed at the nucleotide resolution. This is a strength of our CRAC-based analysis, which provides precise insights into Pol I activity.

We will address these nuances in the revised manuscript to clarify how such differences may impact results and provide context for interpreting our findings in light of previous studies.

Minor points:

Reviewer #1:

In general, the writing style is not clear, and there are some word mistakes or poor descriptions of the results, for example: 

On page 14: "SuperPol accumulation is decreased (compared to Pol I)". 

On page 16: "Compared to WT Pol I, the cumulative distribution of SuperPol is indeed shifted on the right of the graph." 

We clarified and increased the global writing style according to reviewer comment.

There are also issues with the literature, for example: Turowski et al, 2020a and Turowski et al, 2020b are the same article (preprint and peer-reviewed). Is there any reason to include both references? Please, double-check the references.  

This was corrected in this version of the manuscript.

In the manuscript, 5S rRNA is mentioned as an internal control for TMA normalisation. Why are Figure 1C data normalised to 18S rRNA instead of 5S rRNA? 

Data are effectively normalized relative to the 5S rRNA, but the value for the 18S rRNA is arbitrarily set to 100%.

Figure 4 should be a supplementary figure, and Figure 7D doesn't have a y-axis labelling. 

The presence of all Pol I specific subunits (Rpa12, Rpa34 and Rpa49) is crucial for the enzymatic activity we performed. In the absence of these subunits (which can vary depending on the purification batch), Pol I pausing, cleavage and elongation are known to be affected. To strengthen our conclusion, we really wanted to show the subunit composition of the purified enzyme. This important control should be shown, but can indeed be shown in a supplementary figure if desired.

Y-axis is figure 7D is now correctly labelled

In Figure 7C, BMH-21 treatment causes the accumulation of ~140bp rRNA transcripts only in SuperPol-expressing cells that are Rrp6-sensitive (line 6 vs line 8), suggesting that BHM-21 treatment does affect SuperPol. Could the author comment on the interpretation of this result? 

The 140 nt product is a degradation fragment resulting from trimming, which explains its lower accumulation in the absence of Rrp6. BMH21 significantly affects WT Pol I transcription but has also a mild effect on SuperPol transcription. As a result, the 140 nt product accumulates under these conditions.

Reviewer #2:

pp. 14-15: The authors note local differences in peak detection in the 5'-ETS among replicates, preventing a nucleotide-resolution analysis of pausing sites. Still, they report consistent global differences between wild-type and SuperPol CRAC signals in the 5'ETS (and other regions of the rDNA). These global differences are clear in the quantification shown in Figures 2B-C. A simpler statement might be less confusing, avoiding references to a "first and second set of replicates" 

According to reviewer, statement has been simplified in this version of the manuscript.

Figures 2A and 2C: Based on these data and quantification, it appears that SuperPol signals in the body and 3' end of the rDNA unit are higher than those in the wild type. This finding supports the conclusion that reduced pausing (and termination) in the 5'ETS leads to an increased Pol I signal downstream. Since the average increase in the SuperPol signal is distributed over a larger region, this might also explain why even a relatively modest decrease in 5'ETS pausing results in higher rRNA production. This point merits discussion by the authors. 

We agree that this is a very important discussion of our results. Transcription is a very dynamic process in which paused polymerase is easily detected using the CRAC assay. Elongated polymerases are distributed over a much larger gene body, and even a small amount of polymerase detected in the gene body can represent a very large rRNA synthesis. This point is of paramount importance and, as suggested by the reviewer, is now discussed in detail.

A decreased efficiency of cleavage upon backtracking might imply an increased error rate in SuperPol compared to the wild-type enzyme. Have the authors observed any evidence supporting this possibility? 

Reviewer suggested that a decreased efficiency of cleavage upon backtracking might imply an increased error rate in SuperPol compared to the wild-type enzyme. We thank Reviewer #2 to point it as in our opinion, this is an important point what should be added to the manuscript. We have now included new data (panels 5G, 5H and 5I) in the manuscript showing that SuperPol in vitro exhibits an increased error rate compared to the WT enzyme. From these results obtained in vitro, we concluded that SuperPol shows reduced nascent transcript cleavage, associated with more efficient transcript elongation, but to the detriment of transcriptional fidelity.

pp. 15 and 22: Premature transcription termination as a regulator of gene expression is welldocumented in yeast, with significant contributions from the Corden, Brow, Libri, and Tollervey labs. These studies should be referenced along with relevant bacterial and mammalian research. 

According to reviewer suggestion, we referenced these studies.

p. 23: "SuperPol and Rpa190-KR have a synergistic effect on BMH-21 resistance." A citation should be added for this statement. 

This represents some unpublished data from our lab. KR and SuperPol are the only two known mutants resistant to BMH-21. We observed that resistance between both alleles is synergistic, with a much higher resistance to BMH-21 in the double mutant than in each single mutant (data not shown). Comparing their resistance mechanisms is a very important point that we could provide upon request. This was added to the statement.

p. 23: "The released of the premature transcript" - this phrase contains a typo 

This is now corrected.

Reviewer #3:

Figure 1B: it would be opportune to separate the technique's schematic representation from the actual data. Concerning the data, would the authors consider adding an experiment with rrp6D cells? Some RNAs could be degraded even in such short period of time, as even stated by the authors, so maybe an exosome depleted background could provide a more complete picture. Could also the authors explain why the increase is only observed at the level of 18S and 25S? To further prove the robustness of the Pol I TMA method could be good to add already characterized mutations or other drugs to show that the technique can readily detect also well-known and expected changes. 

The precise objective of this experiment is to avoid the use of the Rrp6 mutant. Under these conditions, we prevent the accumulation of transcripts that would result from a maturation defect. While it is possible to conduct the experiment with the Rrp6 mutant, it would be impossible to draw reliable conclusions due to this artificial accumulation of transcripts.

Figure 1C: the NTS1 probe signal is missing (it is referenced in Figure 1A but not listed in the Methods section or the oligo table). If this probe was unused, please correct Figure 1A accordingly. 

We corrected Figure 1A.  

Figure 2A: the RNAPI occupancy map by CRAC is hard to interpret. The red color (SuperPol) is stacked on top of the blue line, and we are not able to observe the signal of the WT for most of the position along the rDNA unit. It would be preferable to use some kind of opacity that allows to visualize both curves. Moreover, the analysis of the behavior of the polymerase is always restricted to the 5'ETS region in the rest of the manuscript. We are thus not able to observe whether termination events also occur in other regions of the rDNA unit. A Northern blot analysis displaying higher sizes would provide a more complete picture. 

We addressed this point to make the figure more visually informative. In Northern Blot analysis, we use a TSS (Transcription Start Site) probe, which detects only transcripts containing the 5' extremity. Due to co-transcriptional processing, most of the rRNA undergoing transcription lacks its 5' extremity and is not detectable using this technique. We have the data, but it does not show any difference between Pol I and SuperPol. This information could be included in the supplementary data if asked.

"Importantly, despite some local variations, we could reproducibly observe an increased occupancy of WT Pol I in 5'-ETS compared to SuperPol (Figure 1C)." should be Figure 2C. 

Thanks for pointing out this mistake. It has been corrected.

Figure 3D: most of the difference in the cumulative proportion of CRAC reads is observed in the region ~750 to 3000. In line with my previous point, I think it would be worth exploring also termination events beyond the 5'-ETS region. 

We agree that such an analysis would have been interesting. However, with the exception of the pre-rRNA starting at the transcription start site (TSS) studied here, any cleaved rRNA at its 5' end could result from premature termination and/or abnormal processing events. Exploring the production of other abnormal rRNAs produced by premature termination is a project in itself, beyond this initial work aimed at demonstrating the existence of premature termination events in ribosomal RNA production.

Figure 4: should probably be provided as supplementary material. 

As l mentioned earlier (see comments), the presence of all Pol I specific subunits (Rpa12, Rpa34 and Rpa49) is crucial for the enzymatic activity we performed. This important control should be shown, but can indeed be shown in a supplementary figure if desired.

"While the growth of cells expressing SuperPol appeared unaffected, the fitness of WT cells was severely reduced under the same conditions." I think the growth of cells expressing SuperPol is slightly affected. 

We agree with this comment and we modified the text accordingly.

Figure 7D: the legend of the y-axis is missing as well as the title of the plot. 

Legend of the y-axis and title of the plot are now present.

The statements concerning BMH-21, SuperPol and Rpa190-KR in the Discussion section should be removed, or data should be provided.

This was discussed previously. See comment above.

Some references are missing from the Bibliography, for example Merkl et al., 2020; Pilsl et al., 2016a, 2016b. 

Bibliography is now fixed

Description of analyses that authors prefer not to carry out:

Does SuperPol mutant produces more functional rRNAs ?

As Reviewer 1 requested, we agree that this point requires clarification.. In cells expressing SuperPol, a higher steady state of (pre)-rRNAs is only observed in absence of degradation machinery suggesting that overproduced rRNAs are rapidly eliminated. We know that (pre)rRNas are unable to accumulate in absence of ribosomal proteins and/or Assembly Factors (AF). In consequence, overproducing rRNAs would not be sufficient to increase ribosome content. This specific point is further address in our lab but is beyond the scope of this article.

Is premature termination coupled with rRNA processing 

We appreciate the reviewer’s insightful comments. The suggested experiments regarding the UTP-A complex's regulatory potential are valuable and ongoing in our lab, but they extend beyond the scope of this study and are not suitable for inclusion in the current manuscript.

Author response:

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

Reviewer #1 (Public review):

Summary:

This study provides a comprehensive single-cell and multiomic characterization of trabecular meshwork (TM) cells in the mouse eye, a structure critical to intraocular pressure (IOP) regulation and glaucoma pathogenesis. Using scRNA-seq, snATAC-seq, immunofluorescence, and in situ hybridization, the authors identify three transcriptionally and spatially distinct TM cell subtypes. The study further demonstrates that mitochondrial dysfunction, specifically in one subtype (TM3), contributes to elevated IOP in a genetic mouse model of glaucoma carrying a mutation in the transcription factor Lmx1b. Importantly, treatment with nicotinamide (vitamin B3), known to support mitochondrial health, prevents IOP elevation in this model. The authors also link their findings to human datasets, suggesting the existence of analogous TM3-like cells with potential relevance to human glaucoma.

Strengths:

The study is methodologically rigorous, integrating single-cell transcriptomic and chromatin accessibility profiling with spatial validation and in vivo functional testing. The identification of TM subtypes is consistent across mouse strains and institutions, providing robust evidence of conserved TM cell heterogeneity. The use of a glaucoma model to show subtype-specific vulnerability, combined with a therapeutic intervention-gives the study strong mechanistic and translational significance. The inclusion of chromatin accessibility data adds further depth by implicating active transcription factors such as LMX1B, a gene known to be associated with glaucoma risk. The integration with human single-cell datasets enhances the potential relevance of the findings to human disease.

We thank the reviewers for their thorough reading of our manuscript and helpful comments.

Weaknesses:

(1) Although the LMX1B transcription factor is implicated as a key regulator in TM3 cells, its role in directly controlling mitochondrial gene expression is not fully explored. Additional analysis of motif accessibility or binding enrichment near relevant target genes could substantiate this mechanistic link. 

We show that the Lmx1b mutation induces mitochondrial dysfunction with mitochondrial gene expression changes but agree with the referee in that we do not show direct regulation of mitochondrial genes by LMX1B. Emerging data suggest that LMX1B regulates the expression of mitochondrial genes in other cell types [1, 2] making the direct link reasonable. Future work that is beyond the scope of the current paper will focus on sequencing cells at earlier timepoints to help distinguish gene expression changes associated with the V265D mutation from those secondary to ongoing disease and elevated IOP. Additional studies, including ATAC seq at more ages, ChIP-seq and/or Cut and Run/Tag (in TM cells) will be necessary to directly investigate LMX1B target genes.

As we studied adult mice, mitochondrial gene expression changes could be secondary to other disease induced stresses. Because we did not intend to say we have shown a direct link, we have now added a sentence to the discussion ensure clarity. 

Lines 932-934: “Although our studies show a clear effect of the Lmx1b mutation on mitochondria, future studies are needed to determine if LMX1B directly modulates mitochondrial genes in V265D mutant TM cells”

(2) The therapeutic effect of vitamin B3 is clearly demonstrated phenotypically, but the underlying cellular and molecular mechanisms remain somewhat underdeveloped - for instance, changes in mitochondrial function, oxidative stress markers, or NAD+ levels are not directly measured. 

We agree that further experiments towards a fuller mechanistic understanding of vitamin B3’s therapeutic effects are needed. Such experiments are planned but are beyond the scope of this paper, which is already very large (7 Figures and 16 Supplemental Figures).

(3) While the human relevance of TM3 cells is suggested through marker overlap, more quantitative approaches, such as cell identity mapping or gene signature scoring in human datasets, would strengthen the translational connection.

We appreciate the reviewer’s suggestion and agree that additional quantitative analyses will further strengthen the translational relevance of TM3 cells. It is not yet clear if humans have a direct TM3 counterpart or if TM cell roles are compartmentalized differently between human cell types. We are currently limited in our ability to perform these comparative analyses. Specifically, we were unable to obtain permission to use the underlying dataset from Patel et al., and our access to the Van Zyl et al. dataset was through the Single Cell Portal, which does not support more complex analyses (ex. cell identity mapping or gene signature scoring). Differences between human studies themselves also affect these comparisons. Future work aimed at resolving differences and standardizing human TM cell annotations, as well as cross species comparisons are needed (working groups exist and this ongoing effort supports 3 human TM cell subtypes as also reported by Van Zyl). This is beyond what we are currently able to do for this paper. We present a comprehensive assessment using readily available published resources.

Reviewer #2 (Public review):

Summary:

This elegant study by Tolman and colleagues provides fundamental findings that substantially advance our knowledge of the major cell types within the limbus of the mouse eye, focusing on the aqueous humor outflow pathway. The authors used single-cell and single-nuclei RNAseq to very clearly identify 3 subtypes of the trabecular meshwork (TM) cells in the mouse eye, with each subtype having unique markers and proposed functions. The U. Columbia results are strengthened by an independent replication in a different mouse strain at a separate laboratory (Duke). Bioinformatics analyses of these expression data were used to identify cellular compartments, molecular functions, and biological processes. Although there were some common pathways among the 3 subtypes of TM cells (e.g., ECM metabolism), there also were distinct functions. For example:

TM1 cell expression supports heavy engagement in ECM metabolism and structure, as well as TGFb2 signaling.

TM2 cells were enriched in laminin and pathways involved in phagocytosis, lysosomal function, and antigen expression, as well as End3/VEGF/angiopoietin signaling.

TM3 cells were enriched in actin binding and mitochondrial metabolism.

They used high-resolution immunostaining and in situ hybridization to show that these 3 TM subtypes express distinct markers and occupy distinct locations within the TM tissue. The authors compared their expression data with other published scRNAseq studies of the mouse as well as the human aqueous outflow pathway. They used ATAC-seq to map open chromatin regions in order to predict transcription factor binding sites. Their results were also evaluated in the context of human IOP and glaucoma risk alleles from published GWAS data, with interesting and meaningful correlations. Although not discussed in their manuscript, their expression data support other signaling pathways/ proteins/ genes that have been implicated in glaucoma, including: TGFb2, BMP signaling (including involvement of ID proteins), MYOC, actin cytoskeleton (CLANs), WNT signaling, etc.

In addition to these very impressive data, the authors used scRNAseq to examine changes in TM cell gene expression in the mouse glaucoma model of mutant Lmxb1-induced ocular hypertension. In man, LMX1B is associated with Nail-Patella syndrome, which can include the development of glaucoma, demonstrating the clinical relevance of this mouse model. Among the gene expression changes detected, TM3 cells had altered expression of genes associated with mitochondrial metabolism. The authors used their previous experience using nicotinamide to metabolically protect DBA2/J mice from glaucomatous damage, and they hypothesized that nicotinamide supplementation of mutant Lmx1b mice would help restore normal mitochondrial metabolism in the TM and prevent Lmx1b-mediated ocular hypertension. Adding nicotinamide to the drinking water significantly prevented Lmxb1 mutant mice from developing high intraocular pressure. This is a laudable example of dissecting the molecular pathogenic mechanisms responsible for a disease (glaucoma) and then discovering and testing a potential therapy that directly intervenes in the disease process and thereby protects from the disease.

Strengths:

There are numerous strengths in this comprehensive study including:

Deep scRNA sequencing that was confirmed by an independent dataset in another mouse strain at another university.

Identification and validation of molecular markers for each mouse TM cell subset along with localization of these subsets within the mouse aqueous outflow pathway.

Rigorous bioinformatics analysis of these data as well as comparison of the current data with previously published mouse and human scRNAseq data.

Correlating their current data with GWAS glaucoma and IOP "hits".

Discovering gene expression changes in the 3 TM subgroups in the mouse mutant Lmx1b model of glaucoma.

Further pursuing the indication of dysfunctional mitochondrial metabolism in TM3 cells from Lmx1b mutant mice to test the efficacy of dietary supplementation with nicotinamide. The authors nicely demonstrate the disease modifying efficacy of nicotinamide in preventing IOP elevation in these Lmx1b mutant mice, preventing the development of glaucoma. These results have clinical implications for new glaucoma therapies.

We thank the reviewer for these generous and thoughtful comments on the strengths of this study.

Weaknesses:

(1) Occasional over-interpretation of data. The authors have used changes in gene expression (RNAseq) to implicate functions and signaling pathways. For example: they have not directly measured "changes in metabolism", "mitochondrial dysfunction" or "activity of Lmx1b".

We thank the reviewer for this feedback. We did not intend to overstate and agree. Our gene expression changes support, but do not by themselves prove, metabolic disturbances. We had felt that this was obvious and did not want to clutter the text. We have revised the manuscript to clarify that our conclusions about metabolic changes and LMX1B activity are based on gene expression patterns rather than direct functional assays and have added EM data (see below under “Recommendations for the authors”).

We have also added the following to the results:

Lines 715-721: “Although the documented gene expression changes strongly suggest metabolic and mitochondrial dysfunction, they do not directly prove it. Using electron microscopy to directly evaluate mitochondria in the TM, we found a reduction in total mitochondria number per cell in mutants (P = 0.015, Figure 6G). In addition, mitochondria in mutants had increased area and reduced cristae (inner membrane folds) in mutants consistent with mitochondrial swelling and metabolic dysfunction (all P < 0.001 compared to WT, Figure 6G-H).”

More detailed EM and metabolic studies are underway but are beyond the scope of this paper.

(2) In their very thorough data set, there is enrichment of or changes in gene expression that support other pathways that have been previously reported to be associated with glaucoma (such as TGFb2, BMP signaling, actin cytoskeletal organization (CLANs), WNT signaling, ossification, etc. that appears to be a lost opportunity to further enhance the significance of this work.

We appreciate the reviewer’s suggestions for enhancing the relevance of our work, we had not initially discussed this due to length concerns. We have now incorporated some of this information into the manuscript (see below under “Recommendations for the authors”).

Reviewer #3 (Public review):

Summary: In this study, the authors perform multimodal single-cell transcriptomic and epigenomic profiling of 9,394 mouse TM cells, identifying three transcriptionally distinct TM subtypes with validated molecular signatures. TM1 cells are enriched for extracellular matrix genes, TM2 for secreted ligands supporting Schlemm's canal, and TM3 for contractile and mitochondrial/metabolic functions. The transcription factor LMX1B, previously linked to glaucoma, shows the highest expression in TM3 cells and appears to regulate mitochondrial pathways. In Lmx1bV265D mutant mice, TM3 cells exhibit transcriptional signs of mitochondrial dysfunction associated with elevated IOP. Notably, vitamin B3 treatment significantly mitigates IOP elevation, suggesting a potential therapeutic avenue.

This is an excellent and collaborative study involving investigators from two institutions, offering the most detailed single-cell transcriptomic and epigenetic profiling of the mouse limbal tissues-including both TM and Schlemm's canal (SC), from wild-type and Lmx1bV265D mutant mice. The study defines three TM subtypes and characterizes their distinct molecular signatures, associated pathways, and transcriptional regulators. The authors also compare their dataset with previously published murine and human studies, including those by Van Zyl et al., providing valuable crossspecies insights.

Strengths: 

(1) Comprehensive dataset with high single-cell resolution

(2) Use of multiple bioinformatic and cross-comparative approaches

(3) Integration of 3D imaging of TM and SC for anatomical context

(4) Convincing identification and validation of three TM subtypes using molecular markers.

We thank the reviewer for their comments on the strengths of this study.

Weaknesses:

(1) Insufficient evidence linking mitochondrial dysfunction to TM3 cells in Lmx1bV265D mice: While the identification of TM3 cells as metabolically specialized and Lmx1b-enriched is compelling, the proposed link between Lmx1b mutation and mitochondrial dysfunction remains underdeveloped. It is unclear whether mitochondrial defects are a primary consequence of Lmx1b-mediated transcriptional dysregulation or a secondary response to elevated IOP. Additional evidence is needed to clarify whether Lmx1b directly regulates mitochondrial genes (e.g., via ChIP-seq, motif analysis, or ATAC-seq), or whether mitochondrial changes are downstream effects.

We agree and refer the reviewer to our responses to the other referees including Reviewer 1, Comment 1 and Reviewer 2 comments 1 and 17. As noted there, these mechanistic questions are the focus of ongoing and future studies. We have revised the text where appropriate to ensure it accurately reflects the scope of our current data.

(2) Furthermore, the protective effects of nicotinamide (NAM) are interpreted as evidence of mitochondrial involvement, but no direct mitochondrial measurements (e.g., immunostaining, electron microscopy, OCR assays) are provided. It is essential to validate mitochondrial dysfunction in TM3 cells using in vivo functional assays to support the central conclusion of the paper. Without this, the claim that mitochondrial dysfunction drives IOP elevation in Lmx1bV265D mice remains speculative. Alternatively, authors should consider revising their claims that mitochondrial dysfunction in these mice is a central driver of TM dysfunction.

We again refer the reviewer to our other response including Reviewer 1, Comment 1 and Reviewer 2 comments 1 and 17.

(3) Mechanism of NAM-mediated protection is unclear: The manuscript states that NAM treatment prevents IOP elevation in Lmx1bV265D mice via metabolic support, yet no data are shown to confirm that NAM specifically rescues mitochondrial function. Do NAM-treated TM3 cells show improved mitochondrial integrity? Are reactive oxygen species (ROS) reduced? Does NAM also protect RGCs from glaucomatous damage? Addressing these points would clarify whether the therapeutic effects of NAM are indeed mitochondrial.

We refer the reviewer to our response to Reviewer 1, Comment 2.

(4) Lack of direct evidence that LMX1B regulates mitochondrial genes: While transcriptomic and motif accessibility analyses suggest that LMX1B is enriched in TM3 cells and may influence mitochondrial function, no mechanistic data are provided to demonstrate direct regulation of mitochondrial genes. Including ChIP-seq data, motif enrichment at mitochondrial gene loci, or perturbation studies (e.g., Lmx1b knockout or overexpression in TM3 cells) would greatly strengthen this central claim.

