Antes de la escena de apertura nos falta la cronología de Webb y Vance para hacernos una mejor idea de cómo murió el periodista y qué pasó en general.

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Document de Synthèse : Interprétariat, Migration et Santé Mentale

Résumé Exécutif

Ce document synthétise les interventions de la soirée-débat organisée le 12 octobre par Médecins du Monde et le CHU de Toulouse.

Le constat central souligne une dégradation de l'accès aux soins pour les personnes précaires en France, exacerbée par une « crise de l'accueil » plutôt que par une simple crise migratoire.

L'interprétariat professionnel y est identifié comme la « clé de voûte » indispensable pour assurer une prise en charge médicale et psychologique digne.

Les points saillants incluent :

• La précarisation croissante des exilés, avec une corrélation forte entre migration et sans-abrisme.

• L'impact délétère des procédures administratives et des conditions d'accueil sur la santé mentale (traumatismes complexes).

• Le rôle précurseur du CHU de Toulouse et de l'unité Iker dans l'intégration de l'interprétariat au sein du parcours de soins.

• La nécessité de structurer et de financer l'interprétariat au-delà des dispositifs hospitaliers spécialisés pour toucher la médecine de ville.

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I. Panorama de la Précarité et de la Migration en France

Une crise de l'accueil institutionnalisée

L'analyse de Nicolas Chambon (Orspere-Samdarra) remplace le terme de « crise migratoire » par celui de « crise de l'accueil ».

Malgré une augmentation constante de la demande d'asile depuis 15 ans, les structures d'hébergement (CADA) restent insuffisantes.

• Statistiques clés de l'hébergement :

◦ 40 % des personnes dans le dispositif national d'accueil ne parlent pas français.   

◦ Dans les Centres d'Hébergement et de Réinsertion Sociale (CHRS), on compte 55 % d'étrangers.  

◦ Dans l'hébergement d'urgence, ce chiffre monte à 77 %.   

◦ L'Aide Médicale d'État (AME) est passée de 75 000 bénéficiaires en 2002 à plus de 400 000 aujourd'hui.

Profils des populations vulnérables

Le public rencontré par les structures de santé est de plus en plus hétérogène, incluant :

• Réfugiés et demandeurs d'asile : Environ 310 000 réfugiés et 140 000 demandeurs d'asile, dont beaucoup sont allophones.

• Mineurs Non Accompagnés (MNA) : Environ 50 000 personnes dans cette situation, en attente de reconnaissance de minorité ou en recours, avec des besoins spécifiques en santé mentale.

• Personnes déboutées : Environ 2/3 des demandeurs d'asile sont déboutés, ce qui entraîne une perte de droits et une précarité extrême.

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II. Dispositifs de Santé et Réponses de Terrain

Le Service de Médecine Sociale du CHU de Toulouse

Sous la direction de Karine Parienté, ce service a développé plusieurs unités spécialisées pour répondre à la barrière linguistique et sociale :

| Unité | Missions Principales | | --- | --- | | Iker | Coordination de l'appui aux professionnels de santé et travailleurs sociaux ; lutte contre l'hospitalo-centrisme ; financement de l'interprétariat en ville. | | PASS (Permanence d'Accès aux Soins de Santé) | Accueil des personnes sans droits (90 % de migrants) ; dispositif mobile pour les personnes à la rue. | | LHSS (Lits Halte Soins de Santé) | Accueil médico-social (séjours d'environ 1 mois) pour les personnes sans domicile nécessitant des soins aigus. | | Santé Sexuelle | Prévention et soins pour les publics à risque, notamment originaires de pays à haute prévalence. |

L'action de Médecins du Monde (MDM) à Toulouse

Yann Laperrière détaille une activité en expansion avec 10 salariés et 100 bénévoles, articulée autour de quatre programmes :

1. CASO (Centre d'Accueil, de Soins et d'Orientation) : Consultations médicales pour les publics sans droits.

2. Actions Mobiles : Médiation en santé dans les bidonvilles, squats et la rue, avec un focus sur les familles.

3. Mission MNA : Programme exploratoire dédié aux mineurs isolés.

4. Santé Mentale et Soutien Psychosocial : Activités collectives (ex: Café du Monde) et évaluations individuelles basées sur les ressources propres des personnes exilées.

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III. Santé Mentale : Traumatismes et Parcours d'Exil

La santé mentale est intrinsèquement liée aux conditions sociales. L'exil est décrit comme un parcours de ruptures successives et de violences.

Pathologies et prévalence

• Trouble de stress post-traumatique (TSPT) : Concerne environ 1/3 de la population réfugiée.

• Troubles dépressifs : Touchent également 1/3 des exilés.

• Traumatismes complexes : Les traumatismes ne sont pas seulement liés au pays d'origine ou au voyage, mais aussi au « non-accueil » en France (vie à la rue, violence en centre d'hébergement).

Les paradoxes du système de soin

Nicolas Chambon souligne deux dérives majeures :

• L'utilitarisme administratif : Le soin est souvent réduit à l'obtention d'un certificat médical pour des procédures (titre de séjour « étranger malade », recours CNDA). Cela peut mener à « ouvrir » un traumatisme pour le récit administratif sans assurer de suivi thérapeutique derrière.

• L'inadaptation des dispositifs modernes : Les nouvelles approches de « santé mentale positive » ou de réhabilitation psychosociale sont souvent inaccessibles aux migrants car elles présupposent une stabilité sociale (logement, droits) que ces publics n'ont pas.

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IV. L'Interprétariat : Un Outil de Soins Fondamental

L'interprétariat est présenté non pas comme un accessoire, mais comme une condition sine qua non de la pratique clinique en contexte de migration.

Enjeux cliniques et éthiques

• Précision du diagnostic : Sans interprète, des erreurs graves surviennent (ex: une patiente diagnostiquée par erreur comme souffrant de troubles psychiatriques alors qu'elle était simplement allophone).

• Reconnaissance de l'altérité : Parler dans sa langue maternelle permet de sortir du « récit de victime » imposé par l'administration pour redevenir sujet de son histoire.

Problématiques de financement et de déploiement

Une étude de l'ARS en région Auvergne-Rhône-Alpes révèle un décalage entre les besoins et l'utilisation des fonds :

• Une enveloppe de 600 000 € destinée aux libéraux est consommée à 60 % par les PASS.

• Les généralistes ne l'utilisent qu'à 2 % et les psychiatres libéraux à 1,8 %.

Conclusion des intervenants : Il est impératif de structurer l'interprétariat comme un outil professionnel, financé et sensibilisé auprès de l'ensemble du corps médical pour garantir un accueil inconditionnel et un soin de qualité.

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Citations Clés

« L'interprétariat est vraiment la clé de voûte de toute cette prise en charge importante. » — Yann Laperrière

« On s'est rendu compte qu'en fait elle n'était pas folle, elle était vietnamienne. » — Nicolas Chambon (citant un psychiatre en 2017)

« Soutenir la situation sociale des personnes, c'est soutenir leur santé mentale et inversement. » — Nicolas Chambon

Briefing Doc : Vivre son adolescence dans un monde en crise

Ce document de synthèse analyse les interventions du Professeur Ludovic Gicquel, pédopsychiatre et responsable de la Maison des Adolescents de la Vienne (Pictadom), lors d'une conférence organisée par la Caisse d'Allocations Familiales (CAF). Il explore les mécanismes biologiques, psychologiques et sociétaux qui définissent l'adolescence contemporaine.

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Synthèse de la direction (Executive Summary)

L'adolescence est une période de métamorphose radicale, marquée par un décalage structurel entre un corps qui s'adultise et un cerveau dont la maturité complète n'est atteinte qu'à l'âge de 25 ans.

Aujourd'hui, ce processus se déroule dans un « bain sociétal » particulièrement anxiogène, caractérisé par une surstimulation numérique et des crises mondiales permanentes.