We refer the reviewer to our response to Reviewer 1, Comment 1.

(5) Focus on LMX1B in Fig. 5F lacks broader context: Figure 5F shows that several transcription factors (TFs)-including Tcf21, Foxs1, Arid3b, Myc, Gli2, Patz1, Plag1, Npas2, Nr1h4, and Nfatc2exhibit stronger positive correlations or motif accessibility changes than LMX1B. Yet the manuscript focuses almost exclusively on LMX1B. The rationale for this focus should be clarified, especially given LMX1B's relatively lower ranking in the correlation analysis. Were the functions of these other highly ranked TFs examined or considered in the context of TM biology or glaucoma? Discussing their potential roles would enhance the interpretation of the transcriptional regulatory landscape and demonstrate the broader relevance of the findings.

Our analysis (Figure 5F) indicates that Lmx1b is the transcription factor most strongly associated with its predicted target gene expression across all TM cells, as reflected by its highest value along the X-axis. While other transcription factors exhibit greater motif accessibility (Y-axis), this likely reflects their broader expression across TM subtypes. In contrast, Lmx1b is minimally expressed in TM1 and TM2 cells, which may account for its lower motif accessibility overall (motifs not accessible in cells where Lmx1b is not / minimally expressed).

Our emphasis on LMX1B is further supported by its direct genetic association with glaucoma. In contrast, the other transcription factors lack clear links to glaucoma and are supported primarily by indirect evidence. Nonetheless, we agree that the transcription factors highlighted in our analysis are promising candidates for future investigation. However, to maintain focus on the central narrative of this study, we have chosen not to include an extended discussion of these additional genes.

(6) In abstract, they say a number of 9,394 wild-type TM cell transcriptomes. The number of Lmx1bV265D/+ TM cell transcriptomes analyzed is not provided. This information is essential for evaluating the comparative analysis and should be clearly stated in the Abstract and again in the main text (e.g., lines 121-123). Including both wild-type and mutant cell counts will help readers assess the balance and robustness of the dataset.

We thank the reviewer for noticing this oversight and have added this value to the abstract and results section. 

Lines 41 and 696: 2,491 mutant TM cells.  

(7) Did the authors monitor mouse weight or other health parameters to assess potential systemic effects of treatment? It is known that the taste of compounds in drinking water can alter fluid or food intake, which may influence general health. Also, does Lmx1bV265D/+ have mice exhibit non-ocular phenotypes, and if so, does nicotinamide confer protection in those tissues as well? Additionally, starting the dose of the nicotinamide at postnatal day 2, how long the mice were treated with water containing nicotinamide, and after how many days or weeks IOP was reduced, and how long the decrease in the IOP was sustained.

Water intake was monitored in both treatment groups, and dosing was based on the average volume consumed by adult mice (lines 1017–1018, young pups do not drink water and so drug is largely delivered through mothers’ milk until weaning and so we do not know an accurate dose for young pups). Mouse health was assessed throughout the experiment through regular monitoring of body weight and general condition.

Depending on genetic context, Lmx1b mutations can cause kidney disease and impact other systems. Non-ocular phenotypes were not the focus of this study and were not characterized.

We added a comment to the method to clarify the NAM treatment timeline. NAM was administered continuously in the drinking water starting at P2 and maintained throughout the experiment. IOP was measured beginning at 2 months and then at monthly time points. NAM lessened IOP at 2 and 3 months. We terminated IOP assessment at 3 months.

Lines 1028-1029: “Treatment was started at postnatal day 2 and continued throughout the experiment.”

(8) While the IOP reduction observed in NAM-treated Lmx1bV265D/+ mice appears statistically significant, it is unclear whether this reflects meaningful biological protection. Several untreated mice exhibit very high IOP values, which may skew the analysis. The authors should report the mean values for IOP in both untreated and NAM-treated groups to clarify the magnitude and variability of the response.

We have added supplemental table 7 with the statistical information. Regarding the high IOP values observed in a subset of untreated V265D mutant mice, we consistently detect individual mutant eyes with IOPs exceeding 30 mmHg across independent cohorts and time points [3-5]. It is important to note that IOP is subject to fluctuation and in disease states such as glaucoma, circadian rhythms can be disrupted with stochastic and episodic IOP spikes throughout the day. This may be occurring in those untreated mice. This is also why we strive to use sample sizes of 40 or more. Additionally, we observe that some mutant eyes with IOPs measured within the normal range have anterior chamber deepening (ACD) - a persistent anatomical change associated with sustained or recurrent high IOP that stretches the cornea and may posteriorly displace the lens. This suggests mutant mice experience transient IOP elevations that are not always captured at a single time point due to the stochastic nature of these fluctuations. To account for this, we include ACD as an additional readout alongside IOP measurements. The reduction in ACD observed in NAM-treated mice provides independent evidence supporting the biological relevance of NAM-mediated IOP reduction.   

(9) Additionally, since NAM has been shown to protect RGCs in other glaucoma models directly, the authors should assess whether RGCs are preserved in NAM-treated Lmx1b V265D/+ mice. Demonstrating RGC protection would support a synergistic effect of NAM through both IOP reduction and direct neuroprotection, strengthening the translational relevance of the treatment.

We again thank the referee. We note the possibility of dual IOP protection and neuroprotection in the manuscript (lines 961–963). The goal of the present study, however, was to determine mechanisms underlying IOP elevation in patients with LMX1B variants. Therefore, we limited our focus to IOP elevation (LMX1B is expressed in the TM but not RGCs). Studies of the RGCs and optic nerve in V265D mutant mice treated with NAM take considerable effort but are underway. They will be reported in a subsequent manuscript. Initial data support protection, but that is a work in progress.  

Additionally, we recently reported a similar pattern of IOP protection to that reported here using pyruvate - in experiments where we analyzed the optic nerve as the focus of the study was assessment of pyruvate as a resilience factor against high genetic risk of glaucoma [4]. In that case, there was statistically significant protection from glaucomatous optic nerve damage, arguing for translational relevance again with a possible synergistic effect through both IOP reduction and direct neuroprotection.

(10) Can the authors add any other functional validation studies to explore to understand the pathways enriched in all the subtypes of TM1, TM2, and TM3 cells, in addition to the ICH/IF/RNAscope validation?

We agree with the reviewer on the importance of further functional validation of pathways active in TM cell subtypes that influence IOP. However, comprehensive investigation of the pathways active in subtypes need to be in future studies. It is beyond the scope of his already large paper.

(11) The authors should include a representative image of the limbal dissection. While Figure S1 provides a schematic, mouse eyes are very small, and dissecting unfixed limbal tissue is technically challenging. It is also difficult to reconcile the claim that the majority of cells in the limbal region are TM and endothelium. As shown in Figure S6, DAPI staining suggests a much higher abundance of scleral cells compared to TM cells within the limbal strip. Additional clarification or visual evidence would help validate the dissection strategy and cellular composition of the captured region.

We appreciate the reviewer’s suggestion and have added additional images to Figure S1 to show our limbal strip dissection. However, we clarify that we do not intend to suggest that TM and endothelial cells are the most abundant populations in these dissected strips.  When we say “are enriched for drainage tissues” we mean in comparison to dissecting the anterior segment as a whole. We have clarified this in the text. In fact, epithelial cells (primarily from the cornea) constituted the largest cluster in our dataset (Figure 1A). Additionally, to avoid misinterpretation, we generally refrain from drawing conclusions about the relative abundance of cell types based on sequencing data. Single-cell and single nucleus RNA sequencing results are sensitive to technical factors that alter cell proportions depending on exact methodological details. In our study, TM cells comprised 24.4% of the single-cell dataset and 11.8% of the single-nucleus dataset, illustrating the impact of methodological variability. 

Lines 163-164: “Individual eyes were dissected to isolate a strip of limbal tissue, which is enriched for TM cells in comparison to dissecting the anterior segment as a whole.”

Reviewer #1 (Recommendations for the authors):

To enhance the reproducibility and transparency of the findings presented in this study, we strongly recommend that the authors make all analysis scripts and computational tools publicly available.

We agree with the reviewer’s emphasis on transparency and are currently building a GitHub page to share our scripts. However, we did not develop any new tools for this study. All tools that we used are publicly available and provided in our methods section. All data will be available as raw data and through the Broad Institute’s Single Cell Portal.

Reviewer #2 (Recommendations for the authors):

The authors are to be commended for a well-written presentation of high-quality data, their comparisons of datasets (other mouse and human scRNAseq data), correlation with clinical glaucoma risk alleles, and curative therapy for the mouse model of Lmx1b glaucoma. There are several minor suggestions that the authors might consider to further improve their manuscript:

(1) Lines 42-43: Although their data strongly support the role of mitochondrial dysfunction in Lmx1b glaucoma, they might want to soften their conclusion "supports a primary role of mitochondrial dysfunction within TM3 cells initiating the IOP elevation that causes glaucoma".

With the inclusion of EM data supporting mitochondrial dysfunction in Lmx1b mutant TM cells, we have revised this sentence to more accurately reflect our findings.

Lines 42-44 (previously lines 42-43): “Mitochondria in TM cells of V265D/+ mice are swollen with a reduced cristae area, further supporting a role for mitochondrial dysfunction in the initiation of IOP elevation in these mice.”

(2) Figure 1: Why is the shape of the "TM containing" cluster in 1A so different than the cluster shown in 1B?

We isolated cells from the 'TM-containing' cluster and performed unbiased reclustering, which alters their positioning in UMAP space. The figure legend has been updated to clarify this point.

Lines 143-144 “A separate UMAP representation of the trabecular meshwork (TM) containing cluster following subclustering.”

(3) Line 160: change "data was" to "data were"

Corrected

(4) S4 Fig C: Please comment on why the Columbia and Duke heatmaps for TM3 are not as congruent as the heatmaps for TM1 and TM2.

We cannot definitively determine the reason for this. However, differences in tissue processing techniques between the Columbia and Duke preparations may contribute. Such variations have been shown to affect cellular transcriptomes in certain contexts. It is possible that TM3 cells are more susceptible to these effects than others. We have added a statement addressing this point to the figure legend.

Lines 238-240: “Because tissue processing techniques can alter gene expression [52], the heatmap variation between institutes likely reflects differences in processing techniques (Methods) and suggests that TM3 cells are more susceptible to these effects than other cell types.”

(5) S9 Fig: It is very difficult to see any staining for TM1 CHIL1 (2nd panel), TM2 End3 (2nd panel), and TM3 Lypd1 (both panels)

We apologize for the difficulty in visualizing these panels. To improve clarity, we have increased the brightness of all relevant marker signals, within standard bounds, to facilitate easier interpretation.

(6) Line 380: "are significantly higher"; since statistical analysis was not reported, please do not use "significantly"

Done

(7) The authors should consider discussing several of their findings that agree with published literature. For example:

Figure 3B: "Wnt protein binding" (PMID: 18274669), "TGFb "binding" (numerous references), "integrin binding" (work of Donna Peters), "actin binding"/"actin filament binding"/"actin filament bundle" (CLANs references)

S10 Fig c: "ossification" (work of Torretta Borres)

S11 Fig A: ID2/ID3 (PMID: 33938911); (B) BMP4 (PMID: 17325163)

S12 Fig A: MYOC in TM1 cells (numerous references)

We appreciate the reviewer’s diligent review and comments regarding these pathways. We have added a comment to the discussion regarding the agreement of these pathways.

Lines 855-858: In addition, the expression of genes that we document generally agrees with the literature. For example, the following genes and signaling molecules have been reported in TM cells, WNT signaling [78], TGF-β signaling [79-85], integrin binding [86-88], actin cytoskeletal networks [89], calcification genes [90, 91], and Myocilin [91-94].

(8) Line 541: was confocal microscopy used to measure the "3D shapes" of nuclei or was this done with a single image to determine sphericity?

This analysis was performed using confocal microscopy and 3D reconstructed models of the TM nuclei. We have added text to clarify this in the figure legend 

Lines 553-556: “To rigorously assess whether TM1 nuclei are more spherical, we analyzed their reconstructed 3D shapes from whole mounts images by confocal microscopy, comparing them to TM3 nuclei using the ‘Sphericity’ tool in Imaris.”

(9) Line 545: please add a close parentheses after "scoring 1"

Done

(10) S15 Fig: (A) There does not appear to be "good agreement" (line 653) between the datasets for TM1. (C) please provide a better explanation on how to interpret these "Confusion Matrix" results.

We understand the referee's concern, the patterns likely appear different to the referee due to limited sampling in snRNA-seq data. Based on our results, TM1 seems particularly susceptible, possibly because these cells do not tolerate the isolation process as well. Although we are confident that TM1 shows good agreement between the two techniques based on our experience, we have revised the language in the text to “generally” to reflect this nuance.

Lines 633-635 (previously line 653): The generated clusters and their marker genes generally agreed with our scRNA-seq analyses (Fig 5A-B, S15A Fig).

We have also added additional clarification for how to interpret the Confusion Matrix. 

Lines 669-672: “Colors indicate the fraction of cells identified in each ATAC cluster (row) which are also identified in each RNA cell type (columns), where darker colors represent stronger correspondence between RNA and ATAC clusters.”

(11) Line 676: The transition from discussing the sc/snRNAseq data to the work in Lmx1b mutant mice is quite abrupt and could use a better transition to introduce this metabolism work.

We have revised this transition for improved flow but prefer to keep all transitions brief due to the paper's length.

Lines 691-694 (previously line 676): To evaluate the utility of our new TM cell atlas, we used it to examine how Lmx1b mutations affect the TM cell transcriptome and to identify potential mechanisms underlying IOP elevation. We selected LMX1B because it causes IOP elevation and glaucoma in humans and was identified as a highly active transcription factor in our TM cell dataset.

(12) Lines 696-697: It appears counter-intuitive that upregulation of ubiquitin pathways would lead to proteostasis (proteosome protein degradation requires ubiquination).

We have clarified that the protein tagging pathway was significantly upregulated. However, polyubiquitin precursor itself was downregulated. In general, the statistical significance of the protein tagging pathway suggests perturbation of the system tagging proteins for degradation. We have clarified this in the text. 

Lines 711-714 (previously lines 696-697): “In addition, mutant TM3 cells showed an upregulation of protein tagging genes. However, there is a downregulation of the polyubiquitin precursor gene (Ubb, P = 4.5E-30), indicating a general dysregulation of pathways that tag proteins for degradation.”

(13) Line 715: Please justify why "perturbed metabolism" was chosen to pursue vs the other differentially expressed pathways

We chose to narrow our focus on TM3 cells because of the enrichment for Lmx1b expression.Most pathways identified in our analysis of TM3 cells implicate mitochondrial metabolism.Therefore, we chose to further explore this avenue. We clarified that perturbed metabolism was the strongest gene expression signature in the text. 

Lines 753-754 (previously line 715): “Our findings most strongly implicate perturbed metabolism within TM3 cells as responsible for IOP elevation in an Lmx1b glaucoma model.”

(14) Line 759: The authors clearly demonstrate that Lmx1b is most expressed in TM3 cells; however, they did not demonstrate that "Lmx1b was most active"

ATAC analysis showed that Lmx1b was most active in TM cells overall. We inferred its activity in TM3 because Lmx1b is most enriched in that subtype. This has been clarified in the text.

Lines 799-800 (previously line 759): “More specifically, we demonstrate that Lmx1b is the most active TM cell TF and is enriched in TM3 cells,…”

(15) Lines 830-835: Please include references documenting increased TGFβ2 concentrations in POAG aqueous humor and TM, effects of TGFβ2 on TM ECM deposition, and TGFβ2 induced ocular hypertension ex vivo and in vivo.

Done.

(16) Line 875: The authors provide no direct evidence for enhances "oxidative stress" in Lmx1b TM3 cells

The mitochondrial abnormalities and changed pathways support oxidative stress, but we have not directly tested this. Experiments are currently underway to evaluate its role, but these additional analyses are beyond the scope of this paper. We removed oxidative stress from the sentence.

Lines 920-922 (previously line 875): “Importantly, in heterozygous mutant V265D/+ mice, TM3 cells had pronounced gene expression changes that implicate mitochondrial dysfunction, but that were absent or much lower in other cells including TM1 and TM2.”

(17) Line 880: Similarly, the authors have not directly assessed effects on metabolism in TM3 cells; they only have shown changes in the expression of mitochondrial genes that may affect metabolism

We have no way to specifically isolating TM3 cells to test this. Future work is underway to test this more broadly in isolated TM cells but is beyond the scope of this is already large paper. Considering our gene expression data and the addition of supporting EM data, we have qualified the text.

Lines 930-931 (previously 880): “Our data extend these published findings by showing that inheritance of a single dominant mutation in Lmx1b similarly affects mitochondria in TM cells.”

(18) Line 892: What markers were used to detect "cell stress"?

We have revised the text. Although our RNA data show stress gene changes, characterization of these markers is beyond the scope of the current study and will be included in a subsequent paper.

Lines 945-948 (previously line 892): “However, these processes were not limited to TM3 cells or even to cell types that express detectable Lmx1b, suggesting that they are secondary damaging processes that are subsequent to the initiating, Lmx1b-induced perturbations in TM3 cells.”

Additional author driven change

While revising and reviewing our data, we identified a coding error that resulted in the WT and V265D mutant group labels being switched in Figure 6. Importantly, the significance of the differentially expressed genes (DEGs), the implicated biological pathways, and the interpretation of pathway directionality in the manuscript remain accurate. The only issue was the incorrect labeling in the figure. We have corrected the labels in Figure 6 to accurately reflect the data. As noted above, all data and code will be made available to ensure full reproducibility of our results.

References

(1) Doucet-Beaupre H, Gilbert C, Profes MS, Chabrat A, Pacelli C, Giguere N, et al. Lmx1a and Lmx1b regulate mitochondrial functions and survival of adult midbrain dopaminergic neurons. Proc Natl Acad Sci U S A. 2016;113(30):E4387-96. Epub 2016/07/14. doi: 10.1073/pnas.1520387113. PubMed PMID: 27407143; PubMed Central PMCID: PMCPMC4968767.

(2) Jimenez-Moreno N, Kollareddy M, Stathakos P, Moss JJ, Anton Z, Shoemark DK, et al. ATG8-dependent LMX1B-autophagy crosstalk shapes human midbrain dopaminergic neuronal resilience. J Cell Biol. 2023;222(5). Epub 2023/04/05. doi: 10.1083/jcb.201910133. PubMed PMID: 37014324; PubMed Central PMCID: PMCPMC10075225.

(3) Cross SH, Macalinao DG, McKie L, Rose L, Kearney AL, Rainger J, et al. A dominantnegative mutation of mouse Lmx1b causes glaucoma and is semi-lethal via LDB1mediated dimerization [corrected]. PLoS Genet. 2014;10(5):e1004359. Epub 2014/05/09. doi: 10.1371/journal.pgen.1004359. PubMed PMID: 24809698; PubMed Central PMCID: PMCPMC4014447.

(4) Li K, Tolman N, Segre AV, Stuart KV, Zeleznik OA, Vallabh NA, et al. Pyruvate and related energetic metabolites modulate resilience against high genetic risk for glaucoma. Elife. 2025;14. Epub 2025/04/24. doi: 10.7554/eLife.105576. PubMed PMID: 40272416; PubMed Central PMCID: PMCPMC12021409.

(5) Tolman NG, Balasubramanian R, Macalinao DG, Kearney AL, MacNicoll KH, Montgomery CL, et al. Genetic background modifies vulnerability to glaucoma-related phenotypes in Lmx1b mutant mice. Dis Model Mech. 2021;14(2). Epub 2021/01/20. doi: 10.1242/dmm.046953. PubMed PMID: 33462143; PubMed Central PMCID: PMCPMC7903917.

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Referee #2

Evidence, reproducibility and clarity

Summary

The manuscript by Peter et al., reports on the neuronal activity and connectivity of iPSC-derived human cortical neurons from Down syndrome (DS) that is caused by caused by trisomy of the human chromosome 21 (TS21).

Major points:

Although the manuscript is potentially interesting, the results appear somehow preliminary and need to be corroborated by control experiments and quantifications of effects to fully sustain the conclusions.

(1) The authors have not assessed the percentage of WT and TS21 cells that acquire a neuronal or glia identity in their cultures. Indeed, the origin of alterations in network activity and connectivity observed in TS21 neurons could simply derive from reduced number of neurons arising from TS21 iPSC. Alternatively, the same alteration in network activity and connectivity could derive from a multitude of other factors including deficits in neuronal development, neurite extension, or intrinsic electrophysiological properties. In the current version of the manuscript, none of these has been investigated.

(2) Electrophysiological properties of TS21 and WT neurons at day 53/54 in vitro indicate an extremely immature stage of development (i.e. RMP between -36 and -27 mV with most of the cells firing a single action potential after current injection) in the utilized culture conditions: This is far from ideal for in vitro neuronal-network studies. Finally, reduced activity of HCN1 channels should be confirmed by specific recordings isolating or blocking the related current.

Main points highlighting the preliminary character of the study.

1) In Figure 1 immunofluorescence images of the neuronal differentiation markers (Tbr1, Ctip2 and Tuj1) are showed. However, no quantification of the percentage of cells expressing these markers for WT and TS21 neurons is reported. On the other hand, simple inspection of the representative images clearly seams to indicate a difference between the two genotypes, with TS21 cultures showing lower number of cells expressing neuronal markers. This quantification should be corroborated by a similar staining for an astrocyte marker (GFAP, but not S100b since is triplicated in DS). This is an extremely important point since it is obvious that any change in the percentage of neurons (or the neuron/astrocyte ratio) in the cultures will strongly affect the resulting network activity (shown in Figure 2) and the connectivity (showed in Figure 4). Possibly, the quantification should be done at the same time points of the calcium imaging experiments.

2) In Figure 2 the authors show some calcium imaging traces of WT and TS21 cultures at different time points. However, they again do not show any quantification of neuronal activity. A power spectra analysis is shown in Supplementary Figure 2, but only for WT cultures, while in Supplementary Figure 3 a comparison between WT and Ts21 power spectra is done, but only at the 50 day time point, while difference in synchrony are assessed at 60 days. At minimum, the author should include in main Figure 2 the quantification of the mean calcium event rate and mean event amplitude at the different time points and the power spectra analysis for both WT and TS21 cultures at the same timepoints.

Of note, the synchronized neuronal activity is present in WT cultures at day 60, but totally lost at subsequent time-points (70 and 80 days). The results of this later time points are different from previous data from the same lab (Kirwan et al., 2015). How might these data be explained? It would be important to rule out any potential issues with the health of the culture that could explain the loss of neuronal activity.It would be beneficial to check cell viability at the different time points to exclude possible confounding factors ? A propidium staining or a MTT assay would strongly improve the soundness of the calcium data.

3) In Figure 3 there is no quantification of the number and/or density of transplanted neurons for WT and TS21, but only representative images. As above, inspection of the representative images seems to show a decrease in cells labeled by the Tbr1 neuronal marker for TS21 cells. Moreover, the in vivo calcium imaging of transplanted WT and TS21 cells lacks most of the quantification normally done in calcium imaging experiments. Are the event rate and event amplitude different between WT and TS21 neurons ? The measure of neuronal synchrony by mean pixel correlation is not well explained, but it looks somehow simplistic. Neuronal synchrony can be more precisely measured by cross-correlation analysis or spike time tiling coefficients on the traces from single-neuron ROI rather than on all pixels in the field of view, as apparently was done here.

4) The results on reduced neuronal connectivity in Figure 3 look very striking. However, these results should be accompanied by control experiments to verify the number of neuronal cells and neurite extension in WT and Ts21 cultures. These two parameters could indeed strongly influence the results. As the cultures appear to grow in clusters, bright-field images and TuJ1 staining of the cultures will also greatly help to understand the degree of morphological interconnection between the clusters.

5) The authors performed RNA-seq experiments on day 50 cultures. Why the authors do not show the complete differential gene expression analysis, but only a small subset of genes? A comprehensive volcano plot and the complete list of identified genes with logFC and FDR values would be helpful. If possible, comparison of the present data (particularly on KCN and HCN expression changes) with published and publicly available expression datasets of other human or human Down syndrome iPSC-derived neurons or human Down syndrome brains will greatly increase the soundness of the present findings. In addition, the gene ontology (GO) results are mentioned in the text, but are not presented. Showing the complete GO analysis for both up and downregulated genes will help the reader to better understand the RNA-seq results. Notably, the results shown in Supplementary Figure on GRIN2A and GRIN2B expression (with values of 300-700 counts versus 2000-4000 counts, respectively) clearly indicate that in both WT and TS21 cultures the NMDA developmental switch has not occurred yet at the 50 days timepoint.

6) The measure of hyperpolarization-activated currents shown in Figure 5 lack proper control experiments. First, the hyperpolarizing current in TS21 cells do not reach a steady-state as the controls. The two curves are therefore hard to compare. To exclude possible difference in kinetic activation, the authors should have prolonged the current injection period (1-2 seconds). Second, to ultimately prove that such currents are mediated by HCN channels in WT cells the authors should perform some control experiments with a specific HCN blocker. A good example of a suitable protocol, with also current blockers to exclude all other possible current contributions, is the one reported in Matt et al Cell. Mol. Life Sci. 68, 125-137 (2011).

7) The manuscript lacks information on the statistical analysis used. Also, the numerosity of samples is not clear. Were the dots shown in some graph technical replicates from a single neuronal induction or were all independent neuronal inductions or a mix of the two ? Please clarify.

8) The method section lacks important information to guarantee reproducibility. Just a few examples: - Only electrophysiology methods for slice are reported, but not for in vitro culture. - Details on Laminin coating is lacking. What concentration was used ? Was poly-ornithine or poly-lysine used before Laminin coating ? - How long cells were switched to BrainPhys medium before calcium imaging ?

Minor point/typos etc.

Introduction

• Page 4 line 6: in the line "Trisomy 21 in humans commonly results in a range in developmental and morphological changes in the forebrain ..." "in" could be replaced by "of".
• Page 5 line 2: please remove "an" before the word "another".
• Page 5 line 2: please replace "ecitatory" with "excitatory"

Results

• Page 10 line 25: The concept of "pixel-wise" appears for the first time in this section and could be better introduced to facilitate the understanding of the experiment.
• In the "results" section, page 11 line 1 and 4, references are made to "Figure 4D" and "4F," but these figures do not appear to be present in the figure section. Upon reviewing the rest of the section, the data seem to refer to "Figure 3D" and "3E."

Discussion

• Page 15 line 20: please replace "synchronised" with "synchronized".
• Page 16 line 11: please replace "T21" with "TS21".

Methods

• Page 19 line 12: "Pens/Strep" has to be replaced by Pen/Strep.
• Page 20 line 20: "Tocris Biocience" has to be replaced by "Tocris Bioscience".
• Page 21 line 2: "Addegene" has to be replaced by "Addgene".

Figures

• Figure 3: the schematic experimental design (Fig. 3A) could be enlarged to match the width of the images/graphs below.
• Figure 5: the reviewer suggests resizing/repositioning the graphs in Fig. 1A so that they match the width of those below.
• Figure S1D: In all the figures of the paper, the respective controls for the TS21 1 and TS21 2 lines are labelled as "WT1/WT2," while in these graphs, they are called "Ctrl1" and "Ctrl2." To ensure consistency throughout the paper, it is suggested to change the names in these graphs.
• Figure S4L: The graph is not very clear, especially regarding the significance reported at -50 pA, please modify the graphical visualization and/or add a legend in the caption.

Significance

Nature and significance of the advance for the field. The results presented in the manuscript are potentially interesting and useful, but not completely novel (currents deregulation has already been highlighted in mouse models of Down Syndrome).

Work in the context of the existing literature. This work follows the line of evidence that characterizes Down Syndrome in human neurons (Huo, H.-Q. et al. Stem Cell Rep. 10, 1251-1266 (2018); Briggs, J. A. et al. Etiology. Stem Cells 31, 467-478 (2013)), both in vitro and in xenotransplanted mice, by corrborating some important findings already found in animal models (Stern, S., Segal, M. & Moses, E. EBioMedicine 2, 1048-1062 (2015); Cramer, N. P., Xu, X., F. Haydar, T. & Galdzicki, Z. Physiol. Rep. 3, e12655 (2015); Stern, S., Keren, R., Kim, Y. & Moses, E. http://biorxiv.org/lookup/doi/10.1101/467522 (2018) doi:10.1101/467522.

Audience. Scientists in the field of pre-clinical biomedical research, especially those working on neurodevelopmental disorders and iPSC-based non-animal models.

Field of expertise. In vitro electrophysiology, Neurodevelopmental disorders, Down Syndrome, ips cells.

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Gyuri's Daily Notes - 2025 Sept Week 4

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/hyperpost/🌐/🎭/gyuri/📓/2025/10/2/💾/

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review):

(1) Their first major claim is that fluid flows alone must be quite strong in order to fragment the cyanobacterial aggregates they have studied. With their rheological chamber, they explicitly show that energy dissipation rates must exceed "natural" conditions by multiple orders of magnitude in order to fragment lab strain colonies, and even higher to disrupt natural strains sampled from a nearby freshwater lake. This claim is well-supported by their experiments and data.

We thank the reviewer for this positive comment. We fully agree, as our fragmentation experiments on division-formed colonies clearly demonstrate their strong mechanical resistance in naturally occurring flows.

(2) The authors then claim that the fragmentation of aggregates due to fluid flows occurs through erosion of small pieces. Because their experimental setup does not allow them to explicitly observe this process (for example, by watching one aggregate break into pieces), they implement an idealized model to show that the nature of the changes to the size histogram agrees with an erosion process. However, in Figure 2C there is a noticeable gap between their experiment and the prediction of their model. Additionally, in a similar experiment shown in Figure S6, the experiment cannot distinguish between an idealized erosion model and an alternative, an idealized binary fission model where aggregates split into equal halves. For these reasons, this claim is weakened.

The two idealized models of colony fragmentation, namely erosion of single cells and fragmentation into equal sizes (or binary fission), lead to distinguishable final size distributions. We believe that our experiments for division-formed colonies support the hypothesis of the erosion mechanism. Specifically, Figure 2E shows that colony fragmentation resulted in a decrease of large colonies and a strong increase of single cells and dimers (two cells). In our view, the strong increase of single cells and dimers provides quite convincing (but indirect) evidence supporting the erosion mechanism. This is described on lines 112-121. To further address the reviewer’s concern, we have included in the revised version of Figure 2 (panels B and D) a direct comparison between these two fragmentation models for large division-formed colonies fragmented at a high dissipation rate of ε = 5.8 m²/s³. Furthermore, we have included the new Supplementary Figure S9, which details the model predictions for the colony size distribution at various time points.

The ideal equal fragments model (i.e., where every fracture event produces two identical fragments with half the original biovolume) does not capture the biovolume transfer from large colonies to single cells, as observed for the experimental results in panel D of Figure 2 and panel E of Figure S9. In contrast, the erosion model, in panel D of Figure 2 and panel D of Figure S9, provides a good prediction of the experimental results within the experimental uncertainty. The different fragmentation models are discussed in lines 226-228 of the revised manuscript and lines 865-873 of the SI.

(3) Their third major claim is that fluid flows only weakly cause cells to collide and adhere in a "coming together" process of aggregate formation. They test this claim in Figure 3, where they suspend single cells in their test chamber and stir them at moderate intensity, monitoring their size histogram. They show that the size histogram changes only slightly, indicating that aggregation is, by and large, not occurring at a high rate. Therefore, they lend support to the idea that cell aggregation likely does not initiate group formation in toxic cyanobacterial blooms. Additionally, they show that the median size of large colonies also does not change at moderate turbulent intensities. These results agree with previous studies (their own citation 25) indicating that aggregates in toxic blooms are clonal in nature. This is an important result and well-supported by their data, but only for this specific particle concentration and stirring intensity. Later, in Figure 5 they show a much broader range of particle concentrations and energy dissipation rates that they leave untested.

We thank the reviewer for this positive comment. We agree that our experimental results show clear evidence that aggregated colonies have a weaker structure in comparison to division-formed colonies, thus supporting the hypothesis that clonal expansion is the main mechanism for colony formation under most natural settings. The range of energy dissipation rates of our experimental setup covers almost entirely the region for which aggregated and division-formed colonies differ in their fragmentation behavior (Zone III of Figure 5). Within this zone, aggregated colonies are fragmented and only the division-formed colonies are able to withstand the hydrodynamic stresses. Furthermore, we show that this fragmentation behavior has a low sensitivity to the total biovolume fraction, as displayed in the Supplementary Figures S2 and S4 and discussed in lines 151-154 and 160-163. We agree that our cone-and-plate setup covers a limited parameter range, and we have added a detailed discussion of these limitations in the revised manuscript, under section Materials and Methods in lines 462-473.

(4) The fourth major result of the manuscript is displayed in Equation 8 and Figure 5, where the authors derive an expression for the ratio between the rate of increase of a colony due to aggregation vs. the rate due to cell division. They then plot this line on a phase map, altering two physical parameters (concentration and fluid turbulence) to show under what conditions aggregation vs. cell division are more important for group formation. Because these results are derived from relatively simple biophysical considerations, they have the potential to be quite powerful and useful and represent a significant conceptual advance. However, there is a region of this phase map that the authors have left untested experimentally. The lowest energy dissipation rate that the authors tested in their experiment seemed to be \dot{epsilon}~1e-2 [m^2/s^3], and the highest particle concentration they tested was 5e-4, which means that the authors never tested Zone II of their phase map. Since this seems to be an important zone for toxic blooms (i.e. the "scum formation" zone), it seems the authors have missed an important opportunity to investigate this regime of high particle concentrations and relatively weak turbulent mixing.

We agree with the reviewer that Zone (II) of Figure 5 is of great importance to dense bloom formation under wind mixing and that this parameter range was not covered by our experiments using a cone-and-plate shear flow. The measuring range of our device was motivated by engineering applications such as artificial mixing of eutrophic lakes using bubble plumes, as well as preliminary experiments which demonstrated that high levels of dissipation rate were required to achieve fragmentation. The range of dissipation rates that can be achieved by the cone-and-plate setup is limited at the lower end by the accumulation of colonies near the stagnation point at the conical tip and at the upper end by the spillage of fluid out of the chamber. We now discuss this measuring range in lines 462-473 of the revised manuscript.

Although our setup does not cover Zone (II), we now refer to recent results in the literature for evidence of aggregation-dominance at Zone (II). The experimental study of Wu et al. (2024) (reference number 64 of the revised manuscript) investigated the formation of Microcystis surface scum layers in wind-mixed mesocosms. Their study identified aggregation of colonies in the scum layer, resulting in increases of colony size at rates faster than cell division. These results agree with our model, and the parameters range investigated fall within the Zone II. We have included in the revised version, lines 328-337, a detailed discussion elucidating the parameter range covered in our experiments and the findings of Wu et al. (2024).

Other items that could use more clarity:

(5) The authors rely heavily on size distributions to make the claims of their paper. Yet, how they generated those size distributions is not clearly shown in the text. Of primary concern, the authors used a correction function (Equation S1) to estimate the counts of different size classes in their image analysis pipeline. Yet, it is unclear how well this correction function actually performs, what kinds of errors it might produce, and how well it mapped to the calibration dataset the authors used to find the fit parameters.

We agree with the reviewer that more details of the correction function should be included. We have included in the revised version of the Supporting Information, in lines 785-796, a more detailed explanation of the correction function. Furthermore, a direct comparison of raw and corrected histograms of the size distribution and its associated uncertainty is presented in the new Supplementary Figure S8.

(6) Second, in their models they use a fractal dimension to estimate the number of cells in the group from the group radius, but the agreement between this fractal dimension fit and the data is not shown, so it is not clear how good an approximation this fractal dimension provides. This is especially important for their later derivation of the "aggregation-to-cell division" ratio (Equation 8)

We agree with the reviewer that more details on the estimation of fractal dimension are needed. The revised version, under Materials and Methods in lines 508-515, now includes the detailed estimation procedure, the number of colonies analysed, and the associated uncertainty.

Reviewer #1 (Recommendations For The Authors):

In light of the weak evidence for claim #2 outlined above, I believe the paper would benefit from a more explicit comparison in Figure 2C of the two models - idealized erosion, and idealized binary fission. With such a comparison, the authors would have stronger footing to claim that one process is more important than the other.

As mentioned in our answer above to comment #2 of public review, we have included in the revised version of Figure 2 (panels B and D) a direct comparison between the erosion and equal fragments (binary fission) models for large division-formed colonies fragmented under ε = 5.8 m²/s³. The comparison is further detailed in the new Supplementary Figure S9 for representative time points. Only the erosion models can recover the biovolume transfer from large colonies to single cells, as observed for the experimental results in Figure 2D and further detailed in Figure S9D. We believe that the revised version of Figure 2 and the new Supplementary Figure S9 provide strong evidence in support of the erosion fragmentation model.

Would the authors comment on their chosen range of experimental dissipation rates? For instance, was their goal more to investigate industrial/engineering applications where the goal is to disrupt the cyanobacteria, but not really typical natural conditions under which the groups might form?

The choice of experimental dissipation rates in our experiment was such that it covers engineering applications such as artificial mixing of eutrophic lakes using bubble plumes. We have now clarified in the Introduction, on lines 37-39, that artificial mixing has been successfully applied in several lakes to suppress cyanobacterial blooms. Furthermore, we have now clarified in the caption of Figure 5 that the bars on the right side indicate typical values of dissipation rates induced by natural wind-mixing, bubble plumes in artificially mixed lakes, and laboratory-scale experiments such as cone-and-plate systems and stirred tanks. The dissipation rates induced by the bubble plumes in artificially mixed lakes could potentially fragment aggregated cyanobacterial colonies and thus disrupt bloom formation. However, our preliminary experiments demonstrated that high levels of dissipation rate were required to achieve fragmentation, therefore we’ve focused on the upper range of values (0.01 to 10 m²/s³).

The dissipation rates generated by the cone-and-plate approach are indeed higher than the dissipation rates under typical natural conditions in lakes. We have now added a detailed discussion of the range of dissipation rates generated by the cone-and-plate approach in the revised manuscript, under section Materials and Methods in lines 462-473, where we also explain that these values are higher than the natural dissipation rates generated by wind action in lakes. However, the more generic insights obtained by our study, shown in Figure 5, are relevant for dissipation rates of natural lakes (e.g., Zone II). Therefore, in our discussion of Figure 5 we have now included the recent findings of Wu et al. (2024) (reference number [64] of the revised manuscript), who studied bloom formation of Microcystis in mesocosm experiments at dissipation rates representative of natural conditions; see also our reply to the next comment.

The authors should consider testing the space of Zone II on their phase map, for instance at very high particle concentrations and even lower rotational speeds, in order to show that their derivations match experiments.

Good point. As mentioned in our answer above to comment #4 of the public review, Zone II lies beyond the measuring range of our experimental setup. Instead, we refer to the recent study of Wu et al. (2024) (reference number [64] of the revised manuscript) which demonstrated that dense scum layers of Microcystis colonies are aggregation-dominated. These mesocosm experiments agree with our model predictions and their parameter range falls within Zone II. We have included in the revised version, lines 328-337, a detailed discussion where we elucidate the parameter range covered in our experiments and compare our predictions for Zone II with the recent findings of Wu et al. (2024).

The authors should show their calibration data and fit for the correction function of equation S1. Additionally, you may consider showing "raw" and "corrected" histograms of the size distribution, to demonstrate exactly what corrections are made.

As mentioned in our answer above to comment #5 of the public review, we have included in the revised version of the Supporting Information the new Supplementary Figure S8, which shows the raw and adjusted histograms of the size distribution, including the associated uncertainties. Furthermore, the correction function is now explained in detail in the new Supporting Information Text in lines 785-796.

The authors might consider commenting on Figure S3 a bit more in the main text. Even at very high dissipation rates, the cyanobacterial groups don't plummet to size 1, but stay in an equilibrium around 10-20x the diameter of a single cell. What might this mean for industrial applications trying to break up the groups?

We agree with the reviewer that further discussion of Figure S3, panels E and F, is warranted. In the revised version of the manuscript, under section Fragmentation of Microcystis colonies occurs through erosion in lines 133-137, we have now included a discussion of this figure. Figure S3F shows that more than 90% of the total biovolume ends up in the category “small colonies” (mostly single cells and dimers); hence, most of the initially large colonies do fragment to single cells or dimers. Only about 5-10% of the biovolume remains as “large colonies” of 10-20 cells. Although it is challenging to draw definitive conclusions about the behavior of these remaining large colonies, as they account for only a minor fraction of the suspension, one hypothesis is that variability in mechanical properties between colonies results in a subset of colonies exhibiting exceptional resistance even to very high dissipation rates (see lines 133-137).

Minor comments:

Typo Caption of Figure 2: Should read [m^2/s^3] for units

Thanks for catching this typo. The units in the caption of Figure 2 has been corrected to [m^2/s^3].

There is no Equation 10 in Materials and Methods as indicated in the rheology section.

We thank the reviewer for pointing out the lack of clarity in this algebraic manipulation. In fact, the yield stress has to be substituted in the current Equation 11 (previously Eq.10), from which the critical dissipation rate must be substituted in Equation 3. The result is the critical colony size (l* = 2.8) mentioned in line 243 of the revised manuscript. The correct equation numbers and algebraic substitutions are now indicated in lines 241-243 of the revised version of the manuscript.

<Reviewer #2 (Public review):

Especially the introduction seems to imply that shear force is a very important parameter controlling colony formation. However, if one looks at the results this effect is overall rather modest, especially considering the shear forces that these bacterial colonies may experience in lakes. The main conclusion seems that not shear but bacterial adhesion is the most important factor in determining colony size. As the importance of adhesion had been described elsewhere, it is not clear what this study reveals about cyanobacterial colonies that was not known before.

We would like to emphasize several key findings that our study reveals about the impacts of fluid flow on cyanobacterial colonies:

(I) Quantification of mechanical strength in cyanobacterial colonies: Our results demonstrate the high mechanical strength of cyanobacterial colonies, as evidenced by the requirement of high shear rates to achieve fragmentation. This is new knowledge, that was not known before for cyanobacterial colonies. To this end, our study highlights the resilience of these colonies against naturally occurring flows and bridges the gap between theoretical assumptions about colony strength and experimentally measured mechanical properties.

(II) The discovery that the mechanical strength of colonies differs between colonies formed by cell division and colonies formed by aggregation. This is again new knowledge, that was not known before for cyanobacterial colonies.

(III) Validation of a hypothesis regarding colony formation: Using a fluid-mechanical approach, we confirm the findings of recent genetic studies (references 25 and 67 of the revised version of the manuscript) which indicated that colony formation occurs predominantly via cell division rather than cell aggregation under natural conditions (except in very dense blooms).

(IV) Practical guidelines for cyanobacterial bloom control: Our findings provide valuable insights into the design of artificial mixing systems applied in several lakes. Artificial mixing of lakes is based on fundamentals of fluid flow, aiming at preventing aggregation of buoyant cyanobacteria in scum layers at the water surface. Our results show that the dissipation rates generated by bubble blumes in artificially mixed lakes can fragment cyanobacterial colonies formed by aggregation, but are not intense enough to cause fragmentation of division-formed colonies (see Figure 5 and lines 348-360).

The agreement between model and experiments is impressive, but the role of the fit parameters in achieving this agreement needs to be further clarified.

The influence of the fit parameters (namely the stickiness α1 and the pairs of colony strength parameters S1,q1,S2,q2) is discussed in the sections Dynamical changes in colony size modelled by a two-category distribution in lines 247-253 and Materials and Methods in lines 559-565. We kept the discussion concise to maintain readability. However, we agree with the reviewer that additional details about the importance of the fit parameters and the sensitivity of the results to these parameters could be beneficial. In the revised version of the section Materials and Methods in lines 560-563, we have included a detailed discussion of the fit parameters.

The article may not be very accessible for readers with a biology background. Overall, the presentation of the material can be improved by better describing their new method.

We apologize for the limited readability of the description of the experimental setup and model used. In the revised version of the manuscript and the SI, we have detailed further the new methods presented here. The modifications include a detailed description of the operating range of the cone-and-plate shear setup (subsection Cone-and-plate shear of the section Materials and Methods, in lines 462-473). Furthermore, we think that incorporation of the recent experimental results of Wu et al. (2024), on lines 331-337 of the manuscript, will appeal to readers with a biology background. Their mesocosm experiments support our model prediction that aggregation is the dominant mechanism for colony formation in region (II) of Figure 5.

Reviewer #2 (Recommendations For The Authors):

(1) The authors seem too modest in claiming technological advance. They should describe the technological advance of combining microscopy with rheometry, in such a way that this invites others to apply this or similar approaches on biological samples. Even though I feel that the advancement of knowledge of this system by their method is relatively modest, there may be more advances in other systems.

We appreciate the positive view of the reviewer towards the importance of this technology and we agree that its advantages should be advertised to researchers investigating similar systems. We have now given more attention to the technological advance of combining microscopic imaging with rheometry in the final paragraph of the Conclusions (lines 386400), where we now also briefly discuss an interesting recent study of marine snow (Song et al. 2023, Song and Rau 2022, reference numbers 70 and 71 of the revised manuscript), which used a similar combination of microscopy and rheometry as in our study. Furthermore, in the Methods section, we now briefly explain how the rheometry can be adjusted to investigate other systems (lines 474-480).

(2) It seems reasonable -also based on what we already know about these aggregates - to assume that the main difference in shear sensitivity between field samples and cultures lies in the production of extracellular polysaccharide substance (EPS). To go beyond what is already known, the study could try to provide more direct and quantitative evidence for EPS involvement. For example, using a chemical quantification of EPS levels, or perturbing EPS levels using digestive enzymes.