Points clés à retenir :

• Santé mentale : Un adolescent sur quatre (11-15 ans) est touché par un trouble anxieux généralisé (sondage Ipsos 2021).

• Neurobiologie : Le cerveau adolescent est régi par les émotions (amygdale) plutôt que par la raison (cortex préfrontal), ce qui explique l'impulsivité.

• Concurrence numérique : Pour la première fois, l'interaction humaine est en concurrence directe avec l'interaction numérique, modifiant le rôle parental de « filtre ».

• Pathologies : 80 à 85 % des adolescents traversent cette période sans encombre majeur, mais les comportements d'auto-régulation dysfonctionnels (scarifications, addictions) sont en forte hausse.

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1. La biologie de la métamorphose

L'adolescence ne doit pas être perçue comme une simple transition, mais comme une étape de développement à part entière avec ses propres spécificités physiologiques.

Le décalage cerveau-corps

Le cerveau adolescent subit un remaniement profond. La maturation cérébrale est postéro-antérieure : elle commence par l'arrière et se termine par le cortex préfrontal, siège de la décision et de la gestion des émotions.

• Le cerveau émotionnel : L'amygdale est hyperactive, provoquant une réactivité émotionnelle intense.

• Le haut débit nerveux : La gaine de myéline s'épaissit, faisant passer la vitesse de l'influx nerveux de 1 à 100 mètres par seconde (passage de l'ADSL à la fibre optique).

• L'élagage synaptique (Pruning) : Le cerveau élimine les connexions inutilisées pour se spécialiser, rendant cette période cruciale pour l'apprentissage.

La puberté et le rapport au corps

La puberté impose un corps « adulte » à un esprit encore infantile. Ce processus est souvent vécu de manière passive : l'adolescent « subit » ses hormones et sa croissance.

• La violence de la métamorphose : Elle est souvent plus marquée chez les filles, créant une discordance entre l'image perçue et l'identité intérieure.

• La parentalité physiologique : Dès la puberté, le corps est apte à la reproduction, créant un décalage avec les normes sociales actuelles où l'autonomie est tardive.

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2. Le « bain sociétal » et l'impact du numérique

L'environnement moderne agit comme un incubateur qui influence directement le développement des jeunes.

Le monde numérique n'est plus un outil, mais un environnement concurrentiel.

La concurrence de l'interaction

L'interaction parentale est désormais en concurrence avec l'interaction numérique.

Le smartphone est décrit comme une « arme de séduction massive » conçue par des industries pour capter le temps de cerveau disponible.

• Le mécanisme de la récompense : Les « likes » activent la dopamine, créant une dépendance à la validation externe (société de l'extimité).

• Le "scrolling" : Un mécanisme hypnotique qui altère la perception du temps et l'effort de concentration.

Les distorsions de la réalité

• Image de soi : Les filtres et les photos retouchées créent des standards de beauté inaccessibles, générant un sentiment permanent d'anormalité.

• Pornographie : L'exposition précoce via smartphone (souvent dès le collège) est un « abus de faiblesse ».

Elle propose une vision performative et violente de la sexualité, souvent incompatible avec les capacités de contenance émotionnelle des jeunes.

| Domaine | Impact du numérique | | --- | --- | | Information | Saturation, difficulté à distinguer l'opinion du fait (Fake News). | | Social | Relations, ruptures et deuils vécus à distance avec une intensité réelle. | | Consommation | L'adolescent n'est plus le client, il est le produit (exploitation des vulnérabilités). |

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3. Santé mentale : de l'adaptation à la rupture

Le Professeur Gicquel distingue le développement normal des trajectoires pathologiques.

Les troubles sont souvent des « aménagements pathologiques d'un développement normal ».

Typologie des états adolescents

1. Remaniements adaptatifs (80-85 %) : L'adolescence se déroule normalement.

2. Troubles adaptatifs (10 %) : Souffrance transitoire et réactionnelle à l'environnement.

3. Troubles structurels (5 %) : Pathologies lourdes (anorexie mentale, schizophrénie, troubles bipolaires, trouble borderline).

Les signaux d'alerte

L'inquiétude parentale est considérée comme le meilleur baromètre. Les symptômes de régulation émotionnelle dysfonctionnelle doivent alerter :

• Scarifications : En hausse de 400 % en 20 ans ; elles servent de « fusible » pour décharger une angoisse psychique insupportable sur le corps.

• Troubles alimentaires : Préoccupations excessives pour la diététique ou perte de poids rapide.

• Déscolarisation : Souvent liée au harcèlement (stigmatisation de la différence).

• Phobie sociale : Incapacité à faire groupe.

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4. Rôle des parents et accompagnement

Dans un monde insécurisant (climat, guerres, économie), le parent doit retrouver son rôle de filtre, tout en acceptant sa propre faillibilité.

Recommandations pour les parents

• Maintenir le dialogue : Ne pas disqualifier les centres d'intérêt numériques (ex: Minecraft, TikTok), mais s'en servir comme base d'échange.

• Cultiver le libre arbitre : Aider l'adolescent à décrypter les images et les sources d'information.

• Accepter l'ingratitude : La séparation-individuation nécessite que l'adolescent se détache des parents, ce qui peut générer des conflits.

• Ne pas être parfait : Les parents ont le droit d'être anxieux ou tristes, à condition de l'expliquer a posteriori pour éviter que l'enfant ne se sente coupable.

Structures de recours (Exemple de la Vienne)

• Pictadom (Maison des Adolescents) : Accueil gratuit, anonyme et sans rendez-vous pour les jeunes et les parents.

• Mon Psy : Dispositif permettant huit consultations de psychologue remboursées sur orientation médicale.

• Unités d'urgence (AZAP) : Prise en charge intensive de jour pour les crises suicidaires ou les troubles borderline.

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Citations clés

« L'adolescent n'est pas un enfant XL ni un adulte miniature. C'est un être à part entière. »

« Le cerveau est adulte à 25 ans, soit 7 ans après le droit de vote. Il y a un décalage non négligeable. »

« Nos enfants sont devenus nos parts de marché. Les adolescents vivent dans une société de consommation qui les consomme. »

« Le parent est un filtre. On ne peut pas filtrer l'environnement si on est soi-même au prise avec des difficultés sans aide. »

DOI: 10.1038/s41431-025-01966-y

Resource: RRID:BDSC_8816

Curator: @maulamb

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DOI: 10.1038/s41431-025-01966-y

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DOI: 10.1038/s41431-025-01966-y

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DOI: 10.1038/s41431-025-01966-y

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DOI: 10.1038/s41431-025-01966-y

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DOI: 10.1038/s41431-025-01966-y

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What is this?

DOI: 10.1371/journal.pbio.3003630

Resource: (Thermo Fisher Scientific Cat# 31872, RRID:AB_2532166)

Curator: @scibot

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DOI: 10.1007/s12035-026-05741-y

Resource: IBM SPSS Statistics (RRID:SCR_016479)

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DOI: 10.1007/s12035-026-05741-y

Resource: Olympus BX61 Upright Wide Field Microscope (RRID:SCR_020343)

Curator: @scibot

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DOI: 10.1007/s12035-026-05741-y

Resource: EthoVision XT (RRID:SCR_000441)

Curator: @scibot

SciCrunch record: RRID:SCR_000441

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DOI: 10.1007/s12035-026-05741-y

Resource: RRID:RRRC_00239

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DOI: 10.1007/s12035-026-05741-y

Resource: Olympus cellSens Software (RRID:SCR_014551)

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What is this?

Esta frase que dice Galí me parece muy acertada; después de todo, si tú mismo no comprendes lo que estás escribiendo, menos lo hará el lector.

Estoy de acuerdo con el autor porque los textos con oraciones muy largas me cansan y me dificultan la comprensión. A veces tengo que releer varias veces para entender la idea principal, lo que demuestra que la claridad es fundamental en la escritura.

El método que sugiere el autor me parece entretenido y eficiente, puesto que este tipo de ejercicios interactivos ayudan más que solo leer.

Agarra cualquier parecido con el humano, primero a nivel fisioólogico y después expande a elementos y cultura compartida con con alos seres vivos.

En particular la dinámica de depresdadores y presas.

Gestion des élèves perturbateurs : approches psychopédagogiques et cadres éthiques

Résumé analytique

Le comportement perturbateur d'un élève ne doit pas être perçu comme une simple transgression, mais comme le symptôme d'un mal-être profond, souvent enraciné dans un vécu personnel ou scolaire difficile.

La gestion efficace de ces situations repose sur la reconnaissance des besoins psychologiques fondamentaux de l'élève (sécurité, reconnaissance, justice, estime de soi) et sur la mise en place d'espaces de parole institutionnalisés.

L'analyse souligne que de nombreux élèves perturbateurs, y compris les harceleurs, sont eux-mêmes en situation de souffrance.

Pour répondre à ces défis, les personnels de direction et les équipes éducatives doivent naviguer entre quatre orientations éthiques :

• la déontologie (la règle),
• le conséquentialisme (l'impact de la sanction),
• la vertu (la confiance) et
• l'éthique du care (le soin).

L'équilibre entre ces dimensions permet de maintenir le lien de confiance entre l'élève et l'institution, évitant ainsi le décrochage ou l'exclusion définitive des profils les plus vulnérables.

La nature du comportement perturbateur : un symptôme de mal-être

Le comportement perturbateur est défini comme une manifestation de symptômes liés à une insatisfaction des besoins psychologiques fondamentaux.

Aider un élève nécessite d'être attentif à ces signes, qu'ils soient émotionnels ou plus subtils.

Les besoins psychologiques fondamentaux

Pour remédier aux comportements problématiques, l'institution doit prendre en considération :

• • Le besoin de sécurité (affective et physique).
• • Le besoin de reconnaissance et de justice.
• • Le besoin d'écoute et d'expression de soi.
• • Le besoin d'estime de soi.

Stratégies d'intervention et espaces de parole

L'intervention repose sur une distinction claire entre les problématiques collectives et individuelles, ainsi que sur la création de structures d'échange formelles.

La règle d'or de la communication

• Problème collectif : Doit faire l'objet d'une discussion collective.

• Problème individuel : Le comportement d'un élève spécifique doit être traité exclusivement avec lui, afin de préserver sa dignité et de favoriser un dialogue constructif.

Institutionnalisation de l'écoute

La mise en place de "cellules d'écoute" ou d'espaces de parole sécurisés est présentée comme une solution aux résultats rapides et significatifs.

• L'écoute active : Les adultes doivent être formés pour permettre à l'élève d'élaborer lui-même le sens de son vécu.

• Efficacité constatée : Des exemples, notamment dans l'académie de Grenoble, montrent qu'une participation à deux ou trois reprises à ces espaces peut transformer le comportement des jeunes.

• Sécurité affective : L'espace doit permettre à l'élève de dire ce qu'il ressent sans crainte immédiate de jugement ou de répression.

Le cadre disciplinaire et le conseil de discipline

Face à des actes graves (comme des injures envers un enseignant), la sanction demeure nécessaire.

Cependant, la procédure doit respecter des principes éthiques et réglementaires stricts.

• Le principe du contradictoire : Avant et pendant le conseil de discipline, toutes les parties doivent pouvoir s'exprimer et clarifier les faits.

• L'analyse de la souffrance : Il est impératif de considérer que l'élève auteur d'actes délictueux est souvent un élève qui souffre.

Le document note par exemple qu'un grand nombre de harceleurs sont eux-mêmes victimes de harcèlement.

• Dialogue avec la famille : La compréhension du contexte familial est cruciale pour identifier les racines du comportement de l'adolescent.

Les quatre orientations de l'éthique professionnelle

Le chef d'établissement et son équipe doivent composer avec quatre dimensions éthiques lors de la prise de décision disciplinaire :

| Orientation éthique | Définition et application | | --- | --- | | Déontologique | Respect strict du règlement intérieur et de la loi. C'est l'approche systématique : "à tel acte correspond telle sanction". Essentiel pour la responsabilité professionnelle du chef d'établissement. | | Conséquentialiste | Attention portée aux conséquences de la sanction sur l'avenir de l'élève. Par exemple, éviter d'informer des parents violents d'une faute mineure pour ne pas infliger une "double peine" à l'enfant. | | Exercice des vertus | Mise sur la patience, la prudence et la confiance. On donne du temps à l'élève pour s'améliorer en privilégiant un blâme ou un avertissement plutôt qu'une exclusion. | | Éthique du Care (Soin) | Posture indispensable vis-à-vis des élèves les plus vulnérables traversant des souffrances psychiques graves. Il s'agit de maintenir la "tête hors de l'eau" pour l'élève par un regard attentif et bienveillant. |

Conclusion : Le rôle de l'arbitrage institutionnel

Le chef d'établissement a la responsabilité première de garantir le respect de la règle et du droit (réflexe déontologique) pour éviter toute faute professionnelle. Toutefois, la réalité du terrain impose une composition entre ces différentes éthiques.

Une décision efficace est souvent hybride : elle rappelle la règle (déontologie), tout en tempérant la sanction au regard du contexte (conséquentialisme) et en demandant à l'équipe pédagogique une "bienveillance attentive" (care).

Cette approche intégrée est présentée comme le seul moyen de préserver la confiance des élèves les plus fragiles envers l'école et les adultes, prévenant ainsi leur exclusion définitive du système scolaire.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors show that genetic deletion of the orphan tumor necrosis factor receptor DR6 in mice does not protect peripheral axons against degeneration after axotomy. Similarly, Schwann cells in DR6 mutant mice react to axotomy similarly to wild-type controls. These negative results are important because previous work has indicated that loss or inhibition of DR6 is protective in disease models and also against Wallerian degeneration of axons following injury. This carefully executed counterexample is important for the field to consider.

Strengths:

A strength of the paper is the use of two independent mouse strains that knock out DR6 in slightly different ways. The authors confirm that DR6 mRNA is absent in these models (western blots for DR6 protein are less convincingly null, but given the absence of mRNA, this is likely an issue of antibody specificity). One of the DR6 knockout strains used is the same strain used in a previous paper examining the effects of DR6 on Wallerian degeneration.

The authors use a series of established assays to evaluate axon degeneration, including light and electron microscopy on nerve histological samples and cultured dorsal root ganglion neurons in which axons are mechanically severed and degeneration is scored in time-lapse microscopy. These assays consistently show a lack of effect of loss of DR6 on Wallerian degeneration in both mouse strains examined.

Therefore, in the specific context of these experiments, the author's data support their conclusion that loss of DR6 does not protect against Wallerian degeneration.

Weaknesses:

(1) The major weaknesses of this paper include the tone of correcting previously erroneous results and the lack of reporting on important details around animal experiments that would help determine whether the results here really are discordant with previous studies, and if so, why.

The authors do not report the genetic strain background of the mice used, the sex distributions of their experimental cohorts, or the age of the mice at the time the experiments were performed. All of these are important variables.