We agree with the reviewer that further characterization of the EPS is highly relevant to understand the mechanical strength of colonies. However, we believe that chemical quantification and/or degradation of EPS lies beyond the scope of our article and should be addressed by future studies.

(3) Assuming EPS is indeed the reason for the differences in shear resistance: the authors speculate the reason why the field samples have more EPS lies in chemical composition (Calcium/nitrogen levels). In addition, there could be grazing that is known to promote aggregation (possibly increasing EPS), or just inherent genetic differences between strains. I am not necessarily expecting the authors to explore this direction experimentally, but it seems certainly feasible and would make the final result less speculative.

We agree with the reviewer that there are more biotic and abiotic factors that can influence EPS amount and composition. The influence of grazing and other relevant factors on cell adhesion is discussed in references [26-29], cited in our introduction in lines 50-53. As discussed in our answer to recommendation #2, we believe that a quantitative investigation of these various factors is beyond the scope of this work and should be addressed in future studies.

(4) A cool finding seems to be the critical relative diameter (Fig 2E), a colony size that seems invariant under shear. I was slightly surprised that the authors seem to take little effort to understand this critical diameter mechanistically (for example by predicting it, or experimentally perturbing it). Again, not a necessary requirement, but this is where the study could harness its technological advantage to provide a more quantitative understanding of something that goes beyond the existing knowledge of the system.

We apologize to the reviewer if our descriptions and discussions of Figure 2 were unclear. One of the key conclusions from our experiments is that the critical relative diameter depends on the dissipation rate, as shown in Figure 2F. This dependence is also incorporated into the model through the constitutive equation (2). Furthermore, we expect the mechanical resistance of colonies, quantified by the critical relative diameter, to be affected by other biotic and abiotic factors that influence EPS amount and composition.

(5) The jump from 0.019 to 1.1 m²/s³ seems large. What was the reason for not exploring intermediate values? The authors should also define low, modest and intense dissipation rates more clearly. Currently, they seem somewhat arbitrarily defined, i.e. 0.019 m²/s³ is described as low (methods) and moderate (results). In Fig 2, the authors further talk about low dissipation rates without a quantitative description.

We thank the reviewer for pointing out the lack of clarity in the choice of parameter range and the nomenclature. Regarding the former, the suspension of division-formed colonies of Microcystis strain V163 displayed negligible fragmentation for dissipation rates between 0.019 to 1.1 m²/s³, as seen in Figures S2A and S3A. Due to the low sensitivity of the fragmentation results in this region, we don’t expect change in behavior for intermediate values. Regarding the nomenclature, we have corrected the inconsistencies throughout the text. We have chosen to name the dissipation rate values as: low for values typical of windmixing, moderate for values typical of the core of bubble plumes, and intense for values typical of propellers. Whenever mentioned in the text, the numerical value of dissipation rate is also included to avoid doubt.

(6.) The structure and narrative of the paper can be improved. The article first describes all lab culture experiments and then the model, while the first figure already shows model fits. Perhaps it would be better to first describe the aggregation experiments, to constrain the appropriate terms of the model, and then move to fragmentation.

We appreciate the recommendation of the reviewer regarding the structure. We have chosen to describe first the fragmentation experiments (Fig. 2), as these can be understood without introducing the aggregation effects. In contrast, the steady state results in the aggregation experiments (Fig. 3) come from the balance between aggregation and fragmentation. Therefore, we judged the current order to be more appropriate. The model fits are combined with the experimental results in Figures 2 and 3 to have a concise display. We have ensured that all the concepts required to understand each figure panel are explained prior to their discussion.

(7) The number of data points that go into the histogram needs to be indicated. The main reason is that the authors report the distribution in terms of the biovolume fraction, suggesting the numerical counts are converted into volume. This to me seems like the most sensible parameter, but I could not find how this conversion is calculated (my apologies if I missed it). This seems especially relevant because a single large colony can impact this histogram quite considerably.

We apologize for the lack of clarity in the calibration and conversion steps of the size distribution. As discussed above in the answer to comment #5 of the reviewer #1, more details of the calibration process have been added to the revised version of the Supporting Information Text in lines 785-796. Furthermore, the new Supplementary Figure S8 presents examples of the raw and adjusted size distribution, including the total number of counted colonies per histogram and the associated uncertainties in the concentration and biovolume distributions.

(8) Over the timescales measured here, colonies could start sinking (or floating), possibly in a size-dependent manner, that could lead to a bias due to boundary effects. Did the authors consider this potential artifact?

The sinking or floating of colonies is a relevant process which was taken into account in the choice of our parameter range for the dissipation rate. The minimum dissipation rate used in our experiments ensures that the upward inertial velocity near stagnation is sufficient to counteract the sedimentation of colonies. A detailed discussion of the choice of the parameter range is now included in the revised version of the Materials and Methods in lines 462-473.

(9) "On the one hand, sequencing of the genetic diversity within Microcystis colonies supports the hypothesis that colony formation undernatural conditions is primarily driven by cell division [25]. On the other hand, cell aggregation can occur on a shorter time scale and may offer improved protection against high grazing pressure [26]." This appears somewhat constructed, as what is described as "on the other hand" is not evidence against the genetic diversity.

We agree that the suggested dichotomy in this text appeared somewhat constructed, and we have now removed the wording “on the one hand” and “on the other hand”. The studies from reference [25] demonstrated that the genetic diversity between independent Microcystis colonies is much greater than the diversity within colonies. If cell aggregation was the dominant mechanism, a similar genetic diversity would be observed between and within colonies, which contrasts the findings from reference [25]. We have adjusted the text in the revised manuscript, in lines 46-54, to clarify this point.

(10) The phase diagram seems largely based on extrapolations that are made outside of the measurement regime (e.g. dark red bars indicating the dissipation rate, Fig 5 - by the way 1 this color scheme could use some better contrast, by the way 2 Fig S7 suggests a wider dissipation rate range as indicated in Fig 5, why?). Hence there seems to be the need to more clearly lineate experimental results, simulations, and extrapolations in the phase diagram.

We agree with the reviewer that further clarifications should be given about the parameter range covered in our experiments and apologize for the lack of readability in the color scheme of Fig 5. In lines 329-337, 346-347, 353-355, we have highlighted the parameters range covered by our experiments as well as the range covered by previous studies of windmixed mesocosm (namely reference [64] of the revised manuscript). Regarding the color scheme of Figure 5, we have modified the legend of the figure to improve readability. The color contrast was increased and leader lines were added to connect the colored bars with the respective label.

(11) Unfortunately, the manuscript did not contain line numbers.

We apologize to the reviewer for the lack of line numbers in our initial version. The revised version of the manuscript now contains line numbers, both in the main text and the supporting information.

(12) Fig 2D. Caption is too minimal. Y-axis could better be named "Fraction of colonies" as both small and large colonies are plotted.

The caption for Figure 2D was extended to better describe the plot. We have kept the y-axis label as “Fraction of small colonies”, since this is the quantity displayed by the three curves in the plot.

(13) An inset should have axis labels.

All the insets in our plots display the same variables as their respective plots. In order to keep the plots light and preserve readability, we therefore prefer to present the axis labels only along the x-axis and y-axis of the main plots, which implies by convention that the same axis labels also apply to the insets. To the best of our knowledge, this is a common approach.

(14) Page 5, first words. Likely Fig 3A, not 2A was meant.

We thank the reviewer for pointing out this readability issue. We intend to compare both Figures 2A and 3A. The text of the revised manuscript, in lines 146-148, has been adjusted with the correct figure numbers.

(15) Introduction, second last paragraph, third last line. "suspension leaded to a broad distribution" I assume you meant "... led to a ..."

We thank the reviewer for pointing out this typo. It has been corrected (line 122).

this is the

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

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

them as well

so each knowledge as it has a certain amount of

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

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

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

Can be connected to any number of other Knowledge Assets

Author response:

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

Reviewer #1 (Public review): 

Summary: 

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

Strengths: 

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

Weaknesses: 

There is insufficient credit and citation of previous work. 

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

Reviewer #2 (Public review): 

Summary: 

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

Strengths: 

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

Weaknesses: 

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

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

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

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

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

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

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

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

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

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

Reviewer #3 (Public review): 

Summary: 

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

Strengths:  

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

Weaknesses: 

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

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

Reviewing Editor Comments

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Incorrect, see Silberberg 2021. 

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

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

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

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

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

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

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

KcsA is not a member of the SKT superfamily. 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Reviewer #1 (Recommendations for the authors): 

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

Specific points: 

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

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

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

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

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

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

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

Incorrect, see Silberberg 2021. 

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

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

Other points: 

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

KcsA is not a member of the SKT superfamily. 

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

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

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

This sentence has been updated as follows:

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

Reviewer #2 (Recommendations for the authors): 

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

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

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

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

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

and

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

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

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

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

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

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

Results sections describing the selectivity filter:

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

Results section describing the CBS:

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

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

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

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

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

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

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

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

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

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

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

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

(8) Label G232D in Figure 2a. 

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

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

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

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

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

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

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

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

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

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

Reviewer #3 (Recommendations for the authors): 

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

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

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

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

(2) Typo: page 14, "diluted" 

This typo has been corrected.

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

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

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

Synthèse des Outils Numériques et des Procédures Associées au Lycée

Résumé

Ce document synthétise l'écosystème des outils numériques utilisés au sein de l'établissement, en se basant sur un tutoriel détaillé.

L'objectif principal est de clarifier le rôle de chaque plateforme et de fournir un guide pratique pour les parents et les élèves. Le point d'entrée fondamental de cet écosystème est le compte EduConnect, qui sert de clé d'accès unique à la quasi-totalité des services.

Sans un compte EduConnect fonctionnel, l'accès aux autres outils est impossible.

Les plateformes principales sont :

1. EduConnect et les Téléservices : Le portail national pour l'identité numérique, indispensable pour les démarches administratives telles que les demandes de bourses, le paiement de la cantine et la mise à jour des informations personnelles.

2. Mon Bureau Numérique (MBN) : L'environnement de travail fourni par la Région Grand Est, servant de portail central pour la vie scolaire quotidienne, incluant le cahier de textes, la messagerie, l'agenda et l'accès aux ressources de la classe.

3. Pronote : Accessible via MBN, cet outil est spécialisé dans le suivi pédagogique : consultation des notes, gestion des absences, emploi du temps mis à jour et prise de rendez-vous pour les rencontres parents-professeurs.

4. Parcoursup : La plateforme nationale pour l'orientation post-bac, essentielle pour les élèves de Terminale et une ressource d'information cruciale pour les élèves de Seconde et Première.

5. Outils Complémentaires : Un système de prise de rendez-vous en ligne pour les psychologues de l'Éducation Nationale (PsyEN) et la fourniture de licences Microsoft Office gratuites pour les élèves.

Un support informatique est disponible en cas de difficulté via l'adresse e-mail X.

Il est souligné qu'une part significative de parents (600) n'a pas encore activé son compte EduConnect, ce qui les prive d'accès à des services essentiels, notamment le vote pour les représentants des parents d'élèves.

1. Le Portail EduConnect : La Clé d'Accès Universelle

EduConnect est le portail d'authentification de l'Éducation Nationale. Il ne s'agit pas d'un outil utilisé au quotidien, mais de la porte d'entrée indispensable à tous les autres services.

Rôle et Importance

• Identifiant Unique : Fournit un identifiant et un mot de passe uniques pour accéder à la majorité des outils numériques de l'établissement.

• Point de Départ Obligatoire : Si le compte EduConnect ne fonctionne pas, aucun autre service n'est accessible.

• Comptes Multiples : Chaque responsable légal (père, mère, responsable 1 et 2) dispose de son propre compte individuel.

Activation et Dépannage

• Problème d'Activation : Un constat a été fait que 600 parents n'ont pas activé leur compte, souvent car un seul des deux parents gère le suivi scolaire.

Cela pose problème, par exemple, pour le vote aux élections des parents d'élèves, où chaque parent peut voter.

• Procédure en cas de problème :

1. Contacter le support informatique du lycée à l'adresse : X.   

2. Vérifier le dossier "spam" de sa messagerie, car les e-mails de réinitialisation peuvent y atterrir.   

3. En dernier recours, l'établissement peut imprimer un document papier avec des identifiants temporaires.

• Complexités Possibles : Des difficultés peuvent survenir en cas de :

◦ Plusieurs enfants scolarisés dans différents établissements.    ◦ Homonymes entre parents d'élèves.    ◦ Changement d'académie.

• Mise à jour des e-mails : Le bon fonctionnement du compte dépend de l'exactitude de l'adresse e-mail enregistrée dans la base de données de l'établissement (base Siècle), car c'est par ce biais que sont envoyés les liens de réinitialisation. Actuellement, une quarantaine d'adresses e-mail sont encore erronées sur environ 2800.

2. Téléservices (Scolarité Services) : La Gestion Administrative

Accessibles via le compte EduConnect, les Téléservices sont la plateforme pour toutes les démarches administratives numérisées.

Fonctionnalités Principales

• Demandes de Bourse : Permet de soumettre une demande de bourse en ligne, sans document papier.

• Fiche de Renseignements : Consultation et demande de modification des coordonnées (adresse postale, e-mail, téléphone). Toute modification doit être validée par le secrétariat de l'établissement.

• Paiement de la Demi-Pension :

◦ Les factures sont publiées sur cette plateforme (la première vers mi-novembre).   

◦ Il est possible de payer chaque facture trimestrielle en deux fois, permettant ainsi un étalement sur six paiements dans l'année.

La mensualisation complète est envisagée pour l'année suivante.

• Documents Officiels : Téléchargement du certificat de scolarité. Ce document est également disponible sur Pronote.

• Orientation : La plateforme sera utilisée pour les phases d'orientation, notamment pour les élèves de Seconde.

3. Mon Bureau Numérique (MBN) : L'Espace de Travail Quotidien

MBN est l'environnement numérique de travail (ENT) fourni par la Région Grand Est. Il s'agit du portail central pour la communication et les informations pédagogiques.

Accès

• La connexion s'effectue via les identifiants EduConnect.

• Après connexion au portail académique, il est nécessaire de sélectionner "Lycée Louis-Vincent" pour accéder à l'espace de l'établissement.

• Il est conseillé d'ajouter le site MBN aux favoris de son navigateur pour un accès plus rapide et écologique.

Fonctionnalités Clés pour les Parents

• Cahier de Textes : Permet de consulter le travail à faire donné aux élèves jour par jour, ainsi que le contenu des séances passées.

• Messagerie (Zimbra) :

◦ Permet de contacter les enseignants de la classe de son enfant, la direction ou d'autres personnels via un assistant destinataire.   

◦ Note importante : Le Proviseur (Olivier Palaise) préfère être contacté sur son adresse académique (olivier.palaise@ac-nancy-metz.fr) plutôt que via la messagerie MBN pour des raisons de gestion de flux (recevant entre 100 et 200 e-mails par jour).

• Agenda de l'Établissement : Un calendrier complet des événements du lycée (Fête de la Science, vacances, sessions d'évaluation Pix, etc.).

• Espace Classe : Section où les enseignants peuvent partager des ressources spécifiques à une classe ou une spécialité (ex: règles pour le travail en mathématiques, liens vers des manuels).

• Accès à Pronote : MBN est le portail d'entrée pour se connecter à Pronote.

Vue Élève : Le Médiacentre

• Les élèves disposent d'un onglet supplémentaire, "Médiacentre", qui donne accès à l'ensemble des manuels scolaires numériques fournis par la Région Grand Est.

• Avantages : Gratuité pour les familles et allègement significatif du poids des cartables.

• Inconvénients : L'utilisation d'un manuel sur écran peut être moins pratique qu'un format papier.

• Autres ressources : Le Médiacentre donne également accès à des outils comme "Do you speak grand test", une plateforme gratuite d'apprentissage des langues.

4. Pronote : Le Suivi Pédagogique Détaillé

Pronote est le logiciel de vie scolaire utilisé pour le suivi précis des résultats et de l'assiduité des élèves.

Accès et Fonctionnalités

• Accès : Se fait en cliquant sur l'icône Pronote depuis Mon Bureau Numérique (MBN).

• Suivi des Notes : Consultation de l'ensemble des notes obtenues. Les parents y ont accès quelques jours après les élèves, pour permettre aux professeurs d'expliquer d'abord les résultats en classe.

• Emploi du Temps : C'est la source la plus fiable et la plus à jour pour l'emploi du temps de l'élève, incluant les absences de professeurs et les modifications de salles.

• Élections des Parents d'Élèves : C'est via Pronote que les parents accèdent à la plateforme de vote électronique. Il est rappelé que les deux parents peuvent voter, et que le vote nécessite un compte EduConnect activé.

https://youtu.be/b7gst-DjVd8?t=1979

• Réunions Parents-Professeurs : La prise de rendez-vous pour les rencontres se fera via cet espace (ex: à partir du 17-18 novembre pour la réunion des classes de Seconde du 29 novembre).

5. Parcoursup : La Plateforme d'Orientation Post-Bac

Parcoursup est l'outil national incontournable pour la gestion des vœux d'affectation dans l'enseignement supérieur.

Utilisation et Calendrier

• Public concerné : Indispensable pour les élèves de Terminale, il est fortement conseillé aux élèves de Première et de Seconde de s'y familiariser pour préparer leurs choix de spécialités et de filières.

• Disponibilité : Même lorsque la plateforme est "fermée" (avant la mise à jour des formations de l'année en cours), elle reste une mine d'informations (vidéos, tutoriels, carte des formations de l'année précédente).

Exploration des Formations

• La recherche d'une formation donne accès à une fiche détaillée contenant des informations capitales.

• Exemple concret - PASS (Parcours d'Accès Spécifique Santé) à Nancy :

Critères de Sélection (2025)

Poids

Détails

Résultats Scolaires

45%

Notes de Première et de Terminale.

Méthodes de Travail

Évaluées via les remarques des enseignants.

Savoir-être

Motivation (lettre), comportement.

Engagement

Activités extra-scolaires valorisées (délégué, SNU, etc.).

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Is this to show how the children's innocence is exploited, or to emphasize how they are slowly being pulled into a cultural system they don't understand?

Author response:

Reviewer #1 (Public review): 

Summary: 

The taxonomic analysis of IRG1 evolution is compelling and fills an important gap in the literature. However, the experimental evidence for IRG1 localization requires greater detail and confirmation. 

Strengths: 

The phylogenetic analysis of IRG1 evolution fills an important gap in the literature. The identification of independent acquisition of metazoan and fungal IRG1 from prokaryotic sources is novel, and the observation that human IRG1 lost mitochondrial matrix localization is particularly interesting, with potentially significant implications for the study of itaconate biology. 

We thank the reviewer for appreciating the novelty of our study in exploring IRG1 evolution.  

Weaknesses: 

The protease protection assay was conducted with MTS-IRG1 but not with wild-type IRG1, which should also be tested. Moreover, no complementary methods, such as microscopy, were employed to validate localization. Beyond humans, the structure and localization of mouse IRG1, highly relevant given the widespread use of the mouse as a model for IRG1 functional studies, are not addressed. 

Regarding submitochondrial localization of IRG1, we want to draw attention to the published data that a protease protection assay for wild-type mammalian IRG1 has been performed by Lian et al. 2023 (Extended Data Fig. 4), which convincingly demonstrated an outer-mitochondrial membrane localization of endogenous mouse IRG1 in mouse DC2.4 cells upon LPS stimulation that induces IRG1 expression. 

Regarding complementary microscopy evidence, the same paper performed two-color,  DNA-paint super-resolution imaging to demonstrate an enrichment of IRG1 to mitochondria with a lack of co-localization of the inner membrane/matrix marker Cox IV. 

Given the direct visualization of sub-mitochondrial localization, we consider applying super-resolution microscopy to revisit the sub-mitochondrial localization of di[erent IRG1 constructs in the study.   

Reference:

Lian H, Park D, Chen M, Schueder F, Lara-Tejero M, Liu J, Galán JE. Parkinson's disease kinase LRRK2 coordinates a cell-intrinsic itaconate-dependent defence pathway against intracellular Salmonella. Nat Microbiol. 2023 Oct;8(10):1880-1895. doi: 10.1038/s41564-023-01459-y. Epub 2023 Aug 28. PMID: 37640963; PMCID: PMC10962312.

Finally, if itaconate is indeed synthesized outside the mitochondrial matrix to safeguard metabolic activity, it is not discussed how this reconciles with its reported inhibitory e[ect on SDH. 

We thank the excellent point raised by the reviewer. Indeed, itaconate has been proposed to inhibit matrix SDH exhibiting anti-inflammation function (Lampropoulou, Cell Metab 2016). While the mitochondrial transport of itaconate has not been fully characterized in vivo or in cells, a specific itaconate transport activity has been shown for the mitochondrial 2-oxoglutarate transporter OGC using in vitro proteoliposome system (Mills et al. Nature 2018). 

We plan to discuss this important point on mitochondrial itaconate transport in the revision. 

Reference: 

Lampropoulou V, Sergushichev A, Bambouskova M, Nair S, Vincent EE, Loginicheva E, Cervantes-Barragan L, Ma X, Huang SC, Griss T, Weinheimer CJ, Khader S, Randolph GJ, Pearce EJ, Jones RG, Diwan A, Diamond MS, Artyomov MN. Itaconate Links Inhibition of Succinate Dehydrogenase with Macrophage Metabolic Remodeling and Regulation of Inflammation. Cell Metab. 2016 Jul 12;24(1):158-66. doi: 10.1016/j.cmet.2016.06.004. Epub 2016 Jun 30. PMID: 27374498; PMCID: PMC5108454.  

Mills EL, Ryan DG, Prag HA, Dikovskaya D, Menon D, Zaslona Z, Jedrychowski MP, Costa ASH, Higgins M, Hams E, Szpyt J, Runtsch MC, King MS, McGouran JF, Fischer R, Kessler BM, McGettrick AF, Hughes MM, Carroll RG, Booty LM, Knatko EV, Meakin PJ, Ashford MLJ, Modis LK, Brunori G, Sévin DC, Fallon PG, Caldwell ST, Kunji ERS, Chouchani ET, Frezza C, Dinkova-Kostova AT, Hartley RC, Murphy MP, O'Neill LA. Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1. Nature. 2018 Apr 5;556(7699):113117. doi: 10.1038/nature25986. Epub 2018 Mar 28. PMID: 29590092; PMCID: PMC6047741.

Reviewer #2 (Public review): 

Summary: 

The authors are trying to explain how the metabolite itaconate evolved, since although it's involved in host defense, it can also limit mitochondrial function. They are trying to probe the trade-o[ between these two functions. 

Strengths: 

The evolutionary aspect is novel; this is the first time to my knowledge that the evolution of IRG1 has been analysed, and there are interesting findings here. The key finding appears to be that subcellular localisation is an important aspect, allowing host defense in some organisms without compromising bioenergetics. This is an interesting finding in the context of immunomebolism, although it needs extra analysis. 