(Response 1) We thank the reviewer for emphasizing the importance of reporting the sex, age, and genetic background of the experimental animals used in our axon protection analyses. We have incorporated this information into the revised manuscript wherever available. The sole exception concerns the genetic background of the conditional DR6 mice generated by Genentech, which remains unknown. The original publication describing these mice (Tam et al., 2012, Dev Cell, PMID 22340501) did not report this information, and we were unable to obtain it directly from Genentech. Details regarding the genetic background of the Wld^S and aPhr1 mutant mice are provided in their respective original publications, which are cited in our manuscript. Because the Gamage et al. study from the Deppmann laboratory did not report the sex or age of the animals used, we were unable to assess whether these variables might contribute to the differences observed between the two studies. Moreover, we are not aware of published evidence identifying sex or age as modifiers of structural axon preservation in axotomized peripheral nerve stumps in mouse models of delayed Wallerian degeneration. Furthermore, in the original publications describing the phenotypes of transgenic Nmnat2 and Wld^S mice, as well as Sarm1 or Phr1 knockout mice, sex and age of the animals used in the Wallerian degeneration assays were not reported (PMIDs 23995269, 12106171, 22678360, 23665224). Although, to our knowledge, no large-scale systematic studies have been conducted, over the last 15 years we have never observed any sex-based differences in Wallerian degeneration phenotypes in these mutants exhibiting prominent axon protection. This topic was discussed informally at conferences, and we are also not aware of other investigators having observed such effects.

In response to the reviewer’s comment regarding “tone”, we made sure that our data and interpretations are presented in a professional, balanced, and objective manner, including a detailed discussion of potential alternative explanations for the discrepant findings.

(2) The DR6 knockout strain reported in Gamage et al. (2017) was on a C57BL/6.129S segregating background. Gamage et al. reported that loss of DR6 protected axons from Wallerian degeneration for up to 4 weeks, but importantly, only in 38.5% (5 out of 13) mice they examined. In the present paper, the authors speculate on possible causes for differences between the lack of effect seen here and the effects reported in Gamage et al., including possible spontaneous background mutations, epigenetic changes, genetic modifiers, neuroinflammation, and environmental differences. A likely explanation of the incomplete penetrance reported by Gamage et al. is the segregating genetic background and the presence of modifier loci between C57BL/6 and 129S. The authors do not report the genetic background of the mice used in this study, other than to note that the knockout strain was provided by the group in Gamage et al. However, if, for example, that mutation has been made congenic on C57BL/6 in the intervening years, this would be important to know. One could also argue that the results presented here are consistent with 8 out of 13 mice presented in Gamage et al.

(Response 2) As noted above, we now provide information on the genetic background of the mice in the revised manuscript, where available. We have not backcrossed the constitutive DR6 knockout mice obtained from the Deppmann laboratory (Gamage et al.) to a C57BL/6 background; our colony was maintained primarily through intercrosses of heterozygous animals. Similarly, the conditional DR6 mutant mice used in this study were also not backcrossed to C57BL/6 mice.

We respectfully hold a different view regarding the reviewer’s final point. We understand it is not appropriate to infer consistency between two datasets by disregarding the subset of results that do not align. By the same logic, it would be flawed to draw conclusions from the Gamage et al. study based solely on the single Wld^S mouse out of five that did not show axon preservation after nerve injury. Selectively omitting conflicting data does not provide a valid basis for establishing phenotype concordance across studies.

To further strengthen our study, we note that we performed additional analyses on three more nerve samples from constitutive DR6 null mice during the revision process and have incorporated the resulting data in Fig. 1.

(3) Age is also an important variable. The protective effects of the spontaneous WldS mutation decrease with age, for example. It is unclear whether the possible protective effects of DR6 also change with age; perhaps this could explain the variable response seen in Gamage et al. and the lack of response seen here.

(Response 3) As discussed above, we now provide the age information for the mice used for the Wallerian degeneration assessments in the respective figure legends. To our knowledge, there are no prior reports suggesting that age is a significant determinant of structural axon preservation in the indicated mutants. Electrophysiological function and neuromuscular junction preservation decrease with age in axotomized Wld^S mice (e.g., PMIDs 12231635, 19158292, 15654865), but these parameters are not subject of our study, and we have not studied them. Unfortunately, a direct comparison of ages between our DR6 mutant mice and those used in Gamage et al. (2017) is not possible, as the earlier study from the Deppmann laboratory did not report this information.

(4) It is unclear if sex is a factor, but this is part of why it should be reported.

(Response 4) We now report the requested sex information for our axon preservation analyses during nerve injury-induced Wallerian degeneration in the DR6 mouse models in Figs. 1 and 2.

(5) The authors also state that they do not see differences in the Schwann cell response to injury in the absence of DR6 that were reported in Gamage et al., but this is not an accurate comparison. In Gamage et al., they examined Schwann cells around axons that were protected from degeneration 2 and 4 weeks post-injury. Those axons had much thinner myelin, in contrast to axons protected by WldS or loss of Sarm1, where the myelin thickness remained relatively normal. Thus, Gamage et al. concluded that the protection of axons from degeneration and the preservation of Schwann cell myelin thickness are separate processes. Here, since no axon protection was seen, the same analysis cannot be done, and we can only say that when axons degenerate, the Schwann cells respond the same whether DR6 is expressed or not.

(Response 5) We appreciate the reviewer’s detailed comments. Our intention was not to directly compare our findings with those of Gamage et al. regarding the myelin behavior at these time points (because we never observed axon protection), but rather to note that we did not observe any DR6-dependent alterations in Schwann cell responses under conditions where axons undergo normal Wallerian degeneration. As the reviewer correctly points out, Gamage et al. analyzed Schwann cell myelin surrounding axons that were protected from degeneration for extended periods, a context fundamentally different from the complete lack of axon protection observed in our DR6-deficient models. Therefore, the specific dissociation between axon preservation and myelin maintenance claimed by Gamage et al. cannot be evaluated in our study. A statement to make this point clearer has been incorporated in the revised manuscript.

We fully agree with the reviewer’s concluding point: in our experiments, once axons degenerate, Schwann cell responses proceed similarly regardless of DR6 expression. This agreement reinforces one of the central conclusions of our work.

(6) The authors also take issue with Colombo et al. (2018), where it was reported that there is an increase in axon diameter and a change in the g-ratio (axon diameter to fiber diameter - the axon + myelin) in peripheral nerves in DR6 knockout mice. This change resulted in a small population of abnormally large axons that had thinner myelin than one would expect for their size. The change in g-ratio was specific to these axons and driven by the increased axon diameter, not decreased myelin thickness, although those two factors are normally loosely correlated. Here, the authors report no changes in axon size or g-ratio, but this could also be due to how the distribution of axon sizes was binned for analysis, and looking at individual data points in supplemental figure 3A, there are axons in the DR6 knockout mice that are larger than any axons in wild type. Thus, this discrepancy may be down to specifics and how statistics were performed or how histograms were binned, but it is unclear if the results presented here are dramatically at odds with the results in Colombo et al. (2018).

(Response 6) Several points raised by the reviewer appear to reflect differences in interpretation of the findings reported in Colombo et al. (2018). That study did not report altered myelination in DR6 null mice at stages when myelination is largely complete (P21). Instead, modest changes were observed at P1, which were reduced by P7, and P21 mutants were reported to be indistinguishable from controls. No analyses of peripheral nerves in older animals were presented, and the authors concluded in the discussion that myelination in young adult DR6 null mice appears normal. In contrast, our analysis of constitutive DR6 null mice at P1 does not reproduce the increase in the number of myelinated fibers per unit area reported by Colombo et al. We obtained similar results in the independent conditional DR6 knockout mouse line. Differences in nerve tissue processing, embedding, staining, or in the microscopic imaging and quantification of thinly myelinated axons in P1 sciatic nerve cross-sections may have contributed to the observed discrepancy. However, because the relevant methodological details were not described in Colombo et al., the underlying reasons for these differences cannot be determined and remain speculative.

(7) Finally, it is important to note that previously reported effects of DR6 inhibition, such as protection of cultured cortical neurons from beta-amyloid toxicity, are not necessarily the same as Wallerian degeneration of axons distal to an injury studied here. The negative results presented here, showing that loss of DR6 is not protective against Wallerian degeneration induced by injury, are important given the interest in DR6 as a therapeutic target, but they are specific to these mice and this mechanism of induced axon degeneration. The extent to which these findings contradict previous work is difficult to assess due to the lack of detail in describing the mouse experiments, and care should be taken in attempting to extrapolate these results to other disease contexts, such as ALS or Alzheimer's disease.

(Response 7) We agree with the reviewer’s point and emphasize that our manuscript carefully differentiates our data regarding the function of DR6 in Wallerian degeneration from the potential involvement of DR6 in other forms of axon degeneration. Our findings do not conflict with previous work on DR6 in the context of in vitro beta-amyloid and prion toxicity as well as in vitro models of ALS and multiple sclerosis. We believe these distinctions are explicitly and appropriately articulated throughout the entire manuscript and in more detail in the discussion section.

Reviewer #1 (Recommendations for the authors):

(1) The authors should include additional information about the mice used, including strain background for both the DR6 mice and the Cre transgenes crossed into the DR6 conditional knockout, the age of the mice when the nerve crush experiments were performed, and the sex distributions of the experimental cohorts. This information is critical for reproducibility in animal experiments, and that point is compounded here, where the major focus of this paper is taking issue with the reproducibility of previous work.

(Response 8) This information has been included in the revision. See above responses.

(2) In the abstract, reference 5 is cited as a study on the response to Schwann cells to injury in a DR6 background, but this probably should be reference 10.

(Response 9) This typo has been corrected.

(3) "Site-by-site comparison" in line 201 should be side-by-side?

(Response 10) This typo has been corrected.

(4) The paper contains a lot of self-evaluative wording, "surprising contrast," "compelling evidence," "robust results." Whether those adjectives apply should be for the reader to decide, and a drier, more objective tone in the presentation would improve the paper.

(Response 11) We agree that excessive self-evaluative wording can weaken objectivity. In the manuscript, such phrasing is used sparingly and intentionally to highlight differences from previously published studies, guide the reader, and convey scholarly judgment. We do not consider this limited use to be counterproductive. The adjectives “surprising,” “compelling,” and “robust” each appear only one to three times across the entire manuscript, and the specific phrase “robust results” does not appear at all.

(5) In Figure 2A, DR6-/-, there is no significant difference, but there is also a lot of variability, and one could argue the authors are seeing axon protection comparable to WldS in 40% of their samples (2/5), which is very similar to Gamage et al.

(Response 12) We respectfully disagree with this reasoning as it relies on selectively emphasizing only a subset of the data. Please also see our response #2 for more detailed discussion.

(6) Overall, the data presented here are convincing and support the conclusions drawn, but the paper needs to focus more on the negative results at hand and less on bashing previous studies, particularly when the results presented here do definitively show that the previous studies were incorrect and plausible explanations for differences in outcome exist.

(Response 13) We have carefully revisited the wording of the manuscript and are confident that our emphasis remains on the central negative finding that DR6 does not regulate axon degeneration and Schwann cell injury responses during Wallerian degeneration. We do not believe the manuscript “bashes” previous studies; nonetheless, we thoroughly re-examined all relevant sections to ensure that our language is neutral, accurate, and non-inflammatory. We believe the current phrasing presents our interpretations in an appropriately balanced, objective, and professional manner.

Reviewer #2 (Public review):

Summary:

This manuscript by Beirowski, Huang, and Babetto revisits the proposed role of Death Receptor 6 (DR6/Tnfrsf21) in Wallerian degeneration (WD). A prior study (Gamage et al., 2017) suggested that DR6 deletion delays axon degeneration and alters Schwann cell responses following peripheral nerve injury. Here, the authors comprehensively test this claim using two DR6 knockout mouse models (the line used in the earlier report plus a CMV-Cre derived floxed ko line) and multiple WD assays in vivo and in vitro, aligned with three positive controls, Sarm1 WldS and Phr1/Mycbp2 mutants. Contrary to the prior findings, they find no evidence that DR6 deletion affects axon degeneration kinetics or Schwann cell dynamics (assessed by cJun expression or [intact+degenerating] myelin abundance after injury) during WD. Importantly, in DRG explant assays, neurites from DR6-deficient mice degenerated at rates indistinguishable from controls. The authors conclude that DR6 is dispensable for WD, and that previously reported protective effects may have been due to confounding factors such as genetic background or spontaneous mutations.

Strengths:

The authors employ two independently generated DR6 knockout models, one overlapping with the previously published study, and confirm loss of DR6 expression by qPCR and Western blotting. Multiple complementary readouts of WD are applied (structural, ultrastructural, molecular, and functional), providing a robust test of the hypothesis.

Comparisons are drawn with established positive controls (WldS, SARM1, Phr1/Mycbp2 mutants), reinforcing the validity of the assays.

By directly addressing an influential but inconsistent prior report, the manuscript clarifies the role of DR6 and prevents potential misdirection of therapeutic strategies aimed at modulating WD in the PNS. The discussion thoughtfully considers possible explanations for the earlier results, including colony-specific second-site mutations that could explain the incomplete penetrance of the earlier reported phenotype of only 36%.

Weaknesses:

(1) The study focuses on peripheral nerves. The manuscript frequently refers to CNS studies to argue for consistency with their findings. It would be more accurate to frame PNS/CNS similarities as reminiscences rather than as consistencies (e.g., line 205ff in the Discussion).

(Response 14) Axon protection in all key genetic models of delayed axon degeneration, including Wld^S, SARM1, Phr1/Mycbp2 mutants, has been demonstrated in both the peripheral and central nervous systems. This observation supports the view that core molecular mechanisms regulating axon degeneration are conserved across neuronal populations throughout the entire nervous system. We have scrutinized the wording in our manuscript and are not aware that we frequently refer to CNS studies in regards to axon degeneration. Nevertheless, we have replaced the term “consistent” to avoid potential ambiguity when we discuss the earlier study showing normal Wallerian degeneration in the optic nerves from DR6 knockout mice.

(2) The DRG explant assays are convincing, though the slight acceleration of degeneration in the DR6 floxed/Cre condition is intriguing (Figure 4E). Could the authors clarify whether this is statistically robust or biologically meaningful?

(Response 15) We thank the reviewer for noting this aspect of our in vitro data in Fig. 4. The difference observed in the DR6 floxed/Cre condition is statistically significant at the 6h time point following disconnection, as indicated by the p value shown in Fig. 4E. However, a similarly statistically significant acceleration of axon degeneration was not observed in DRG axotomy experiments using constitutive DR6 knockout preparations, although a trend toward more rapid axon breakdown is apparent at 6 h post-axotomy (Fig. 4B). These observations may suggest reduced stability of DR6-deficient axons in this specific neuron-only in vitro context. Further investigation would be required to determine the biological significance of this effect. In contrast, our in vitro quantitative analyses of the initiation and early phases of Wallerian degeneration (Fig. 2) revealed no evidence of accelerated axon disintegration in the DR6 mutant mouse models, highlighting potential differences between in vitro and in vitro systems.

(3) In the summary (line 43), the authors refer to Hu et al. (2013) (reference 5) as the study that previously reported AxD delay and SC response alteration after injury. However, this study did not investigate the PNS, and I believe the authors intended to reference Gamage et al. (2017) (reference 10) at this point.

(Response 16) Thanks for pointing this out. We have corrected this typo in the revised manuscript.

(4) In line 74ff of the results section, the authors claim that developmental myelination is not altered in DR6 mutants at postnatal day 1. However, the variability in Figure S2 appears substantial, and the group size seems underpowered to support this claim. Colombo et al. (2018) (reference 11) reported accelerated myelination at P1, but this study likewise appears underpowered. Possible reasons for these discrepancies and the large variability could be that only a defined cross-sectional area was quantified, rather than the entire nerve cross-section.