Weaknesses: 

The work concerning sub-mitochondrial localisation is confusing and needs better analysis. 

We thank the reviewer for the constructive feedback. As in our response to reviewer 1, we want to draw attention to the published data in which the outer mitochondrial membrane localization of IRG1 has been demonstrated by protease protection assay and explored using super-resolution imaging by Lian et al. 2023 (Extended Data Fig. 4). Given the direct visualization of sub-mitochondrial localization by super-resolution imaging, we plan to revisit and to apply the method to di[erent IRG1 constructs used in the paper.

Reviewer #3 (Public review): 

Summary: 

IRG1 is highly expressed in activated human and mouse myeloid cells. It encodes the mitochondrial enzyme cis-aconitate decarboxylase 1 (ACOD1) that generates itaconate. Itaconate has anti-microbial activity and acts immunoregulatory by interfering with cellular metabolism, signaling to cytokine production, and multiple other processes. 

The authors perform a phylogenetic analysis of IRG1 to obtain insight into the evolution of itaconate biosynthesis. Combining BLAST with human IRG1 and a MmgE/Ptrp domain search, they find CAD in all domains of life, but the presence of IRG1 homologs is patchy in eukaryotes, indicating that itaconate biosynthesis is not essential. The phylogenetic analysis showed a more distant relationship of fungal and metazoan CAD/IRG1 to many prokaryotic sequences, suggesting independent acquisition of these metazoan and fungal CAD genes. In metazoans, three subbranches of paleo-IRG1 (in mollusks/early chordates) and two paralogous vertebrate forms (IRG1 and IRG1-like) were identified, with the latter derived from paleo-IRG1, and by genome duplication. While most jawed vertebrates have both IRG1 and IRG1L, metatherian and eutherian mammals have lost IRG1L and contain only IRG1. 

Interestingly, sequence analysis of both paralogues showed that many IRG1L genes contain an N-terminal mitochondrial targeting sequence (MTS) that is absent from most IRG1 sequences. Limited proteolysis of submitochondrial localization confirmed that zebrafish IRG1L is only sensitive to proteases in the presence of high Triton X-100, indicative of association with mitochondrial matrix. In contrast, a recent paper from the Galan lab (Lian 2003 Nature Microbiology) reported that human IRG1 is not localized to the mitochondrial matrix, although enriched in mitochondria. Here, the authors generated a matrix-targeted human IRG1 by adding the N-terminal MTS and found that it localizes to the matrix based on a limited proteolysis assay. The loss of MTS-containing IRG1L from most mammals appears, therefore, to indicate that itaconate generation is directed to the cytoplasm, potentially reducing inhibition of TCA cycle activity in the mitochondria. 

Next, the authors confirmed that the recombinant IRG1L protein has CAD activity in vitro. The last part of the manuscript addresses the expression of paleo-IRG1 in oysters and amphioxus, where they found high mRNA levels in oyster hemocytes which was further increased by poly(I:C), which was also the case in amphioxus tissues after feeding of LPS or poly(I:C), indicating a role for paleo-IRG1/itaconate in early metazoan innate immunity. 

Strengths 

(1) Phylogenetic perspective largely lacking so far in the IRG1/itaconate field. 

(2) Manuscript clearly written and understandable across disciplines. 

(3) Phylogenetic analyses complemented by biochemical and gene expression analyses to link to function. 

(4) Lack of MTS in IRG1 and change in localization from mitochondria, highly relevant antimicrobial and cellular e[ects of itaconate. 

We thank the reviewer for the positive comments with the strengths.  

Weaknesses: 

(1) Biochemical and functional analysis of di[erent CAD mRNA and proteins lacks depth. 

We plan to explore two types of experiments: 

First, we plan to purify di[erent CAD recombinant proteins; and if successful, we will test their in vitro enzymatic activity in synthesize itaconate. The positive data will also answer question (3) below.

Second, we plan to measure itaconate level in oyster hemocytes after PAMP stimulation, to demonstrate an in vivo itaconate production activity by paleo-IRG1. The data will also address question (4) below. 

(2) The submitochondrial localization assay lacks a native human IRG1 control. 

As in our response to reviewer 1, we believe Lian et al. 2023. provided strong evidence supporting an outer mitochondrial membrane localization of wild-type endogenous, mouse IRG1. Given the direct visualization using suer-resolution imaging, we plan to revisit submitochondrial localization of di[erent IRG1 constructs using super-resolution imaging. 

(3) CAD activity shown for IRG1L but not paleo-IRG1. 

We plan to purify di[erent CAD recombinant proteins; and if successful, we will test their in vitro enzymatic activity in producing itaconate.

(4) Itaconate production by early metazoans after PAMP stimulation? 

We plan to measure itaconate level in oyster hemocytes after PAMP stimulation, to demonstrate an in vivo itaconate production activity by paleo-IRG1.

(5) No measurement of energy metabolism (trade-o[s?). 

Because PAMP signaling might trigger other downstream e[ects that also impair mitochondrial function, for instance nitric oxide that inhibits complex IV, we plan to avoid PAMP condition and direct test the e[ect of itaconate production. We plan to compare the impact on mitochondrial bioenergetics, if the same CAD enzymes (thus with the same activity) can be expressed at the same level intra-mitochondrially and extramitochondrially, for instance in the case of MTS-hACOD1 and hACOD1.

Author response:

Reviewer #1 (Public Review):

The authors describe a new computational pipeline designed to identify smFISH probes with improved RNA detection compared to preexisting approaches. smFISH is a powerful and relatively straightforward technique to detect single RNAs in cells at subcellular resolution, which is critical for understanding gene expression regulation at the RNA level. However, existing methods for designing smFISH oligos suffer from several limitations, including off-target binding that produces high background signals, as well as a restricted number of probes that are sufficiently specific to target shorter-than-average mRNAs. To address these challenges, the authors developed TrueProbes, a computational method that aims to minimize off-target-mediated background fluorescence.

Overall, the study addresses a technically relevant problem. If improved, this would allow researchers to study gene expression regulation more effectively using single-molecule FISH. However, based on the current presentation of data, it is not yet clear that TrueProbes offers significant advantages over preexisting pipelines. In the following section, I describe some concerns, which should be adequately addressed.

Major Comments:

(1) The manuscript currently presents only one example in which different pipelines were tested to generate probes (targeting ARF4). While the images suggest that both TrueProbes and Stellaris outperform the other pipelines, the comparison is potentially misleading because the number of probes used differs substantially. I recommend that the authors include at least three independent examples in which an equal number of probes are designed across pipelines, so that signal-to-noise can be assessed in a controlled and comparable way. This would allow the probe number to be held constant while directly evaluating performance.

This is an important observation. We have already addressed this issue in Figures 3E-G and Supplementary Figure 4E-G, where we plotted the number of OFF-targets for each ON-target probe. If we select longer genes to ensure an equal number of designed probes with strong signals, we will still end up with the same number of ON-target probes. Consequently, Figures 3B-D and 3E-G would show similar trends, albeit with different values on the y-axis. Additionally, we will conduct an analysis using Stellaris at its highest probe design stringency setting to compare the software under its strictest design conditions. Additional experiments are outside the scope of the current manuscript.

(2) It is also unclear how many biological replicates were performed for the ARF4 experiments. If only a single replicate was included, it is difficult to conclude that TrueProbes consistently outperforms other pipelines in a robust and reproducible manner. I suggest the authors include data from at least three biological replicates with appropriate statistical analysis, and ideally extend this to additional smFISH targets as outlined in Comment 1.

Three biological replicates were utilized for the ARF4 experiments. As stated in the original submission, the average data from all three replicates is presented in Figure 4, while the data for each individual replicate can be found in Figure S5. Statistical analyses were conducted for both the pooled data in Figure 4 and the individual data in Figure S5. The results of all statistical calculations are detailed in Supplemental Table 1. We will update the text to clearly indicate the number of biological replicates and the outcomes of the statistical analysis.

(3) No controls are presented to demonstrate that the TrueProbes-designed smFISH spots are specifically detecting ARF4. The current experiment primarily measures signal-to-noise, but it remains possible that some detected spots do not correspond to ARF4 mRNAs. Since one of the major criteria used by TrueProbes is to limit cross-hybridization, the authors should perform ARF4 knockdown experiments and demonstrate that nearly all ARF4 smFISH signal is lost. A similar approach should be applied to the additional examples recommended in Comment 1.

Thank you for your suggestion. Currently, we lack the expertise in our lab to conduct such experiments, so they are beyond the scope of this manuscript. However, we will create additional supplementary figures to demonstrate that the likelihood of false positives is low, based on the assumption that current publicly available BLAST algorithms, genome annotations, and reference transcription expression data are accurate.

We will include a comparison in our supplementary materials showing the off-target RNA that can bind the highest number of probes simultaneously for each software. Additionally, we will perform a correlation analysis to illustrate the relationship between spot intensity for different software and the number of probes they design. This will help us estimate how the number of probes bound to RNA correlates with expected spot intensity ranges.

Using this information, along with autofluorescence background intensity measurements from no-probe controls, we will estimate the minimum number of probes that need to bind to targets to be detected as single spots. If this minimum is higher than the maximum number of simultaneous off-target probe bindings, we anticipate that the detected spot signal will primarily reflect ARF4 rather than other transcripts.

(4) In the limitations of the study, the authors note that "RNA secondary and tertiary structures are not included, which may lead to inaccuracies if binding sites are structurally occluded." However, I am not convinced that this is a true limitation, since formamide in the smFISH protocol should denature secondary structures and allow oligo access to the RNA. I recommend that the authors comment on this point and clarify whether secondary structure poses a practical limitation in smFISH probe design.

Thank you for pointing this out. We will revise the manuscript to clarify: "We did not include RNA secondary and tertiary structures in the model because the use of formamide in RNA-FISH experiments denatures these structures, allowing oligonucleotides to access the RNA."

(5) The authors also correctly acknowledge in their limitations that "RNA-protein interactions, which can modulate accessibility of the transcript, are not modeled." I suggest referencing relevant studies on this issue, particularly Buxbaum et al. (2014, Science), which would provide important context.

Thank you for highlighting the literature that supports this limitation. We will include Buxbaum et al. (2014, Science) and additional studies that discuss how RNA-protein interactions can affect RNA-FISH experiments.

Reviewer #2 (Public review):

Summary:

Hughes et al present a new single-molecule RNA fluorescence in situ hybridization (smFISH) probe design software, termed "TrueProbes" in this manuscript. They claim that all existing smFISH (and variants) probe design software packages have limitations that ultimately impact experimental performance. The author's claim to address the majority of these limitations in TrueProbes by introducing multiple computational steps to ensure high-quality probe design. The manuscript's goal is clear, and the authors provide some evidence by designing and targeting one gene. Overall, the manuscript lacks rigorous evidence to support the claims, does not demonstrate its suitability for a variety of smFISH-type experiments, and some of the provided quantification data are unclear. While TrueProbes clearly has potential, more data is required, or the authors should tone down the claims.

We appreciate the reviewer’s thoughtful feedback. We will revise the text to ensure that all claims are backed by computational or experimental evidence. For claims that do not have supporting results, we will relocate them to the discussion section as potential future extensions. Since our probe design is open access, both we and the community can further develop our codes as needed.

Strengths:

(1) The problem is well-articulated in the abstract and the introduction.

(2) Figures 3 and 4 follow a consistent color scheme where each probe design method has its own color, which helps the reader visually compare methods.

(3) The authors compared multiple probe design software packages both computationally and experimentally.

(4) TrueProbes does produce visually and quantitatively better results when compared to 2 of the 4 existing smFISH probe design packages (Paintshop and MERFISH panel designer).

(5) The authors introduce a comprehensive steady-state thermodynamic model to help optimally guide probe design.

We like to thank the reviewer for pointing out the strength of the manuscript.

Weaknesses:

(1) The abstract describes the problem well and introduces the solution (the TrueProbes software), but fails to provide specific ways in which the TrueProbes software performs better. The authors state that "...[TrueProbes] consistently outperformed alternatives across multiple computational metrics and experimental validation assays", but specific, quantitative evidence of improved performance would strengthen the statement.

Thank you for acknowledging the clarity of the abstract and introduction. We will revise the abstract to provide more specific details on how TrueProbes outperforms other software. Additionally, we will include specific computational and experimental metrics that demonstrate TrueProbes' improved performance compared to other software.

(2) The text claims that TrueProbes outperforms all other probe design software, but Figure 3 indicates that TrueProbes has neither the greatest number of on-target binding nor the lowest number of off-target binding. The data in Figure 3 does not support the claims made in the text. Specifically, the authors claim that "RNA FISH Experimental Results Demonstrate that Off Target and Binding Affinity Inclusive Probe Design Improve RNA FISH Signal Discrimination" (lines 217-218). However, despite their claim that Stellaris and Oligostan-HT produce more off-target probes when evaluated with the TrueProbes framework, the experiment results are nearly identical. The authors should consider modifying their claims or performing new experiments that more clearly demonstrate their claims.

In Figure 3, we aim to convey two main points. 

The first point is to compare the number of ON-target probes designed by each software using their most stringent design criteria (Figure 3A). Currently, we are using a medium strict design criterion for Stellaris (level 3). As shown in the new supplementary figure XX, when we apply the most stringent design criteria for Stellaris (level 5), the number of ON-target probes decreases to XX probes. This clearly indicates that, based on theoretical calculations, TrueProbes can design more probes than any of its competitors.

The second point is to compare the number of OFF-targets produced by each probe design. To illustrate this, we used two different metrics. In Figures 3B-D, we compare the total number of probes bound to OFF-target RNA. However, since each software generates a different number of ON-target probes, the number of OFF-targets may vary simply due to the differences in ON-target probe counts. Therefore, we introduced a second metric to compare OFF-targets. In Figures 3E-G, we present the number of OFF-targets normalized by the number of ON-targets. Using this metric, TrueProbes shows the lowest number of OFF-targets. We will updat the manuscript to clarify this point.

Regarding the experiments and their comparison to theoretical calculations: The theoretical calculations consider only the reference DNA and RNA genomes along with the oligonucleotide sequences for the probes. We then use a thermodynamic model to identify ON- and OFF-targets. Thus, these theoretical calculations represent an upper bound on the maximum possible number of ON-targets and the minimum number of OFF-targets. All other design software evaluated in this manuscript relies on the same or less reference data and makes certain assumptions. None of these methods quantitatively compare their computational designs with experimental results; they simply design probes based on unverified assumptions, conduct experiments, and present spot data to conclude that their probe designs are effective.

We will update the manuscript to clarify the goals of the theoretical model and its relationship to the experiments. Future work will be necessary to enhance our theoretical model to fully account for additional aspects of RNA-FISH experiments (e.g., formaldehyde crosslinking, hybridization conditions, washing steps) to better predict the experimental data shown in Figure 4. We will also adjuste our claims to accurately reflect the current capabilities of our theoretical framework and its relation to experimental outcomes.

(3) The bar graphs in Figure 3 do not seem to agree with the probability graphs in Figure 4. For example, Figure 3 indicates that Stellaris probes have higher off-target binding than TrueProbes; however, in Figure 4, their probability graphs lie almost on top of each other.

The predictions in Figure 3 regarding the number of probe off-target binding events, based on reference gene expression data, do not necessarily encompass all the information required to predict RNA-FISH signal intensity. Therefore, these predictions should not be expected to translate directly into the experimental results shown in Figure 4, particularly concerning the background signal.

While our software aims to minimize off-target probe binding, this does not automatically lead to a reduction in off-target background signal. Numerous other factors influence the spot background and overall signal-to-noise ratio (SNR) performance, beyond just probe-target binding interactions. Although we strive to minimize off-target background through probe binding, this approach is not designed to directly predict the SNR. Extending the computational analysis of probe binding dynamics to RNA-FISH signal intensity dynamics is beyond the scope of this study.

We have revised our text to clearly separate computational results from experimental results into two distinct sections. We will use different terminology to describe the outcomes of computational performance versus experimental performance, reducing potential confusion between these two aspects. Additionally, we will clarify our conceptual overview in Figure 1 regarding traditional probe design limitations related to sensitivity and specificity. We will specify how the signal from the number of probes bound to ON-target RNA, relative to those bound to OFF-targets and cellular autofluorescence, translates—either linearly or non-linearly—into the signal-to-noise ratio.

(4) The authors performed validation for only one gene (ARF4), because "...it had the highest gene expression (in TPM units) and the fewest isoforms among all candidate genes for the Jurkat cell line" (lines 176-177). While the results do look good, this is a minimal use case and does not really showcase the power of their method. One experiment that could be helpful would be two-color (or more) smFISH in tissue, where the chances for off-target binding contributing to higher errors are much greater than in an adherent cell line.

Thank you for highlighting these valuable experiments. Currently, our lab lacks the expertise to generate tissue samples beyond culturing cells. Additionally, implementing a two-color probe design in tissues containing different cell types with unknown expression levels presents further challenges. Due to these limitations, designing and conducting two-color experiments in tissue samples is beyond the scope of the current manuscript, but we plan to pursue this in the future.

(5) A common strategy for both smFISH and highly multiplexed methods is to use secondary DNA oligos with dye molecules instead of direct conjugation. Given that this is a primary design goal of PaintSHOP and the Zhuang lab's MERFISH probe design code, it would be helpful to demonstrate that TrueProbes can design a two-layer probe strategy for high-quality RNA-FISH labeling.

Thank you for bringing this to our attention. TrueProbes is currently designed and tested specifically for primary smRNA-FISH probes. Our focus is on demonstrating a new approach to designing these probes without the added complexities of secondary probes and multiplexing. Future work will expand on this foundation to incorporate secondary probe detection and transcript multiplexing.

(6) The authors claim, "For every probe set, TrueProbes can simulate expected smRNA FISH outcomes including optimal probe, RNA, and salt concentrations and optionally account for probe secondary structure, hybridization temperature, multiple targets, fluorophore choice, DNA, nascent RNA, and photon count statistics (Figures S2A, S2B). The model can be used to generate predictions for temperature and cell line sensitivity, multi-target discrimination, multiple fluorophore colocalization; when provided transcript expression levels and probe/background intensity, it can start to generate predictions for spot intensity, background, signal to noise ratio, and false negative rates (Figure S2C)." (lines 156-163). Figure S2 is a flow chart and does not provide evidence for any of these items. The authors should provide evidence for these claims, either as a figure or an example script in their software repository. If that is not possible, then it should be removed.

The supplemental information of the article will be updated to include figures that illustrate predictions for each capability currently offered by TrueProbes, along with the scripts used to generate these predictions. Any capabilities that do not have corresponding scripts will be removed from this section and instead referred to as potential improvements or future additions to the TrueProbes framework in the discussion section.

(7) All thermodynamic equations are performed at steady state. The authors do not justify this assumption, and there is no discussion of the potential impacts of either low molecule numbers or violations of the well-mixed assumption. Can the authors please include a discussion on the potential impacts non non-steady state dynamics?

Thermodynamic equations are calculated at steady state because RNA-FISH hybridization reactions typically last from eight to twenty hours. This duration allows probes adequate time to localize to their targets and reach binding equilibrium, based on current estimates of DNA oligonucleotide association and dissociation rate constants. We will address the potential violation of the well-mixed assumption in the assumptions and limitations section, specifically discussing how RNA localization can affect the spatial distribution of both on-target and off-target probes within cells, which may disrupt the well-mixed condition.

Low molecule numbers are not a significant concern, as probe DNA oligonucleotide concentrations in RNA-FISH protocols are much higher than the number of transcripts present in cells, by several orders of magnitude.

The assumptions and limitations section will be revised to clearly state: “Probe hybridization reactions were computed at steady state because most RNA-FISH protocols utilize probe hybridization incubation steps lasting over eight hours, which should provide sufficient time to reach equilibrium based on current estimates of forward and reverse reaction rate constants. Predictions from the equilibrium model may be less accurate for RNA-FISH experiments with shorter hybridization times, where non-steady state dynamics can result in different transient outcomes depending on the duration of hybridization.”

Reviewer #3 (Public review):

Summary:

This manuscript introduces a new platform termed "TrueProbes" for designing mRNA FISH probes. In comparison to existing design strategies, the authors incorporate a comprehensive thermodynamic and kinetic model to account for probe states that may contribute to nonspecific background. The authors validate their design pipeline using Jurkat cells and provide evidence of improved probe performance.

Strengths:

A notable strength of TrueProbes is the consideration of genome-wide binding affinities, which aims to minimize off-target signals. The work will be of interest to researchers employing mRNA FISH in certain human cell lines.

Weaknesses:

However, in my view, the experimental validation is not sufficient to justify the broad claims of the platform. Given the number of assumptions in the model, additional experimental comparisons across probe design methods, ideally targeting transcripts with different expression levels, would be necessary to establish the general superiority of this approach.

We will revise our text to make our claims more specific and clearer, avoiding overgeneralizations and ensuring that all claims are adequately supported by the data we present.

Author response:

Reviewer #1 (Public review):

Summary:

The manuscript reports a series of experiments designed to test whether optogenetic activation of infralimbic (IL) neurons facilitates extinction retrieval and whether this depends on animals' prior experience. In Experiment 1, rats underwent fear conditioning followed by either one or two extinction sessions, with IL stimulation given during the second extinction; stimulation facilitated extinction retrieval only in rats with prior extinction experience. Experiments 2 and 3 examined whether backward conditioning (CS presented after the US) could establish inhibitory properties that allowed IL stimulation to enhance extinction, and whether this effect was specific to the same stimulus or generalized to different stimuli. Experiments 5 - 7 extended this approach to appetitive learning: rats received backward or forward appetitive conditioning followed by extinction, and then fear conditioning, to determine whether IL stimulation could enhance extinction in contexts beyond aversive learning and across conditioning sequences. Across studies, the key claim is that IL activation facilitates extinction retrieval only when animals possess a prior inhibitory memory, and that this effect generalizes across aversive and appetitive paradigms.

Strengths:

(1) The design attempts to dissect the role of IL activity as a function of prior learning, which is conceptually valuable.

We thank the Reviewer for their positive assessment.

(2) The experimental design of probing different inhibitory learning approaches to probe how IL activation facilitates extinction learning was creative and innovative.

We thank the Reviewer for their positive assessment.

Weaknesses:

(1) Non-specific manipulation.

ChR2 was expressed in IL without distinction between glutamatergic and GABAergic populations. Without knowing the relative contribution of these cell types or the percentage of neurons affected, the circuit-level interpretation of the results is unclear.

ChR2 was intentionally expressed in the infralimbic cortex (IL) without distinction between local neuronal populations for two reasons. First, this manuscript aimed to uncover some of the features characterizing the encoding of inhibitory memories in the IL, and this encoding likely engages interactions among various neuronal populations within the IL. Second, the hypotheses tested in the manuscript derived from findings that indiscriminately stimulated the IL using the GABA_A receptor antagonist picrotoxin, which is best mimicked by the approach taken. We agree that it is also important to determine the respective contributions of distinct IL neuronal populations to inhibitory encoding; however, the global approach implemented in the present experiments represents a necessary initial step. This rationale will be incorporated into the revised manuscript, which will also make reference to the need to identify the relative contributions of the various neuronal populations within the IL. 