(Response 17) We confirm that the quantification of thinly myelinated axons was performed on entire sciatic nerves from P1 mouse pups, as described in the methods section in our original manuscript. The data shown in Fig. S2 were obtained from 5-9 pups per experimental group. Sample sizes were determined based on a priori power analyses using pilot data, which indicated that a minimum of five biological replicates was sufficient to detect statistically significant differences with acceptable confidence. Comparable sample sizes have been used in our previous studies and by other groups to assess early postnatal myelination (e.g., PMIDs 21949390, 28484008). Several published studies have reported analyses using 3-4 animals per group (e.g., PMIDs 28484008, 25310982, 29367382). For comparison, the study by Colombo et al. used 3-8 pups for the analysis presented in their Fig. 3. We note that the apparent variability in Fig. S2 may be accentuated by the scaling of the y-axis, which was chosen to ensure that individual data points are clearly resolved and visible.

(5) The authors stress the data of Gamage et al. (2017) on altered SC responses in DR6 mutants after injury. They employed cJun quantification to show that SC reprogramming after injury is not altered in DR6 mutants. This approach is valid and the conclusion trustworthy. Here, the addition of data showing the combined abundance of intact and degenerated myelin does not add much insight. However, Gamage et al. (2017) reported altered myelin thickness in a subset of axons at 14 days after injury, which is considerably later than the time points analyzed in the present study. While, in the Reviewer's view, the thin myelin observed by Gamage et al. in fact resembles remyelination, the authors may wish to highlight the difference in the time points analyzed.

(Response 18) We consider the additional quantification of the area occupied by intact myelin and myelin debris to provide complementary information that supports the c-Jun-based conclusion that Schwann cell injury responses are normal in DR6-deficient nerves following lesion. We agree with this reviewer that the thin myelin observed by Gamage et al. resembles remyelination, raising the possibility that axon regeneration occurred into the distal nerve stump at the studied 14d post-injury time point (see their Fig. 3). This may have been interpreted as axon protection in this study. In our study, it was impossible to examine such myelin effects since axon protection was never observed in any of the DR6 mutant models at any of the time point we investigated. We have incorporated appropriate additional text to highlight this difference. See also response #5 above.

Reviewer #3 (Public review):

Summary:

The authors revisit the role of DR6 in axon degeneration following physical injury (Wallerian degeneration), examining both its effects on axons and its role in regulating the Schwann cell response to injury. Surprisingly, and in contrast to previous studies, they find that DR6 deletion does not delay the rate of axon degeneration after injury, suggesting that DR6 is not a mediator of this process.

Overall, this is a valuable study. As the authors note, the current literature on DR6 is inconsistent, and these results provide useful new data and clarification. This work will help other researchers interpret their own data and re-evaluate studies related to DR6 and axon degeneration.

Strengths:

(1) The use of two independent DR6 knockout mouse models strengthens the conclusions, particularly when reporting the absence of a phenotype.

(2) The focus on early time points after injury addresses a key limitation of previous studies. This approach reduces the risk of missing subtle protective phenotypes and avoids confounding results with regenerating axons at later time points after axotomy.

Weaknesses:

(1) The study would benefit from including an additional experimental paradigm in which DR6 deficiency is expected to have a protective effect, to increase confidence in the experimental models, and to better contextualize the findings within different pathways of axon degeneration. For example, DR6 deletion has been shown in more than one study to be partially axon protective in the NGF deprivation model in DRGs in vitro. Incorporating such an experiment could be straightforward and would strengthen the paper, especially if some of the neuroprotective effects previously reported are confirmed.

(Response 19) We thank the reviewer for these suggestions. We would like to highlight that our study addresses the role of DR6 in Wallerian degeneration, whereas in vitro NGF deprivation has been used to model developmental axon pruning. Previous work indicates fundamental biological differences between these regressive pathways regulating the stereotyped removal of axon segments. We feel that studying this alternative form of axon degeneration is beyond the scope of the current work and could be addressed in a separate manuscript. Although additional tests will be needed, we note that our preliminary data using samples from both DR6 knockout mouse models suggest no axon protection after NGF-deprivation in DRG neuron preparations in our hands (deprivation of the growth factor and administration of anti-NGF antibody).

(2) The quality of some figures could be improved, particularly the EM images in Figure 2. As presented, they make it difficult to discern subtle differences.

(Response 20) We have pseudocolored intact (turquoise) and degenerated (magenta) myelinated fibers on the high-resolution semithin micrographs (not electron micrographs) in the new Fig. 2 to make the distinction between the two fiber categories clearer.

Reviewer #3 (Recommendations for the authors):

(1) Line 121: The authors mention toluidine blue staining, but it does not appear to be shown in Figure S5.

(Response 21) This appears to be a misunderstanding. Fig. S5A shows the ultrastructure of dedifferentiated Schwann cells in transmission electron micrographs, while Figs. S5B and C show quantification of the area occupied by myelin sheaths and myelin debris profiles on osmium tetroxide and toluidine blue stained nerve sections from the two DR6 mutant models, based on semithin light microscopy. These are two different aspects of the analysis. The text has been modified in the revised manuscript to make the distinction clearer.

(2) Line 175: The authors should add NMNAT2 to the list of enzymes implicated in the regulation of Wallerian degeneration in mammals.

(Response 22) Nmnat2 and a literature reference (Milde et al., 2013) has been incorporated in the discussion of the revised manuscript to address this point.

(3) Line 201: Please correct the typo "site-by-site" to "side-by-side."

(Response 23) This typo has been corrected.

Author response:

We appreciate that the reviewers provided an overall positive assessment of our manuscript and offered constructive suggestions for improvement. All three reviewers noted that a key strength of our study is the implementation of a gut microbiome model for the characterization of interbacterial antagonism pathways such as the type VI secretion system (T6SS) that approaches natural complexity. They note our work represents a significant advance in microbiome research, and generates resources that will be of use to many researchers in the field. Two of the reviewers point out that the complexity of our model limits the nature of measurements we can make, and suggest we temper the strength of the some of the conclusions we draw. As noted in more detail below, in our revised manuscript, we will be more precise in the wording we use to characterize our findings, and we will be more explicit about what the measurements we are able to make allow us to conclude about the physiological role of the T6SS in the gut microbiome.

Reviewer #1 (Public review):

Summary:

In this study, the authors investigate the physiological role of the Type VI secretion system (T6SS) in a naturally evolved gut microbiome derived from wild mice (the WildR microbiome). Focusing on Bacteroides acidifaciens, the authors use newly developed genetic tools and strain-replacement strategies to test how T6SS-mediated antagonism influences colonization, persistence, and fitness within a complex gut community. They further show that the T6SS resides on an integrative and conjugative element (ICE), is distributed among select community members, and can be horizontally transferred, with context-dependent effects on colonization and persistence. The authors conclude that the T6SS stabilizes strain presence in the gut microbiome while imposing ecological and physiological constraints that shape its value across contexts.

This study is likely to have a significant impact on the microbiome field by moving experimental tests of T6SS function out of simplified systems and into a naturally co-evolved gut community. The WildR system, together with the strain replacement strategy, ICE-seq approach, and genetic toolkit, represents a powerful and reusable platform for future mechanistic studies of microbial antagonism and mobile genetic elements in vivo.

The datasets, including isolate genomes, metagenomes, and ICE distribution maps, will be a valuable community resource, particularly for researchers interested in strain-resolved dynamics, horizontal gene transfer, and ecological context dependence. Even where mechanistic resolution is incomplete, the work provides a strong experimental foundation upon which such questions can be directly addressed.

Overall, this study occupies a space between system building and mechanistic dissection. The authors demonstrate that the T6SS influences persistence and community structure in vivo, but the physiological basis of these effects remains unresolved. Interpreting the results as evidence of fitness costs or selective advantage, therefore, requires caution, as multiple ecological and host-mediated processes could produce similar abundance trajectories.

Placing the findings within the broader literature on microbial antagonism, particularly work emphasizing measurable costs, benefits, and tradeoffs, would help readers better contextualize what is directly demonstrated here versus what remains an open question. Viewed in this light, the principal contribution of the study is to show that such questions can now be addressed experimentally in a realistic gut ecosystem.

We thank the reviewer for this thoughtful summary of our study. We were glad to read they conclude our work will have a significant impact on the microbiome field and that the resources we have developed will be of value to the community.

Strengths:

A major strength of this study is that it directly interrogates the physiological role of the T6SS in a naturally evolved gut microbiome, rather than relying on simplified pairwise or in vitro systems. By working within the WildR community, the authors advance beyond descriptive surveys of T6SS prevalence and address function in an ecologically relevant context.

The authors provide clear genetic evidence that Bacteroides acidifaciens uses a T6SS to antagonize co-resident Bacteroidales, and that loss of T6SS function specifically compromises long-term persistence without affecting initial colonization. This temporal separation is well designed and supports the conclusion that the T6SS contributes to maintenance rather than establishment within the community.

Another strength is the identification of the T6SS on an integrative and conjugative element (ICE) and the demonstration that this element is distributed among, and exchanged between, community members. The use of ICE-seq to track distribution and transfer provides strong support for horizontal mobility and adds mechanistic depth to the study.

Finally, the transfer of the T6SS-ICE into Phocaeicola vulgatus and the observation of context-dependent colonization benefits followed by decline is a compelling result that moves the study beyond simple "T6SS is beneficial" narratives and highlights ecological contingency.

We appreciate this detailed and nuanced characterization of the strengths of our study.

Weaknesses:

Despite these strengths, there is a mismatch between the precision of the claims and the precision of the measurements, particularly regarding fitness costs, physiological burden, and the mechanistic role of the T6SS.

We acknowledge that in some places, our manuscript could benefit from greater precision in the language we use when linking the outcomes we observe in our study to their potential underlying causes. Specific revisions we propose to address this concern are described below.

First, while the authors conclude that the T6SS "stabilizes strain presence" and that its value is constrained by fitness costs, these costs are not directly measured. Persistence, abundance trajectories, and eventual loss are informative outcomes, but they do not uniquely identify fitness tradeoffs. Decline could arise from multiple non-exclusive mechanisms, including community restructuring, host-mediated effects, incompatibilities of the ICE in new hosts, or ecological retaliation, none of which are disentangled here.

We agree that multiple mechanisms could explain why P. vulgatus carrying a T6SS-encoding ICE declines over time. Our use of the term “fitness cost” to describe this trend was not meant to imply any particular underlying mechanism, but was rather our attempt to characterize the phenotypic outcome we observed in simplified terms. We note that ecological context is an important determinant of the fitness cost or benefit of any given trait, and our study sheds light on the importance of the presence of the WildR community and the mouse intestinal environment to the fitness contribution of the ICE to P. vulgatus. Nonetheless, to avoid implying an overly simplistic interpretation of our results, we propose to modify the language used in the manuscript when describing the contribution of the T6SS to species persistence in WildR-colonized mice.

Second, the manuscript frames the T6SS as having a defined physiological role, yet the data do not resolve which physiological processes are under selection. The experiments demonstrate that T6SS activity affects persistence, but they do not distinguish whether this occurs via direct killing, resource release, niche modification, or higher-order community effects. As a result, "physiological role" remains underspecified and risks being conflated with ecological outcome.

We acknowledge that our study does not fully resolve the physiological processes under selection that mediate role of the T6SS in maintaining B. acidifaciens populations in WildR-colonized mice. Indeed, several of the outcomes of T6SS activity the reviewer lists, such as target cell killing and nutrient release, are inextricably linked and thus inherently difficult to disentangle. We note that we did attempt to measure higher-order community effects of T6SS activity with metagenomic sequencing, but acknowledge that this approach may not have been sufficiently sensitive to detect small community shifts mediated by a relatively low-abundance species. To address the concern that our current framing implies more of a mechanistic understanding that our study achieves, we propose to substitute “ecological” for “physiological” where appropriate when summarizing our key findings.

Third, although the authors emphasize context dependence, the study offers limited quantitative insight into what aspects of context matter. Differences between native and recipient hosts, or between early and late colonization phases, are described but not mechanistically interrogated, making it difficult to generalize beyond the specific cases examined.

We are not entirely clear what the reviewer means by “differences between native and recipient hosts”, but we agree that additional quantitative studies will be needed to address the generalizability of our findings. Future studies are also needed to address the mechanistic basis for the difference in the benefit conferred by the T6SS that we observed between P. vulgatus and B. acidifaciens.

Fourth is the lack of engagement with recent experimental literature demonstrating functional roles of the T6SS beyond simple interference competition. While the authors focus on persistence and competitive outcomes, they do not adequately situate their findings within recent work demonstrating that T6SS-mediated antagonism can serve additional physiological functions, including resource acquisition and DNA uptake, thereby linking killing to measurable benefits and tradeoffs. The absence of this literature makes it difficult to place the authors' conclusions about physiological role and fitness cost within the current conceptual framework of the field. Without this context, the physiological interpretation of the results remains incomplete, and alternative functional explanations for the observed dynamics are underexplored.

We thank the reviewer for specifically highlighting the potential pertinence of this literature to our study. Indeed, we did not cite studies indicating a link between T6SS activity and the uptake of DNA and other resources released by targeted cells. As we note above, the release of intracellular contents from target cells is an inevitable consequence of the delivery of lytic effectors. Thus, distinguishing between fitness benefits conferred from the elimination of competitor species and those arising from scavenging the nutrients released during this process is not straightforward. Measuring the benefits deriving from the uptake of certain released molecules, such as DNA, was not immediately feasible in the system employed in this study and instead we focused on the direct lytic consequences of the effectors delivered via the T6SS. We will revise our Discussion to include reference to how downstream consequences of T6SS activity on target cells could impact the community, and thus the adaptive role of the T6SS in the microbiome.

A further limitation concerns the taxonomic scope of the functional analysis. The authors state that the role of the T6SS in the murine environment is functionally investigated using genetically tractable Bacteroides species, citing the lack of genetic tools for Mucispirillum schaedleri. While this is a reasonable, practical choice, it means that a substantial fraction of T6SS-encoding species in the WildR community are not experimentally interrogated. Consequently, conclusions about the role of the T6SS in the murine gut necessarily reflect the subset of taxa that are genetically accessible and may not fully capture community-level or niche-specific functions of T6SS activity. Given that M. schaedleri is represented as a metagenome-assembled genome, its isolation and genetic manipulation would be technically challenging. Nonetheless, explicitly acknowledging this limitation and slightly tempering claims of generality would strengthen the manuscript.

The reviewer points out that studying the T6SS activity in M. schadleri would potentially expand the generality of our claims. We agree that having an isolate of this species along with genetic tools for its manipulation would allow us to probe the importance of the T6SS in the gut microbiome more broadly. At the suggestion of the reviewer, we will add explicit mention for the need to develop such tools, an endeavor that lies outside of the scope of the current study.

Finally, several interpretations would benefit from more cautious language. In particular, claims invoking fitness costs, selective advantage, or physiological burden should be explicitly framed as inferences from persistence dynamics, rather than as direct measurements, unless supported by additional quantitative fitness or growth assays.