(2) Extinction retrieval test conflates processes

The retrieval test included 8 tones. Averaging across this many tone presentations conflate extinction retrieval/expression (early tones) with further extinction learning (later tones). A more appropriate analysis would focus on the first 2-4 tones to capture retrieval only. As currently presented, the data do not isolate extinction retrieval.

It is unclear when retrieval of what has been learned across extinction ceases and additional extinction learning occurs. In fact, it is only the first stimulus presentation that unequivocally permits a distinction between retrieval and additional extinction learning, as the conditions for this additional learning have not been fulfilled at that presentation. However, confining evidence for retrieval to the first stimulus presentation introduces concerns that other factors could influence performance. For instance, processing of the stimulus present at the start of the session may differ from that present at the end of the previous session, thereby affecting what is retrieved. Such differences between the stimuli present at the start and end of an extinction session have been long recognized as a potential explanation for spontaneous recovery (Estes, 1955). More importantly, whether the test data presented confound retrieval and additional extinction learning or not, the interpretation remains the same with respect to the effects of a prior history of inhibitory learning on enabling the facilitative effects of IL stimulation. Finally, it is unclear how these facilitative effects could occur in the absence of the subjects retrieving the extinction memory formed under the stimulation. Nevertheless, the revised manuscript will provide the trial-by-trial performance during the post-extinction retrieval tests and discuss this issue.

(3) Under-sampling and poor group matching.

Sample sizes appear small, which may explain why groups are not well matched in several figures (e.g., 2b, 3b, 6b, 6c) and why there are several instances of unexpected interactions (protocol, virus, and period). This baseline mismatch raises concerns about the reliability of group differences.

Efforts were made to match group performance upon completion of each training stage and before IL stimulation. Unfortunately, these efforts were not completely successful due to exclusions following post-mortem analyses. However, we acknowledge that the unexpected interactions deserve further discussion, and this will be incorporated into the revised manuscript (see also comment from Reviewer 2). Although we cannot exclude that sample sizes may have contributed to some of these interactions, we remain confident about the reliability of the main findings reported, especially given their replication across the various protocols. Overall, the manuscript provides evidence that IL stimulation does not facilitate brief extinction in the absence of prior inhibitory experience in five different experiments, replicating previous findings (Lingawi et al., 2018; Lingawi et al., 2017). It also replicates these previous findings by showing that prior experience with either fear or appetitive extinction enables IL stimulation to facilitate subsequent fear extinction. Furthermore, the facilitative effects of such stimulation following fear or appetitive backward conditioning are replicated in the present manuscript.  

(4) Incomplete presentation of conditioning data.

Figure 3 only shows a single conditioning session despite five days of training. Without the full dataset, it is difficult to evaluate learning dynamics or whether groups were equivalent before testing.

We apologize, as we incorrectly labeled the X axis for the backward conditioning data set in Figures 3B, 4B, 4D and 5B. It should have indicated “Days” instead of “Trials”. This error will be corrected in the revised manuscript.

(5) Interpretation stronger than evidence.

The authors conclude that IL activation facilitates extinction retrieval only when an inhibitory memory has been formed. However, given the caveats above, the data are insufficient to support such a strong mechanistic claim. The results could reflect non-specific facilitation or disruption of behavior by broad prefrontal activation. Moreover, there is compelling evidence that optogenetic activation of IL during fear extinction does facilitate subsequent extinction retrieval without prior extinction training (Do-Monte et al 2015, Chen et al 2021), which the authors do not directly test in this study.

As noted above, the revised manuscript will show that the interpretations of the main findings stand whether ore the test data confounds retrieval with additional extinction learning. The revised manuscript will also clarify the plotting of the data for the backward conditioning stages. We do agree that further discussion of the unexpected interactions is necessary, and this will also be incorporated into the revised manuscript. However, the various replications of the core findings provide strong evidence for their reliability and the interpretations advanced in the original manuscript. The proposal that the results reflect non-specific facilitation or disruption of behavior seems highly unlikely. Indeed, the present experiments and previous findings (Lingawi et al., 2018; Lingawi et al., 2017) provide multiple demonstrations that IL stimulation fails to produce any facilitation in the absence of prior inhibitory experience with the target stimulus. Although these demonstrations appear inconsistent with previous studies (Do-Monte et al., 2015; Chen et al., 2021), this inconsistency is likely explained by the fact that these studies manipulated activity in specific IL neuronal populations. Previous work has already revealed differences between manipulations targeting discrete IL neuronal populations as opposed to general IL activity (Kim et al., 2016). Importantly, as previously noted, the present manuscript aimed to generally explore inhibitory encoding in the IL that, as we will acknowledge, is likely to engage several neuronal populations within the IL. Adequate statements on these matters will be included in the revised manuscript.

Impact:

The role of IL in extinction retrieval remains a central question in the fear learning literature. However, because the test used conflates extinction retrieval with new learning and the manipulations lack cell-type specificity, the evidence presented here does not convincingly support the main claims. The study highlights the need for more precise manipulations and more rigorous behavioral testing to resolve this issue.

As noted in our responses, the interpretations of the data presented remain identical whether the test data conflate extinction retrieval with additional extinction learning or not. Although we agree that it is important to establish the role of specific IL neuronal populations in extinction learning, this was beyond the scope of the manuscript and the findings reported remain valuable to our understanding of inhibitory encoding within the IL.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors examine the mechanisms by which stimulation of the infralimbic cortex (IL) facilitates the retention and retrieval of inhibitory memories. Previous work has shown that optogenetic stimulation of the IL suppresses freezing during extinction but does not improve extinction recall when extinction memory is probed one day later. When stimulation occurs during a second extinction session (following a prior stimulation-free extinction session), freezing is suppressed during the second extinction as well as during the tone test the following day. The current study was designed to further explore the facilitatory role of the IL in inhibitory learning and memory recall. The authors conducted a series of experiments to determine whether recruitment of IL extends to other forms of inhibitory learning (e.g., backward conditioning) and to inhibitory learning involving appetitive conditioning. Further, they assessed whether their effects could be explained by stimulus familiarity. The results of their experiments show that backward conditioning, another form of inhibitory learning, also enabled IL stimulation to enhance fear extinction. This phenomenon was not specific to aversive learning, as backward appetitive conditioning similarly allowed IL stimulation to facilitate extinction of aversive memories. Finally, the authors ruled out the possibility that IL facilitated extinction merely because of prior experience with the stimulus (e.g., reducing the novelty of the stimulus). These findings significantly advance our understanding of the contribution of IL to inhibitory learning. Namely, they show that the IL is recruited during various forms of inhibitory learning, and its involvement is independent of the motivational value associated with the unconditioned stimulus.

Strengths:

(1) Transparency about the inclusion of both sexes and the representation of data from both sexes in figures.

We thank the Reviewer for their positive assessment.

(2) Very clear representation of groups and experimental design for each figure.

We thank the Reviewer for their positive assessment.

(3) The authors were very rigorous in determining the neurobehavioral basis for the effects of IL stimulation on extinction. They considered multiple interpretations and designed experiments to address these possible accounts of their data.

We thank the Reviewer for their positive assessment.

(4) The rationale for and the design of the experiments in this manuscript are clearly based on a wealth of knowledge about learning theory. The authors leveraged this expertise to narrow down how the IL encodes and retrieves inhibitory memories.

We thank the Reviewer for their positive assessment.

Weaknesses:

(1) In Experiment 1, although not statistically significant, it does appear as though the stimulation groups (OFF and ON) differ during Extinction 1. It seems like this may be due to a difference between these groups after the first forward conditioning. Could the authors have prevented this potential group difference in Extinction 1 by re-balancing group assignment after the first forward conditioning session to minimize the differences in fear acquisition (the authors do report a marginally significant effect between the groups that would undergo one vs. two extinction sessions in their freezing during the first conditioning session)?

As noted (see response to Reviewer 1), efforts were made daily to match group performance across the training stages, but these efforts were ultimately hampered by the necessary exclusions following post-mortem analyses. This will be made explicit in the revised manuscript. Regarding freezing during Extinction 1, as noted by the Reviewer, the difference, which was not statistically significant, was absent across trials during the subsequent forward fear conditioning stage. Likewise, the protocol difference observed during the initial forward fear conditioning was absent in subsequent stages. We are therefore confident that these initial differences (significant or not) did not impact the main findings at test. Importantly, these findings replicate previous work using identical protocols in which no differences were present during the training stages. These considerations will be addressed in the revised manuscript.

(2) Across all experiments (except for Experiment 1), the authors state that freezing during the initial conditioning increased across "days". The figures that correspond to this text, however, show that freezing changes across trials. In the methods, the authors report that backward conditioning occurred over 5 days. It would be helpful to understand how these data were analyzed and collated to create the final figures. Was the freezing averaged across the five days for each trial for analyses and figures?

We apologize, as noted above, we incorrectly labeled the X axis for the backward conditioning data sets in Figures 3B, 4B, 4D and 5B. It should have indicated “Days” instead of “Trials”. The data shown in these Figures use the average of all trials on a given day. This will be clarified in the methods section of the revised manuscript. The labeling errors on the Figures will be corrected.

(3) In Experiment 3, the authors report a significant Protocol X Virus interaction. It would be useful if the authors could conduct post-hoc analyses to determine the source of this interaction. Inspection of Figure 4B suggests that freezing during the two different variants of backward conditioning differs between the virus groups. Did the authors expect to see a difference in backward conditioning depending on the stimulus used in the conditioning procedure (light vs. tone)? The authors don't really address this confounding interaction, but I do think a discussion is warranted.

We agree with the Reviewer that further discussion of the Protocol x Virus interaction that emerged during the backward conditioning and forward conditioning stages of Experiment 3 is warranted. This will be provided in the revised manuscript. Briefly, during both stages, follow-up analyses did not reveal any differences (main effects or interactions) between the two groups trained with the light stimulus (Diff-EYFP and Diff-ChR2). By contrast, the ChR2 group trained with the tone (Back-ChR2) froze more overall than the EYFP group (Back-EYFP), but there were no other significant differences between the two groups. Based on these analyses, the Protocol x Virus interaction appears to be driven by greater freezing in the ChR2 group trained with the tone rather than a difference in the backward conditioning performance based on stimulus identity. Consistent with this, the statistical analyses did not reveal a main effect of Protocol during either the backward conditioning stage or the stimulus trials during the forward conditioning stage. Nevertheless, during this latter stage, a main effect of Protocol emerged during baseline performance, but once again, this seems to be driven by the Back-ChR2 group. Critically, it is unclear how greater stimulus freezing in the Back-ChR2 group during forward conditioning would lead to lower freezing during the post-extinction retrieval test.  

(4) In this same experiment, the authors state that freezing decreased during extinction; however, freezing in the Diff-EYFP group at the start of extinction (first bin of trials) doesn't look appreciably different than their freezing at the end of the session. Did this group actually extinguish their fear? Freezing on the tone test day also does not look too different from freezing during the last block of extinction trials.

We confirm that overall, there was a significant decline in freezing across the extinction session shown in Figure 4B. The Reviewer is correct to point out that this decline was modest (if not negligible) in the Diff-EYFP group, which was receiving its first inhibitory training with the target tone stimulus. It is worth noting that across all experiments, most groups that did not receive infralimbic stimulation displayed a modest decline in freezing during the extinction session since it was relatively brief, involving only 6 or 8 tone alone presentations. This was intentional, as we aimed for the brief extinction session to generate minimal inhibitory learning and thereby to detect any facilitatory effect of infralimbic stimulation. This issue will be clarified and explained in the revised version of the manuscript.

(5) The Discussion explored the outcomes of the experiments in detail, but it would be useful for the authors to discuss the implications of their findings for our understanding of circuits in which the IL is embedded that are involved in inhibitory learning and memory. It would also be useful for the authors to acknowledge in the Discussion that although they did not have the statistical power to detect sex differences, future work is needed to explore whether IL functions similarly in both sexes.

In line with the Reviewer’s suggestion (see also Reviewer 3), the revised manuscript will include a discussion of the broader implications of the findings regarding inhibitory brain circuitry and will acknowledge the need to further explore sex differences and IL functions.

Reviewer #3 (Public review):

Summary:

This is a really nice manuscript with different lines of evidence to show that the IL encodes inhibitory memories that can then be manipulated by optogenetic stimulation of these neurons during extinction. The behavioral designs are excellent, with converging evidence using extinction/re-extinction, backwards/forwards aversive conditioning, and backwards appetitive/forwards aversive conditioning. Additional factors, such as nonassociative effects of the CS or US, are also considered, and the authors evaluate the inhibitory properties of the CS with tests of conditioned inhibition.

Strengths:

The experimental designs are very rigorous with an unusual level of behavioral sophistication.

We thank the Reviewer for their positive assessment.

Weaknesses:

(1) More justification for parametric choices (number of days of backwards vs forwards conditioning) could be provided.

All experimental parameters were based on previously published experiments showing the capacity of the backward conditioning protocols to generate inhibitory learning and the forward conditioning protocols to produce excitatory learning. Although this was mentioned in the methods section, we acknowledge that further explanation is required to justify the need for multiple days of backward training. This will be provided in the revised manuscript.

(2) The current discussion could be condensed and could focus on broader implications for the literature.

The revised manuscript will make an effort to condense the discussion and focus on broader implications for the literature.

References

Chen, Y.-H., Wu, J.-L., Hu, N.-Y., Zhuang, J.-P., Li, W.-P., Zhang, S.-R., Li, X.-W., Yang, J.-M., & Gao, T.-M. (2021). Distinct projections from the infralimbic cortex exert opposing effects in modulating anxiety and fear. J Clin Invest, 131(14), e145692. https://doi.org/10.1172/JCI145692

Do-Monte, F. H., Manzano-Nieves, G., Quiñones-Laracuente, K., Ramos-Medina, L., & Quirk, G. J. (2015). Revisiting the role of infralimbic cortex in fear extinction with optogenetics. J Neurosci, 35(8), 3607-3615. https://doi.org/10.1523/JNEUROSCI.3137-14.2015

Estes, W. K. (1955). Statistical theory of spontaneous recovery and regression. Psychol Rev, 62(3), 145-154. https://doi.org/10.1037/h0048509

Kim, H.-S., Cho, H.-Y., Augustine, G. J., & Han, J.-H. (2016). Selective Control of Fear Expression by Optogenetic Manipulation of Infralimbic Cortex after Extinction. Neuropsychopharmacology, 41(5), 1261-1273. https://doi.org/10.1038/npp.2015.276

Lingawi, N. W., Holmes, N. M., Westbrook, R. F., & Laurent, V. (2018). The infralimbic cortex encodes inhibition irrespective of motivational significance. Neurobiol Learn Mem, 150, 64-74. https://doi.org/10.1016/j.nlm.2018.03.001

Lingawi, N. W., Westbrook, R. F., & Laurent, V. (2017). Extinction and Latent Inhibition Involve a Similar Form of Inhibitory Learning that is Stored in and Retrieved from the Infralimbic Cortex. Cereb Cortex, 27(12), 5547-5556. https://doi.org/10.1093/cercor/bhw322

Reviewer #2 (Public review):

Summary:

Okuno et al. investigate the structure-function relationship in the fruit fly Drosophila melanogaster. To do so, they combine published data from two recent synapse-level connectomes ("hemibrain" and "FlyWire") with a dataset comprising functional whole-brain calcium imaging and behavioural data. First, they investigate the applicability of fMRI pre-processing techniques on data from calcium imaging. They then cross-correlate this pre-processed functional data with structural data extracted from the connectomes, including a comparison to humans. The authors proceed to compare the two connectomes and find significant differences, which they attribute to differences in the accuracy of the synapse detections. Next, they present a novel algorithm to quantify whether neurons are segregated (pre- and postsynapses are spatially separate) or unsegregated (pre- and postsynapses are mixed). Using this approach, they find that unsegregated neurons may contribute more to function than segregated neurons. Applying a general linear model to the functional dataset suggests that activity in two brain areas (Wedge and AVLP) is suppressed during walking. The authors identify a GABAergic neuron in the connectome that could be responsible for this effect and suggest it may provide feedback to the fly's "compass" in the central complex.

Strengths:

The study tackles a relevant question in connectomics by exploring the relationship between structural and functional connectivity in the Drosophila brain. The authors apply a range of established and adapted analytical methods, including fMRI-style preprocessing and a novel synaptic segregation index. The effort to integrate multiple datasets and to compare across species reflects a broad and methodical approach.

Weaknesses:

The manuscript would benefit from a clearer overarching narrative to unify the various analyses, which currently appear somewhat disjointed. While the technical methods are extensive, the writing is often convoluted and lacks crucial details, making it difficult to follow the logic and interpret key findings. Additionally, the conclusions are relatively incremental and lack a compelling conceptual advance, limiting the overall impact of the work.

(1) The introduction currently contains a number of findings and conclusions that would be better placed in the results and discussion to clearly delineate past findings from new results and speculations.

(2) The narrative would benefit greatly from some clear statements along the lines of "we wanted to find out X, therefore we did Y".

(3) More concise terminology would be helpful. For example, the connectomes are currently referred to as either "hemibrain", "FlyEM", "whole-brain", or "FlyWire".

(4) The abstract claims "a new, more robust method to quantify the degree of pre- and post-synaptic segregation". However, the study fails to provide evidence that this method is indeed more robust than existing methods.

(5) The authors define unsegregated neurons as having mixed pre- and postsynapses in the same space. However, this ignores the neurons' topology: a neuron can exhibit a clearly defined dendrite with (mostly) postsynapses and a clearly defined axon with (mostly) presynapses, which then occupy the same space. This is different from genuinely unsegregated neurons with no distinct dendritic and axonal compartments, such as CT1.

(6) It is not entirely clear where the marmoset dataset originates from. Was it generated for this study? If not, why is there a note in the Ethics Declaration?

(7) On the differences between hemibrain and FlyWire: What is the "18.8 million post-synapses" for FlyWire referring to? The (thresholded) FlyWire synapse table has 130M connections (=postsynapses). Subsetting that synapse cloud to the hemibrain volume still gives ~47M synapses. Further subsetting to only connections between proofread neurons inside the hemibrain volume gives 19.4M - perhaps the authors did something like that? Similarly, the hemibrain synapse table contains 64M postsynapses. Do the 21M "FlyEM" post-synapses refer to proofread neurons only? If the authors indeed used only (post-)synapses from proofread neurons, they need to make that explicit in results and methods, and account for differences in reconstruction status when making any comparisons. For example, the mushroom body in the hemibrain got a lot more attention than in FlyWire, which would explain the differences reported here. For that reason, connection weights are often expressed as, e.g., a fraction of the target's inputs instead of the total number of synapses when comparing connectivity across connectomic datasets. Furthermore, in Figure 3b, it looks like the FlyWire synapse cloud was not trimmed to the exact hemibrain boundaries: for example, the trimmed FlyWire synapse cloud seems to extend further into the optic lobes than the hemibrain volume does.

por eso la doxi y la mino estan bien vergitas

lla diferencia con las viejas

datito pendejo de resistencias

Élections au Conseil de la Vie Lycéenne 2025 : Analyse des Programmes

Résumé

Le présent document synthétise les professions de foi des candidats aux élections du Conseil de la Vie Lycéenne (CVL) du lycée Louis Vincent pour le mandat 2025.

Les élections se dérouleront du 30 septembre au 2 octobre via la plateforme Pronote, où cinq binômes seront élus pour un mandat de deux ans.

L'objectif commun déclaré par l'ensemble des candidats est d'améliorer les conditions et la qualité de vie au sein de l'établissement.

L'analyse des programmes révèle plusieurs thèmes transversaux et récurrents :

1. Convivialité et Événements : La quasi-totalité des candidats propose l'organisation de journées à thème (pyjama, sans sac, carnaval, maillot de foot), de ventes de nourriture et de boissons chaudes (notamment en hiver), et de grands événements fédérateurs comme un festival de fin d'année ouvert à tous les niveaux, des tournois sportifs interclasses ou un bal des terminales.

2. Solidarité et Actions Caritatives : Un fort accent est mis sur la solidarité, avec des propositions de collectes (alimentaires, produits d'hygiène, fournitures scolaires) et de levées de fonds dont les bénéfices seraient reversés à des associations caritatives (Restos du cœur, un cahier un crayon) ou à des lycéens dans le besoin de manière anonyme.

3. Culture et Pédagogie : De nombreux programmes ambitionnent de dynamiser la vie culturelle du lycée.

Les idées incluent une meilleure utilisation de la salle de cinéma pour des projections pédagogiques ou des séances gratuites, l'organisation de sorties culturelles, la création de clubs (lecture, débat), et la mise en place de systèmes de tutorat et de parrainage entre élèves.

4. Bien-être et Cadre de Vie : L'amélioration du bien-être des élèves est une priorité centrale.

Les propositions visent à créer des espaces de détente et de travail plus calmes et accueillants, à aménager des espaces verts (potager, jardin partagé), à gérer le stress via des ateliers, et à améliorer les infrastructures pratiques comme l'ajout de casiers.

5. Communication et Représentation : Plusieurs binômes souhaitent renforcer le lien entre le CVL et les élèves par la gestion active d'un compte Instagram, la mise en place d'une boîte à idées, la publication de comptes-rendus mensuels et une promesse générale d'écoute.

Un candidat, Baptiste, fonde même l'intégralité de sa démarche sur l'absence de programme prédéfini afin de représenter directement les demandes des élèves.

En somme, les candidatures de 2025 témoignent d'une volonté partagée de rendre le lycée Louis Vincent plus dynamique, solidaire, culturellement riche et attentif au bien-être de chaque élève.

Présentation des Candidats et de Leurs Plateformes

Le tableau suivant récapitule les différents binômes candidats, leur niveau de classe et les points saillants de leur programme.

Candidats (Titulaires et Suppléants)

Classe(s)

Qualités Revendiquées

Points Clés du Programme

VM & CR

Terminale 6 & Première 2

Créatives, motivées, sérieuses, expérience et énergie

Projet "H24" (camping au lycée), journées à thème (maillot de foot, sans sac, pyjama).

SP et OP

Seconde 12 & Seconde 13

Bienveillants, honnêtes, altruistes, intègres, ouverts au dialogue

Ventes de snacks/boissons chaudes, 2ème service à la cantine, création d'un lieu de travail/repos, tournois sportifs interclasses.

MV et KM

Non spécifié

-

4 axes : écologie (coin nature, potager), vie scolaire (journées à thème, menus spéciaux), culture (concours de talents, tutorat, sorties), bien-être (coin calme).

MO et LO

Première STL & Première 11

Engagée, sympathique, responsable, réfléchi, à l'écoute, sérieux, ambitieux

Récolte caritative, grande journée conviviale de fin d'année, vente de gourdes écoresponsables, communication améliorée (compte-rendu, boîte à idées).

AI et IP

Terminale 2 & Première 7

Inspirés par les anciens CVL

Vente de fournitures scolaires, vente de thé/chocolat chaud pour des associations, festival de fin d'année pour tous les niveaux.

AP et GL

Première 2 & Première 4

Expérience (délégué, ancien CVC)

Ventes de pâtisseries/boissons, gestion active du compte Instagram du CVL (sondages, menus), ajout de casiers pour tous, décorations du lycée.

RS et LH

Seconde 4 & Seconde 1

À l'écoute

Utilisation de la salle de cinéma à but pédagogique, organisation de goûters thématiques pour financer des associations ou des sorties, décorations festives.

HS et JPM

Terminale & Première 6

-

Optimisation des TV de l'accueil (horaires de bus, menus), dynamisation de la web TV, séances de cinéma gratuites, collecte de fournitures, vente de pulls du lycée, changement de la sonnerie via vote.

RB et NL

Seconde 4 & Seconde 6

-

Grand repas mensuel à la cantine, collecte de produits d'hygiène/vêtements pour les SDF, semaines à thème (cinéma), tutorat entre 15h et 18h.

B

Seconde

-

Absence de programme défini. Volonté de représenter directement les idées et besoins des 1600 élèves. Souhaite renouveler les initiatives type "Journées du Patrimoine".

SV et LL

Terminale 4 & Première 9

Souriantes, à l'écoute, motivées

Ateliers de gestion du stress, espaces de détente, tutorat Terminales/Secondes, création d'espaces verts (potager), sorties culturelles, amélioration des salles de travail.

Analyse Détaillée des Thèmes de Campagne

1. Convivialité, Événements et Vie Sociale

Ce thème est le plus largement partagé par les candidats, qui souhaitent briser la routine et renforcer les liens entre les élèves.

• Journées à Thème : Une proposition quasi unanime.

◦ Journée pyjama : Proposée par Victorine & Capucine, et Mabine & Victoria.   

◦ Journée sans sac : Proposée par Victorine & Capucine, Mabine & Victoria, Henry & Maxime, et Sacha & Lili.  

◦ Journée en maillot de foot : Proposée par Victorine & Capucine. 

◦ Journée carnaval : Proposée par Mabine & Victoria, et Sacha & Lili.  

◦ Journée de l'élégance : Proposée par Sacha & Lili.

• Événements de Fin d'Année :

◦ Festival pour tous les niveaux : Avazov & Isaac proposent un événement avec stands de nourriture, concours de talents et concerts pour inclure les Secondes et Premières, souvent exclus du bal des terminales.  

◦ Grande journée conviviale : Méina & Lilian suggèrent une journée avec jeux, sport et musique pour élèves, professeurs et personnel.  

◦ Bal des terminales et "Perçant" : Arnaud & Grégoire s'engagent à organiser ces événements traditionnels.

• Ventes de Nourriture et Boissons :

◦ Ventes de snacks et boissons chaudes (hiver) : Proposées par Sacha & Oscar, Avazov & Isaac.    ◦ Ventes de pâtisseries : Proposées par Arnaud & Grégoire.  

◦ Goûters thématiques (Noël, Halloween) : Proposés par Roman & Louison.

• Événements Sportifs et Sociaux :

◦ Tournois sportifs interclasses : Proposés par Sacha & Oscar.  

◦ Grand repas mensuel à la cantine : Suggéré par Romain & Noa pour favoriser les rencontres.

2. Solidarité, Écologie et Actions Caritatives

De nombreux candidats placent l'engagement solidaire et écologique au cœur de leur projet.

• Collectes et Dons :

◦ Récoltes caritatives générales : Proposées par Méina & Lilian, avec une redistribution anonyme possible aux lycéens dans le besoin.  

◦ Collectes pour des associations (SDF, etc.) : Romain & Noa proposent de récolter des produits d'hygiène, de la nourriture et des vêtements chauds. Mabine & Victoria souhaitent organiser des collectes solidaires régulières.  

◦ Collecte de fournitures scolaires : Henry & Maxime suggèrent que les terminales fassent don de leur matériel (ex: calculatrices) aux nouveaux élèves.

• Financement d'Associations :

◦ Plusieurs binômes (Avazov & Isaac, Roman & Louison) proposent que les bénéfices des ventes de nourriture soient reversés à des associations comme les Restos du cœur ou Un cahier, un crayon.

• Écologie et Cadre de Vie :

◦ Espaces verts : Mabine & Victoria et Sacha & Lili veulent créer un "coin nature", un jardin partagé ou un potager pédagogique.  

◦ Gourdes écoresponsables : Méina & Lilian proposent de créer et vendre une gourde en matériaux recyclés.

3. Culture, Éducation et Entraide

L'accès à la culture et le soutien scolaire sont des axes de développement majeurs.

• Utilisation de la Salle de Cinéma : Un projet récurrent.

◦ Usage pédagogique : Roman & Louison souhaitent encourager les professeurs à utiliser la salle pour des projections en lien avec les cours.  

◦ Accès gratuit à la culture : Henry & Maxime et Mabine & Victoria veulent organiser des séances de cinéma gratuites et régulières pour tous.

• Tutorat et Parrainage :

◦ Aide aux devoirs : Romain & Noa suggèrent un système de tutorat entre 15h et 18h pour les élèves en difficulté.   

◦ Parrainage Secondes/Terminales : Sacha & Lili proposent un parrainage pour faciliter l'intégration des Secondes, une idée également mentionnée par Mabine & Victoria.

• Enrichissement Culturel :

◦ Sorties culturelles et sportives : Mabine & Victoria et Sacha & Lili souhaitent en organiser davantage.  

◦ Rencontres avec d'anciens élèves : Mabine & Victoria proposent d'inviter des anciens pour partager leur parcours.   

◦ Concours de talents : Mabine & Victoria veulent lancer un concours annuel (photo, écriture, musique, sport).  

◦ Clubs : L'idée de clubs (lecture, débat) est avancée par Mabine & Victoria.

4. Améliorations Pratiques et Bien-être

Les candidats sont attentifs aux aspects concrets de la vie lycéenne et à la santé mentale des élèves.

• Espaces de Vie et de Travail :

◦ Création d'un lieu de repos/travail : Sacha & Oscar constatent que la Maison des Lycéens est souvent encombrée et bruyante et proposent un nouvel espace.  

◦ Aménagement d'un coin calme et détente : Une priorité pour Mabine & Victoria et Sacha & Lili, qui souhaitent aussi mettre en place des ateliers de gestion du stress.

• Infrastructures et Services :

◦ Ajout de casiers : Arnaud & Grégoire insistent sur le manque de casiers, notamment pour les demi-pensionnaires aux journées longues.  

◦ Deuxième service à la cantine : Proposé par Sacha & Oscar.    ◦ Vente de fournitures scolaires : Avazov & Isaac suggèrent une vente au foyer pour les oublis avant une évaluation.

• Ambiance Sonore et Visuelle :

◦ Changement de la sonnerie : Henry & Maxime proposent un vote via Instagram pour changer la sonnerie plusieurs fois par an.  

◦ Décorations du lycée : Arnaud & Grégoire et Roman & Louison veulent décorer l'établissement pour les fêtes (Noël, Halloween).

5. Communication et Démocratie Lycéenne

Améliorer la transmission de l'information et l'écoute des élèves est un enjeu clé.

• Outils de Communication Numérique :

◦ Gestion du compte Instagram du CVL : Arnaud & Grégoire veulent le rendre plus actif avec des sondages et des informations pratiques (menus de la cantine).   

◦ Utilisation des télévisions de l'accueil : Henry & Maxime veulent y diffuser des informations utiles (horaires de bus, actualités du CVL).

• Mécanismes de Participation :

◦ Boîte à idées : Proposée par Méina & Lilian pour que chaque élève puisse soumettre des propositions.  

◦ Comptes-rendus mensuels : Également une idée de Méina & Lilian pour plus de transparence sur les actions du CVL.

• Philosophie de Représentation :

◦ La quasi-totalité des candidats se disent "à l'écoute".  

◦ Baptiste se distingue par une approche radicale, refusant un programme pour se faire le porte-parole direct et exclusif des demandes formulées par les élèves.

• quantra y añadir link

The author points out that cultural capital encompasses linguistic style, demeanor, behavioral habits, and appearance—characteristics seemingly unrelated to academic performance that nonetheless influence students' school achievements and recognition. For instance, students from upper-middle-class families are more likely to master expressions and behavioral norms aligned with mainstream school culture, making them more easily perceived by teachers as “polite,” “intelligent,” or “promising.” Conversely, students from lower socioeconomic backgrounds may be underestimated despite equal effort, due to differences in their manner of expression or conduct. This demonstrates how the education system subtly favors specific social groups, making cultural capital a key mechanism for reproducing social inequality.

This passage combines the theory of capital with Chantelle's actual situation, allowing me to have a clearer understanding of how structural inequality operates in schools.

I think that success in school isn’t just about talent, it’s shaped by the resources students have. For example, a wealthy student might afford SAT prep or private tutoring, while another gains help through family connections. Even knowing how to speak or behave in ways teachers value gives some students an advantage.

* Soar = elevar. * Amount = cantidad. * Strengthened = fortificadas , fortalecidas. * coverage = cobertura, reportaje. * Medios = medios, noticias * So on = y Asi sucesivamente, y además.

The image of a smiling woman from Antilles, standing between two bushels of bananas, was the first image used in 1912 to advertise Banania, a banana-flavored chocolate drink most widely distributed in France. Three years later, she was replaced with a smiling African man holding a spoon of the drink like a child and accompanied with the slogan, “Y’a bon” — a phrase of pidgin French that translates to, “It’s good.” https://hyperallergic.com/380053/the-racist-caricatures-of-african-soldiers-that-soothed-french-colonial-anxieties/

contemporarous equation math

Note de Synthèse : Rapport sur la Santé Mentale des Mineurs

Le rapport: https://hyp.is/go?url=https%3A%2F%2Fwww.assemblee-nationale.fr%2Fdyn%2F17%2Frapports%2Fdde%2Fl17b1700_rapport-information.pdf&group=world

Résumé

Ce document de synthèse présente les conclusions et recommandations clés du rapport parlementaire sur la santé mentale des mineurs.

Le constat principal est une dégradation alarmante de la santé psychique des enfants et adolescents en France, avec 1,6 million de mineurs souffrant d'un trouble psychique, et une augmentation particulièrement marquée de +70% chez les 10-14 ans entre 2017 et 2023.

Le système de soins est totalement débordé, avec seulement un jeune sur deux ayant accès à un suivi et des délais d'attente atteignant 18 mois pour un premier rendez-vous en Centre Médico-Psychologique (CMP).

Le rapport identifie deux crises interdépendantes : d'une part, une offre de soins illisible, sous-financée et en manque criant de personnel, et d'autre part, une prévention quasi inexistante, notamment dans le cadre scolaire.

Une surreprésentation dramatique des enfants de l'Aide Sociale à l'Enfance (ASE) au sein des services de pédopsychiatrie (jusqu'à 40% des lits pour 2% de la population mineure) met en lumière la faillite du suivi de ces publics particulièrement vulnérables.

Les 54 recommandations formulées visent à une refonte structurelle du système. Les priorités incluent :

1. La réorganisation du parcours de soins via une "gradation" claire, renforçant le premier recours (médecins généralistes, pédiatres) pour désengorger les CMP.

2. Le renforcement des CMP comme pivots du système, avec des budgets pérennes, un maillage territorial assurant un accès en 30 minutes, et une approche pluridisciplinaire.

3. L'investissement massif dans la prévention, de la périnatalité (unités parents-bébés) à la santé scolaire (atteindre les ratios européens de médecins et psychologues).

4. Une meilleure prise en charge des enfants de l'ASE avec un repérage systématique, un suivi de santé effectif et la création de structures mixtes soin-hébergement.

5. La lutte contre la pénurie de professionnels par la revalorisation des salaires, la réforme de la tarification des actes et des mesures pour améliorer l'attractivité de la pédopsychiatrie.

Enfin, le rapport souligne l'impact majeur de facteurs environnementaux comme la pression scolaire (Parcoursup), l'addiction aux écrans et les inégalités sociales, appelant à des réponses politiques plus larges.

1. Le Constat Alarmant d'une Crise de Santé Publique

Le rapport parlementaire, fruit de six mois de travail incluant 37 auditions et quatre déplacements, dresse un tableau sans équivoque de la détérioration de la santé mentale des mineurs en France.

A. Chiffres Clés de la Dégradation

• Prévalence des troubles : Selon le rapport de la Cour des comptes de 2023, 1,6 million d'enfants et d'adolescents souffrent d'un trouble psychique.

• Augmentation significative : Entre 2017 et 2023, une forte augmentation des troubles a été observée, notamment :

◦ +70 % chez les 10-14 ans.    ◦ +46 % chez les 15-20 ans.

• Actes auto-infligés : La DREES note une augmentation des hospitalisations pour tentatives de suicide et gestes auto-infligés chez les 10-14 ans.

• Précocité des troubles : La moitié des troubles psychiatriques se déclarent avant l'âge de 15 ans, soulignant l'urgence d'une intervention précoce.

B. La Vulnérabilité Extrême des Enfants Protégés

Un des constats les plus marquants du rapport est la surreprésentation des enfants relevant de l'Aide Sociale à l'Enfance (ASE) dans les services de pédopsychiatrie.

• Occupation des lits : Jusqu'à 40-50 % des lits d'hospitalisation à temps plein sont occupés par des enfants placés, alors qu'ils ne représentent que 2 % des mineurs en France.

• Facteurs de risque : La maltraitance subie par ces enfants multiplie par deux les risques de suicide et par quatre les risques de psychotrauma.

• Cercle vicieux : Le suivi médical défaillant de ces mineurs entraîne une prise en charge trop tardive, souvent via une hospitalisation prolongée qui se substitue à un lieu de vie, aggravant leur état.

En parallèle, les foyers sont démunis face à des jeunes avec des troubles lourds non pris en charge.

2. Un Système de Soins Débordé et Illisible

Face à la hausse de la demande, l'offre de soins est criante de manque de moyens et d'organisation, laissant de nombreuses familles sans solution.

A. La Saturation des Structures

• Accès aux soins limité : Sur les 1,6 million de jeunes présentant des troubles, seuls 750 000 à 850 000 sont suivis, soit environ un sur deux.

• Délais d'attente insoutenables : L'attente pour un premier rendez-vous en Centre Médico-Psychologique (CMP) peut atteindre 12 à 18 mois, parfois plus.

Cette attente laisse le temps à la situation de s'aggraver, conduisant à une prise en charge aux urgences, elles-mêmes engorgées.

B. Recommandation : Instaurer une Gradation des Soins

Pour rendre l'offre plus lisible et efficiente, le rapport préconise une meilleure organisation du parcours de soins.

• Premier niveau : Les médecins généralistes et pédiatres devraient assurer la première consultation, la détection et l'orientation.

Cela nécessite un renforcement de leur formation en psychiatrie infanto-juvénile.

Ce niveau pourrait aussi inclure des psychologues et les Maisons des Adolescents.

• Deuxième niveau (pivot) : Les Centres Médico-Psychologiques (CMP) se concentreraient sur les cas nécessitant une expertise pluridisciplinaire, tout en conservant un accueil inconditionnel sans adressage obligatoire pour ne pas éloigner les plus fragiles.

• Troisième niveau : Des centres de gestion de crise de courte durée, adossés aux urgences pédiatriques ou hôpitaux psychiatriques, pour les situations les plus aiguës.

C. Recommandation : Renforcer le Secteur de la Pédopsychiatrie

Le rapport insiste sur la nécessité de renforcer l'existant plutôt que de disperser les moyens dans des dispositifs innovants non pérennes.

• Stabilité financière : Pérenniser et flécher les budgets de la pédopsychiatrie au sein des hôpitaux et privilégier les dotations pluriannuelles aux appels à projets.

• Priorité au soin : Réorienter les moyens financiers et humains vers le soin direct plutôt que vers des plateformes de diagnostic qui, malgré leurs avancées, peuvent favoriser une approche médicamenteuse au détriment de la relation thérapeutique.

• Maillage territorial : Revoir le maillage des CMP pour que chaque enfant puisse y accéder en moins de 30 minutes de son domicile, en adaptant le découpage des secteurs aux évolutions démographiques (un secteur couvre aujourd'hui jusqu'à 500 000 habitants contre 200 000 prévus initialement).

3. L'Urgence d'une Prévention Efficace

Le rapport souligne que la prévention est le parent pauvre de la politique de santé mentale, alors qu'elle est déterminante.

A. La Période des 1000 Premiers Jours

La période allant de la conception aux deux ans de l'enfant est fondamentale.

• Soutien à la parentalité : Les "Maisons des bébés" sont des dispositifs essentiels pour prévenir les troubles précoces du lien parent-enfant.

• Psychiatrie périnatale : Les unités d'hospitalisation conjointe mère-bébé, comme celle de Toulouse, permettent de détecter les signes de souffrance dès les premiers jours et de réparer le lien d'attachement. Il est recommandé de développer ces unités et de former les professionnels à la théorie de l'attachement.

B. Le Rôle Fondamental de la Santé Scolaire

L'Éducation Nationale est un lieu stratégique pour la détection précoce, mais ses moyens sont gravement fragilisés.

Professionnel

Ratio Actuel

Ratio Recommandé (UE)

Recommandation du Rapport

Psychologue scolaire

1 pour 1 600 élèves

1 pour 800 élèves

1 pour 800 élèves

Médecin scolaire

1 pour 13 000 élèves

-

1 pour 5 000 élèves

Infirmier scolaire

1 pour 9 établissements

-

1 par établissement

• Conséquence directe : Le bilan de santé obligatoire à 6 ans est réalisé à 12 ans dans 80 % des cas, entraînant une accumulation de difficultés non détectées à l'entrée au collège.

Le rapport préconise une trajectoire de recrutement ambitieuse pour atteindre les ratios cibles.

4. La Crise des Ressources Humaines et la Question de l'Attractivité

La pénurie de professionnels qualifiés est au cœur de la crise du système.

• Pénurie de pédopsychiatres : La densité nationale est de 6,7 pédopsychiatres pour 100 000 jeunes, et la profession est vieillissante (moyenne d'âge de 60 ans).

La discipline est peu attractive pour les internes (127 inscrits pour 157 postes ouverts en 2023) car jugée trop proche des sciences humaines et aux conditions de travail difficiles.

• Manque de valorisation : Les professions (psychologues, paramédicaux) sont peu valorisées et faiblement rémunérées.

Une consultation en pédopsychiatrie est un acte long et complexe (minimum 1h) mais rémunéré seulement 67 € en moyenne, poussant les professionnels vers le secteur non conventionné ou la patientèle adulte.

• Recommandations :

• ◦ Revaloriser la rémunération des psychologues et personnels paramédicaux en CMP.  

• ◦ Augmenter significativement le tarif des consultations en pédopsychiatrie.  

• ◦ Réformer le codage des actes pour valoriser le temps de concertation avec les familles et partenaires. 

• ◦ Instaurer un quotient départemental pour les stages en internat afin de mieux répartir les futurs médecins sur le territoire.

5. Facteurs Environnementaux et Sociétaux Aggravants

Le rapport identifie plusieurs facteurs externes qui pèsent lourdement sur la santé mentale des jeunes.

• Médicalisation excessive : La consommation de psychotropes (type Ritaline) est en forte augmentation (490 000 enfants de 3 à 17 ans concernés).

Ces médicaments sont souvent prescrits par des médecins généralistes peu formés, posant la question du bon usage.

Le rapport recommande de privilégier les soins thérapeutiques à la prescription.

• Impact des écrans : L'addiction aux écrans et l'exposition à des contenus violents, pornographiques ou au cyberharcèlement sont unanimement citées comme aggravant les troubles anxio-dépressifs.

Le rapport soutient la majorité numérique, la mise en place de "pauses numériques" dans les établissements scolaires et appelle à des campagnes de prévention massives.

• Pression scolaire : Le système d'orientation, et notamment Parcoursup, est décrit comme créant un "climat extrêmement anxiogène".

Les rapporteurs proposent, avec des nuances, de réformer en profondeur (Nathalie Colin-Osterlé) ou de supprimer (autre rapporteure) ce dispositif et toute forme de sélection à l'université.

• Déterminants sociaux : Les enfants de familles défavorisées ont un risque trois fois plus important de développer un trouble mental et ont plus de difficultés d'accès aux soins.

Le rapport appelle à investir dans les services publics et les politiques de lutte contre les inégalités sociales pour agir sur les causes profondes.

audio

DOI: 10.1016/j.immuni.2025.09.008

Resource: (Bio X Cell Cat# BE0001-1, RRID:AB_1107634)

Curator: @scibot

SciCrunch record: RRID:AB_1107634

What is this?

I think this story is optimistic. it shows that even though people might try to enforce and keep gender norms the same. That does not allow people to grow in the ways they should best. At first that was very difficult for the parents to see, but I think they grew into it. A moment I see optimism is in the paragraph before this, where X says its a Y.

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My hyperpost work diary sharded per Peergos Accounts

I think this should be $X_{t-p-h+1}$ (so X, not Y)

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

This manuscript presents a study on expectation manipulation to induce placebo and nocebo effects in healthy participants. The study follows standard placebo experiment conventions with the use of TENS stimulation as the placebo manipulation. The authors were able to achieve their aims. A key finding is that placebo and nocebo effects were predicted by recent experience, which is a novel contribution to the literature. The findings provide insights into the differences between placebo and nocebo effects and the potential moderators of these effects.

Specifically, the study aimed to:

(1) assess the magnitude of placebo and nocebo effects immediately after induction through verbal instructions and conditioning

(2) examine the persistence of these effects one week later, and

(3) identify predictors of sustained placebo and nocebo responses over time.

Strengths:

An innovation was to use sham TENS stimulation as the expectation manipulation. This expectation manipulation was reinforced not only by the change in pain stimulus intensity, but also by delivery of non-painful electrical stimulation, labelled as TENS stimulation.

Questionnaire-based treatment expectation ratings were collected before conditioning and after conditioning, and after the test session, which provided an explicit measure of participants' expectations about the manipulation.

The finding that placebo and nocebo effects are influenced by recent experience provides a novel insight into a potential moderator of individual placebo effects.

We thank the reviewer for their thorough evaluation of our manuscript and for highlighting the novelty and originality of our study.

Weaknesses:

There are a limited number of trials per test condition (10), which means that the trajectory of responses to the manipulation may not be adequately explored.

We appreciate the reviewer’s comment regarding the number of trials in the test phase. The trial number was chosen to ensure comparability with previous studies addressing similar research questions with similar designs (e.g. Colloca et al., 2010). Our primary objective was to directly compare placebo and nocebo effects within a within-subject design and to examine their persistence one week after the first test session. While we did not specifically aim to investigate the trajectory of responses within a single testing session, we fully agree that a comprehensive analysis of the trajectories of expectation effects on pain would be a valuable extension of our work. We have now acknowledged this limitation and future direction in the revised manuscript.

The paragraph reads as follows: “It is important to note that our study was designed in alignment with previous studies addressing similar questions (e.g., Colloca et al., 2010). Our primary aim was to directly compare placebo and nocebo effects in a within-subject design and assess their persistence of these effects one week following the first test session. One limitation of our approach is the relatively short duration of each session, which may have limited our ability to examine the trajectory of responses within a single session. Future studies could address this limitation by increasing the number of trials for a more comprehensive analysis.”

On day 8, one stimulus per stimulation intensity (i.e., VAS 40, 60, and 80) was applied before the start of the test session to re-familiarise participants with the thermal stimulation. There is a potential risk of revealing the manipulation to participants during the re-familiarization process, as they were not previously briefed to expect the painful stimulus intensity to vary without the application of sham TENS stimulation.

We thank the reviewer for the opportunity to clarify this point. Participants were informed at the beginning of the experiment that we would use different stimulation intensities to re-familiarize them with the stimuli before the second test session. We are therefore confident that participants perceived this step as part of a recalibration rather than associating it with the experimental manipulation. We have added this information to the revised version of the manuscript.

The paragraph now reads as follows: “On day 8, one stimulus per stimulation intensity (i.e., VAS 40, 60 and 80) was applied before the start of the test session to re-familiarise participants with the thermal stimulation. Note that participants were informed that these pre-test stimuli were part of the recalibration and refamiliarization procedure conducted prior to the second test session.”

The differences between the nocebo and control conditions in pain ratings during conditioning could be explained by the differing physiological effects of the different stimulus intensities, so it is difficult to make any claims about expectation effects here.

We appreciate the reviewer’s comment and agree that, despite the careful calibration of the three pain stimuli, we cannot entirely rule out the possibility that temporal dynamics during the conditioning session were influenced by differential physiological effects of the varying stimulus intensities (e.g., intensity-dependent habituation or sensitization). We have addressed this in the revision of the manuscript, but we would like to emphasize that the stronger nocebo effects during the test phase are statistically controlled for any differences in the conditioning session.

The paragraph now reads: “This asymmetry is noteworthy in and of itself because it occurred despite the equidistant stimulus calibration relative to the control condition prior to conditioning. It may be the result of different physiological effects of the stimuli over time or amplified learning in the nocebo condition, consistent with its heightened biological relevance, but it could also be a stronger effect of the verbal instructions in this condition.”

A randomisation error meant that 25 participants received an unbalanced number of 448 trials per condition (i.e., 10 x VAS 40, 14 x VAS 60, 12 x VAS 80).

We agree that this is indeed unfortunate. However, we would like to point out that all analyses reported in the manuscript have been controlled for the VAS ratings in the conditioning session, i.e., potential effects of the conditioned placebo and nocebo stimuli. Moreover, we have now conducted additional analyses, presented here in our response to the reviewers, to demonstrate that this imbalance did not systematically bias the results. Importantly, the key findings observed during the test phase remain robust despite this issue.

Specifically, when excluding these 25 participants from the analyses, the reported stronger nocebo compared to placebo effects in the test session on day 1 remain unchanged. Likewise, the comparison of placebo and nocebo effects between days 1 and 8 shows the same pattern when excluding the participants in question. The only exception is the interaction between effect (placebo vs nocebo) x session (day 1 vs day 8), which changed from a borderline significant result (p = .049) to insignificant (p = .24). However, post hoc tests continued to show the same pattern as originally reported: a significant reduction in the nocebo effect from day 1 to day 8 and no significant change in the placebo effect.

Reviewer #2 (Public review):

Summary:

Kunkel et al aim to answer a fundamental question: Do placebo and nocebo effects differ in magnitude or longevity? To address this question, they used a powerful within-participants design, with a very large sample size (n=104), in which they compared placebo and nocebo effects - within the same individuals - across verbal expectations, conditioning, testing phase, and a 1-week follow-up. With elegant analyses, they establish that different mechanisms underlie the learning of placebo vs nocebo effects, with the latter being acquired faster and extinguished slower. This is an important finding for both the basic understanding of learning mechanisms in humans and for potential clinical applications to improve human health.

Strengths:

Beyond the above - the paper is well-written and very clear. It lays out nicely the need for the current investigation and what implications it holds. The design is elegant, and the analyses are rich, thoughtful, and interesting. The sample size is large which is highly appreciated, considering the longitudinal, in-lab study design. The question is super important and well-investigated, and the entire manuscript is very thoughtful with analyses closely examining the underlying mechanisms of placebo versus nocebo effects.

We thank the reviewer for their positive evaluation of our manuscript and for acknowledging the methodological rigor and the significant implications for clinical applications and the broader research field.

Weaknesses:

There were two highly addressable weaknesses in my opinion:

(1) I could not find the preregistration - this is crucial to verify what analyses the authors have committed to prior to writing the manuscript. Please provide a link leading directly to the preregistration - searching for the specified number in the suggested website yielded no results.

We thank the reviewer for pointing this out. We included a link to the preregistration in the revised manuscript. This study was pre-registered with the German Clinical Trial Register (registration number: DRKS00029228; https://drks.de/search/de/trial/DRKS00029228).

(2) There is a recurring issue which is easy to address: because the Methods are located after the Results, many of the constructs used, analyses conducted, and even the main placebo and nocebo inductions are unclear, making it hard to appreciate the results in full. I recommend finding a way to detail at the beginning of the results section how placebo and nocebo effects have been induced. While my background means I am familiar with these methods, other readers will lack that knowledge. Even a short paragraph or a figure (like Figure 4) could help clarify the results substantially. For example, a significant portion of the results is devoted to the conditioning part of the experiment, while it is unknown which part was involved (e.g., were temperatures lowered/increased in all trials or only in the beginning).

We thank the reviewer for their helpful comment and agree that the Results section requires additional information that would typically be provided by the Methods section if it directly followed the Introduction. In response, we have moved the former Figure 4 from the Methods section to the beginning of the Results section as a new Figure 1, to improve clarity. Further, we have revised the Methods section to explicitly state that all trials during the conditioning phase were manipulated in the same way.

Recommendations for the Authors:

Reviewer #1 (Recommendations for the authors):

(1) Given that the authors are claiming (correctly) that there is only limited work comparing placebo/nocebo effects, there are some papers missing from their citations:

Nocebo responses are stronger than placebo responses after subliminal pain conditioning - - Jensen, K., Kirsch, I., Odmalm, S., Kaptchuk, T. J. & Ingvar, M. Classical conditioning of analgesic and hyperalgesic pain responses without conscious awareness. Proc. Natl. Acad. Sci. USA 112, 7863-7 (2015)

We thank the reviewer and have now included this relevant publication into the introduction of the revised manuscript.

Hird, E.J., Charalambous, C., El-Deredy, W. et al. Boundary effects of expectation in human pain perception. Sci Rep 9, 9443 (2019). https://doi.org/10.1038/s41598-019-45811-x

We thank the reviewer for suggesting this relevant publication. We have now included it into the discussion of the revised manuscript by adding the following paragraph:

“Recent work using a predictive coding framework further suggests that nocebo effects may be less susceptible to prediction error than placebo effects (Hird et al., 2019), which could contribute to their greater persistence and strength in our study.”

(2) The trial-by-trial pain ratings could have been usefully modelled with a computational model, such as a Bayesian model (this is especially pertinent given the reference to Bayesian processing in the discussion). A multilevel model could also be used to increase the power of the analysis. This is a tentative suggestion, as I appreciate it would require a significant investment of time and work - alternatively, the authors could acknowledge it in the Discussion as a useful future avenue for investigation, if this is preferred.

We thank the reviewer for this thoughtful suggestion. While we agree that computational modelling approaches could provide valuable insights into individual learning, our study was not designed with this in mind and the relatively small number of trials per condition and the absence of trial-by-trial expectancy ratings limit the applicability of such models. We have therefore chosen not to pursue such analysis but highlight it in the discussion as a promising direction for future research.

“Notably, the most recent experience was the most predictive in all three analyses; for instance, the placebo effect on day 8 was predicted by the placebo effect on day 1, not by the initial conditioning. This finding supports the Bayesian inference framework, where recent experiences are weighted more heavily in the process of model updating because they are more likely to reflect the current state of the environment, providing the most relevant and immediate information needed to guide future actions and predictions24. Interestingly, while a change in pain predicted subsequent nocebo effects, it seemed less influential than for placebo effects. This aligns with findings that longer conditioning enhanced placebo effects, while it did not affect nocebo responses10 and the conclusion that nocebo instruction may be sufficient to trigger nocebo responses. Using Bayesian modeling, future studies could identify individual differences in the development of placebo and nocebo effects by integrating prior experiences and sensory inputs, providing a probabilistic framework for understanding the underlying mechanisms.”

(3) The paper is missing any justification of sample size, i.e. power analysis - please include this.

We apologize for the missing information on our a priori power analysis. As there is a lack of prior studies investigating within-subjects comparisons of placebo and nocebo effects that could inform precise effect size estimates for our research question, we based our calculation on the ability detect small effects. Specifically, the study was powered to detect effect sizes in the range of d = 0.2 - 0.25 with α = .05 and power = .9, yielding a required sample size of N = 83-129. We have now added this information to the methods section of the revised manuscript.

(4) "On day 8, one stimulus per stimulation intensity (i.e., VAS 40, 60 and 80) was applied before the start of the test session to re-familiarise participants with the thermal stimulation."

What were the instructions about this? Was it before the electrode was applied? This runs the risk of unblinding participants, as they only expect to feel changes in stimulus intensity due to the TENS stimulation.

We thank the reviewer for pointing out the potential risk of unblinding participants due to the re-familiarization process prior to the second test session. We would like to clarify that we followed specific procedures to prevent participants from associating this process with the experimental manipulation. The re-familiarisation with the thermal stimuli was conducted after the electrode had been applied and re-tested to ensure that both stimulus modalities were re-introduced in a consistent and neutral context. Participants were explicitly informed that both procedures were standard checks prior to the actual test session (“We will check both once again before we begin the actual measurement.”). For the thermal stimuli, we informed participants that they would experience three different intensities to allow the skin to acclimate (e.g., “...we will test the heat stimuli in 3 trials with different temperatures, allowing your skin to acclimate to the stimuli. …”), without implying any connection to the experimental conditions.

Importantly, this re-familiarization procedure mirrored what participants had already experienced during the initial calibration session on day 1. We therefore assume that participants interpreted as a routine technical step rather than part of the experimental manipulation. We have now clarified this procedure in the methods section of the revised manuscript.

(5) "For a comparison of pain intensity ratings between time-points, an ANOVA with the within-subject factors Condition (placebo, nocebo, control) and Session (day 1, day 8) was carried out. For the comparison of placebo and nocebo effects between the two test days, an ANOVA with the with-subject factors Effect (placebo effect, nocebo effect) and Session (day 1, day 8) was used."

It seems that one ANOVA is looking at raw pain scores and one is looking at difference scores, but this is a bit confusing - please rephrase/clarify this, and explain why it is useful to include both.

We thank the reviewer for highlighting this point. Our primary analyses focus on placebo and nocebo effects, which we define as the difference in pain intensity ratings between the control and the placebo condition (placebo effect) and the nocebo and the control condition (nocebo effect), respectively.

To examine whether condition effects were present at each time-point, we first conducted two separate repeated measures ANOVAs - one for day 1 and one for day 8 - with the within-subject factor CONDITION (placebo, nocebo, control).

To compare the magnitude and persistence of placebo and nocebo effects over time, we then calculated the above-mentioned difference scores and submitted these to a second ANOVA with within-subject factors EFFECT (placebo vs. nocebo effect) and SESSION (day 1 vs. day 8). We have now clarified this approach on page 19 of the revised manuscript. To avoid confusion, the Condition x Session ANOVA has been removed from the manuscript.

(6) Please can the authors provide a figure illustrating trial-by-trial ratings during test trials as well as during conditioning trials?

In response to the reviewer’s point, we now provide the trial-by-trial ratings of the test phases on days 1 and 8 as an additional figure in the Supplement (Figure S1) and would like to clarify that trial-by-trial pain intensity ratings of the conditioning phase are displayed in Figure 2C of the manuscript,

(7) "Separate multiple linear regression analyses were performed to examine the influence of expectations (GEEE ratings) and experienced effects (VAS ratings) on subsequent placebo and nocebo effects. For day 1, the placebo effect was entered as the dependent variable and the following variables as potential predictors: (i) expected improvement with placebo before conditioning, (ii) placebo effect during conditioning and (iii) the expected improvement with placebo before the test session at day 1"

The term "placebo effect during conditioning" is a bit confusing - I believe this is just the effect of varying stimulus intensities - please could the authors be more explicit on the terminology they use to describe this? NB changes in pain rating during the conditioning trials do not count as a placebo/nocebo effect, as most of the change in rating will reflect differences in stimulation intensity.

We agree with the reviewer that the cited paragraph refers to the actual application of lower or higher pain stimuli during the conditioning session, rather than genuinely induced placebo or nocebo effect. We thank the reviewer for this helpful observation and have revised the terminology, accordingly, now referring to these as “pain relief during conditioning” and “pain worsening during conditioning”.

(8) Supplementary materials: "The three temperature levels were perceived as significantly different (VAS ratings; placebo condition: M= 32.90, SD= 16.17; nocebo condition: M= 56.62, SD= 17.09; control condition: M= 80.84, SD= 12.18"

This suggests that the VAS rating for the control condition was higher than for the nocebo condition. Please could the authors clarify/correct this?

We thank the reviewer for spotting this error. The values for the control and the nocebo condition had accidentally been swapped. This has now been corrected in the manuscript: control condition: M= 56.62, SD= 17.09; nocebo condition: M= 80.84, SD= 12.18.

(9) "To predict placebo responses a week later (VAScontrol - VASplacebo at day 8), the same independent variables were entered as for day 1 but with the following additional variables (i) the placebo effect at day 1 and (ii) the expected improvement with placebo before the test session at day 8."

Here it would be much clearer to say 'pain ratings during test trials at day 1".

We agree with the reviewer and have revised the manuscript as suggested.

(10) For completeness, please present the pain intensity ratings during conditioning as well as calibration/test trials in the figure.

Please see our answer to comment (6).

(11) In Figure 1a, it looks like some participants had rated the control condition as zero by day 8. If so, it's inappropriate to include these participants in the analysis if they are not responding to the stimulus. Were these the participants who were excluded due to pain insensitivity?

On day 8, the lowest pain intensity ratings observed were VAS 3 in the placebo condition and VAS 2 in the control condition, both from the same participant. All other participants reported minimum values of VAS 11 or higher (all on a scale from 0-100). Thus, no participant provided a pain rating of VAS 0, and all ratings indicated some level of pain perception in response to the stimulus. We did not define an exclusion criterion based on day 8 pain ratings in our preregistration, and we did not observe any technical issues with the stimulation procedure. To avoid post-hoc exclusions and maintain consistency with our preregistered analysis plan, we therefore decided to include all participants in the analysis.

(12) "Comparison of day 1 and day 8. A direct comparison of placebo and nocebo effects on day 1 and day 8 pain intensity ratings showed a main effect of Effect with a stronger nocebo effect (F(1,97)= 53.93, 131 p< .001, η2= .36) but no main effect of Day (F(1,97)= 2.94, p= .089, η2 = .029). The significant Effect x Session interaction indicated that the placebo effect and the nocebo effect developed differently over time (F(1,97)= 3.98, p= .049, η2 = .039)"

This is confusing as it talks about a main effect of "day" and then interaction with "session" - are they two different models? The authors need to clarify.

We thank the reviewer for pointing this out. In our analysis, “Session” is the correct term for the experimental factor, which has two factor levels, “day 1” and “day 8”. This has now been corrected in the revised manuscript.

Reviewer #2 (Recommendations for the authors):

(1) More information on how "size of the effect" in Figures 1b and 2b was calculated is needed; this can be in the legend. If these are differences between control and each condition, then they were reversed for one condition (nocebo?), which is ok - but this should be clearly explained.

We agree with the reviewer and have now revised the figure legends to improve clarity. The legends now read:

1b: “Figure 1. Pain intensity ratings and placebo and nocebo effects during calibration and test sessions. (A) Mean pain intensity ratings in the placebo, nocebo and control condition during calibration, and during the test sessions at day 1 and day 8. (B) Placebo effect (control condition - placebo condition, i.e., positive value of difference) and nocebo effect (nocebo condition - control condition, i.e., positive value of difference) on day 1 and day 8. Error bars indicate the standard error of the mean, circles indicate mean ratings of individual participants. *: p < .001, : p < .01, n.s.: non-significant.”

2b: “Figure 2. Mean and trial-by-trial pain intensity ratings, placebo and nocebo effects during conditioning. (A) Mean pain intensity ratings of the placebo, nocebo and control condition during conditioning. (B) Placebo effect (control condition - placebo condition, i.e., positive value of difference) and nocebo effect (nocebo condition - control condition, i.e., positive value of difference) during conditioning. (C) Trial-by-trial pain intensity ratings (with confidence intervals) during conditioning. Error bars indicate the standard error of the mean, circles indicate mean ratings of individual participants. ***: p < .001.”

(2) In the methods, I was missing a clear understanding of how many trials there were in the conditioning phase, and then how many in the other testing phases. Also, how long did the experiment last in total?

We apologize that the exact number of trials in the testing phases was not clear in the original manuscript. We now indicate on page 18 of the revised manuscript that we used 10 trials per condition in the test sessions. We have also added information on the duration of each test day (i.e., three hours on day 1 and one hour on day 8) on page 15.

(3) In expectancy ratings, line 186 - are improvement and worsening expectations different from expected pain relief? It is implied that these are two different constructs - it would be helpful to clarify that.

We agree that this is indeed confusing and would like to clarify that both refer to the same construct. We used the Generic rating scale for previous treatment experiences, treatment expectations, and treatment effects (GEEE questionnaire, Rief et al. 2021) that discriminates between expected symptom improvement, expected symptom worsening, and expected side effects due to a treatment. We now use the terms “expected pain relief” and “expected pain worsening” throughout the whole manuscript.

(4) In the last section of the Results, somatosensory amplification comes out of nowhere - and could be better introduced (see point 2 above).

We agree with the reviewer that introducing the concept of somatosensory amplification and its potential link to placebo/nocebo effects only in the Methods is unhelpful, given that this section appears at the end of the manuscript. We therefore now introduce the relevant publication (Doering et al., 2015) before reporting our findings on this concept.

(5) In line 169, if the authors want to specify what portion of the variance was explained by expectancy, they could conduct a hierarchical regression, where they first look at R2 without the expectancy entered, and only then enter it to obtain the R2 change.

We fully agree that hierarchical regression can be a useful approach for isolating the contribution of variables. However, in our case, expectancy was assessed at different time points (e.g., before conditioning and before the test session on day 1), and there was no principled rationale for determining the order in which these different expectancy-related variables should be entered into a hierarchical model.

That said, in response to the reviewer’s suggestion, we have now conducted hierarchical regression analyses in which all expectancy-related variables were entered together as a single block (see below). These analyses largely confirmed the findings reported so far and are provided here in the response to the reviewers below. Given the exploratory nature of this grouping and the lack of an a priori hierarchy, we feel that the standard multiple regression models remain the most appropriate for addressing our research question because it allows us to evaluate the total contribution of expectancy-related predictors while also examining the individual contribution of each variable within the block. We would therefore prefer to retain these as the primary analyses in the manuscript.

Results of the hierarchical regression analyses:

Day 1 - Placebo response: In step 1, we entered the difference in pain intensity ratings between the control and the placebo condition during conditioning as a predictor. In step 2, we added the two variables reflecting expectations (i.e., expected improvement with placebo (i) before conditioning and (ii) before the test session on day 1). This allowed us to assess whether expectation-related variables explained additional variance beyond the effect of conditioning.

The overall regression model at step 1 was significant, F(1, 102) = 13.42, p < .001, explaining 11.6% of the variance in the dependent variable (R² = .116). Adding the expectancy-related predictors in step 2 did not lead to a significant increase in explained variance, ΔR² = .007, F(2, 100) = 0.384, p = .682. Thus, the conditioning response significantly predicted placebo-related pain reduction on day 1, but additional information on expectations did not account for further variance.

Day 1 - Nocebo response: The equivalent analysis was run for the nocebo response on day 1. In step 1, the pain intensity difference between the nocebo and the control condition was entered as a predictor before adding the two expectancy ratings (i.e., expected worsening with nocebo (i) before conditioning and (ii) before the test session on day 1).

In step 1, the regression model was not statistically significant, F(1, 102) = 2.63, p = .108, and explained only 2.5% of the variance in nocebo response (R² = .025). Adding the expectation-related predictors in Step 2 slightly increased the explained variance by ΔR² = .027, but this change was also non-significant, F(2, 100) = 1.41, p = .250. The overall variance explained by the full model remained low (R² = .052). These results suggest that neither conditioning nor expectation-related variables reliably predicted nocebo-related pain increases on day 1.

Day 8 - Placebo response: For the prediction of the placebo effect on day 8, the following variables reflecting perceived effects were entered as predictors in step 1: the difference in pain intensity ratings between the control and the placebo condition (i) during conditioning and (ii) on day 1. In step 2, the variables reflecting expectations were added: the expected improvement with placebo (i) before conditioning, (ii) before the test session on day 1 and (iii) before the test session on day 8.

In step 1, the model was statistically significant, F(3, 95) = 14.86, p < .001, explaining 23.8% of the variance in the placebo response (R² = .238, Adjusted R² = .222). In step 2, the addition of the expectation-related predictors resulted in a non-significant improvement in model fit, ΔR² = .051, F(3, 92) = 2.21, p = .092. The overall variance explained by the full model increased modestly to 29.0%.

Day 8 - Nocebo response: For the equivalent analyses of nocebo responses on day 8, the following variables were included in step 1: the difference in pain intensity ratings between the nocebo and the control condition (i) during conditioning and (ii) on day 1. In step 2, we entered the variables reflecting nocebo expectations including expected worsening with nocebo (i) before conditioning, (ii) before the test session on day 1 and (iii) before the test session on day 8. In step 1, the model significantly predicted the day 8 nocebo response, F(3, 95) = 6.04, p = .003, accounting for 11.3% of the variance (R² = .113, Adjusted R² = .094). However, the addition of expectation-related predictors in Step 2 resulted in only a negligible and non-significant improvement, ΔR² = .006, F(3, 92) = 0.215, p = .886. The full model explained just 11.9% of the variance (R² = .119).

Typos:

(6) Abstract - 104 heathy xxx (word missing).

(7) Line 61 - reduce or decrease - I think you meant increase.

Thank you, we have now corrected both sentences.

References

Colloca L, Petrovic P, Wager TD, Ingvar M, Benedetti F. How the number of learning trials affects placebo and nocebo responses. Pain. 2010

Doering BK, Nestoriuc Y, Barsky AJ, Glaesmer H, Brähler E, Rief W. Is somatosensory amplification a risk factor for an increased report of side effects? Reference data from the German general population. J Psychosom Res. 2015