We agree with the reviewer that invoking fitness costs, selective advantages or physiological burdens should be done cautiously, and in our revised manuscript we will carefully re-evalute our usage of those terms. However, we would also argue invoking fitness costs and benefits when describe strain persistence dynamics in mice has substantial precedent in the literature ((Feng et al. 2020, Brown et al. 2021, Park et al. 2022, Segura Munoz et al. 2022), to list a handful of representative examples published by different groups). It is unclear to us what additional in vivo growth measurements could be taken to substantiate our claim that the T6SS provides a fitness benefit to B. acidifaciens during prolonged gut colonization, or that carrying the ICE imposes a fitness cost on P. vulgatus during long-term colonization. Our in vitro experiments evaluating the competitiveness conferred by T6SS activity provide a measure of insight into its fitness benefits, but as our in vivo strain persistence data and the work of many others show, in vitro measurements do not necessarily capture in vivo parameters.

Reviewer #2 (Public review):

Summary:

In this study, the authors set out to determine how a contact-dependent bacterial antagonistic system contributes to the ability of specific bacterial strains to persist within a complex, native gut community derived from wild animals. Rather than focusing on simplified or artificial models, the authors aimed to examine this system in a biologically realistic setting that captures the ecological complexity of the gut environment. To achieve this, they combined controlled laboratory experiments with animal colonization studies and sequencing-based tracking approaches that allow individual strains and mobile genetic elements to be followed over time.

Strengths:

A major strength of the work is the integration of multiple complementary approaches to address the same biological question. The use of defined but complex communities, together with in vivo experiments, provides a strong ecological context for interpreting the results. The data consistently show that the antagonistic system is not required for initial establishment but plays a critical role in long-term strain persistence. This insight that moves beyond traditional invasion-based views of microbial competition. The observation that transferable genetic elements can confer only temporary advantages, and may impose longer-term costs depending on community context, adds important nuance to current understanding of microbial fitness.

We thank the reviewer for the positive feedback and are glad they agree our study provides new insight into the role of interbacterial antagonism in natural communities.

Weaknesses:

Overall, there is not a lack of evidence, but a deliberate trade-off between ecological realism and mechanistic resolution, which leaves some causal pathways open to interpretation.

The reviewer makes a good point that the complexity of the experimental system we employ precludes some lines of experimentation that would yield more mechanistic information. As the reviewer notes, we were aware of the tradeoff between mechanistic resolution and ecological realism when selecting our experimental system. Our deliberate choice to favor biological complexity over mechanistic clarity in this study stemmed from our perception that a major gap in understanding of the T6SS and other antagonism pathways lies in defining their ecological function in complex microbial communities.

Reviewer #3 (Public review):

Summary:

Shen et al. investigate the contribution of the type VI secretion system of Bacteroidales in the gut microbiome assembly and targeting of closely related species. They demonstrate that B. acidifaciens relies on T6SS-mediated antagonism to prevent displacement by co-resident Bacteroidales and other members of the microbiome, allowing B. acidifaciens to persist in the gut.

Strengths:

Using a gnotobiotic model colonized with a wild-mouse microbiome is a significant strength of this study. This approach allows tracking of microbiome changes over time and directly examining targeting by Bacteroidales carrying T6SS in a more natural setting. The development of ICE-seq for mapping the distribution of the T6SS in the microbiome is remarkable, enabling the study of how this bacterial weapon is transferred between microbiome members without requiring long-read metagenomics methods.

We thank the reviewer for their enthusiasm toward our study.

Weaknesses:

Some conclusions are based on only four mice per condition. The author should consider increasing the sample size.

We agree that in some experiments it would be beneficial to increase the sample size from four mice. However, the experiments we performed for this study are time and resource intensive. Additionally, the experiments on which we base our primary conclusions were all independently replicated with similar results. Given these factors, we determined that the extra confidence that might be afforded by increasing our sample size did not merit the delay in publication and investment in resources that would be required.

Overall, the authors successfully achieved their objectives, and their experimental design and results support their findings. As mentioned in the discussion, it would be important to investigate the role of the T6SS in resilience to disturbances in the microbiome, such as antibiotics, diet, or pathogen invasion. This work represents a step forward in understanding how contact-dependent competition influences the gut microbiome in relevant ecological contexts.

We agree that investigating the role of the T6SS during perturbations of the microbiome is a key next step for this work and thank the reviewer for highlighting this important future direction.

References

Brown, E. M., H. Arellano-Santoyo, E. R. Temple, Z. A. Costliow, M. Pichaud, A. B. Hall, K. Liu, M. A. Durney, X. Gu, D. R. Plichta, C. A. Clish, J. A. Porter, H. Vlamakis and R. J. Xavier (2021). "Gut microbiome ADP-ribosyltransferases are widespread phage-encoded fitness factors." Cell Host Microbe 29(9): 1351-1365 e1311.

Feng, L., A. S. Raman, M. C. Hibberd, J. Cheng, N. W. Griffin, Y. Peng, S. A. Leyn, D. A. Rodionov, A. L. Osterman and J. I. Gordon (2020). "Identifying determinants of bacterial fitness in a model of human gut microbial succession." Proc Natl Acad Sci U S A 117(5): 2622-2633.

Park, S. Y., C. Rao, K. Z. Coyte, G. A. Kuziel, Y. Zhang, W. Huang, E. A. Franzosa, J. K. Weng, C. Huttenhower and S. Rakoff-Nahoum (2022). "Strain-level fitness in the gut microbiome is an emergent property of glycans and a single metabolite." Cell 185(3): 513-529 e521.

Segura Munoz, R. R., S. Mantz, I. Martinez, F. Li, R. J. Schmaltz, N. A. Pudlo, K. Urs, E. C. Martens, J. Walter and A. E. Ramer-Tait (2022). "Experimental evaluation of ecological principles to understand and modulate the outcome of bacterial strain competition in gut microbiomes." ISME J 16(6): 1594-1604.

La Bienveillance en Milieu Scolaire : Enjeux, Défis et Pratiques Professionnelles

Résumé Exécutif

Ce document synthétise les échanges issus d'une table ronde portant sur le concept de bienveillance à l'école.

Loin d'être synonyme de laxisme ou de complaisance, la bienveillance est définie comme une condition essentielle de l'équité et de l'efficacité du système éducatif, particulièrement pour les élèves les plus vulnérables.

Elle repose sur une tension constructive entre exigence et soutien, visant le développement à long terme de l'élève.

Sa mise en œuvre nécessite une clarification conceptuelle pour lever les résistances professionnelles, l'adoption de gestes professionnels spécifiques (feedback positif, écoute active) et une réinvention des espaces et des modalités d'évaluation.

En somme, la bienveillance est un levier de réussite qui engage tant la posture individuelle de l'enseignant que la stratégie collective de l'établissement.

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1. Définition et Clarification Conceptuelle

La bienveillance en éducation souffre souvent de représentations simplistes ou erronées. Il est nécessaire d'en préciser les contours épistémologiques :

• Étymologie et intention : Littéralement, la bienveillance consiste à « vouloir du bien à autrui ».

C'est une disposition favorable qui vise la réussite et la réalisation personnelle de l'autre.

• Temporalité (Court terme vs Long terme) : La bienveillance peut impliquer de sacrifier le confort immédiat pour le bien de l'élève à long terme.

Ainsi, la fermeté, l'exigence ou même une sanction peuvent être des actes bienveillants s'ils sont explicités et pratiqués dans le respect de l'élève.

• Distinction fondamentale : Elle ne doit pas être confondue avec :

• ◦ Le laxisme.  
• ◦ La complaisance.   
• ◦ La mansuétude.

• Cadre institutionnel : La notion est devenue une valeur centrale de l'Éducation nationale depuis la circulaire de 2014, bien qu'elle fût déjà présente dans le secteur privé et les services publics.

La DGESCO (2013) l'associe à un ensemble d'attitudes physiques, morales et psycho-affectives positives et constantes (respect, confiance, encouragement).

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2. Analyse des Résistances Professionnelles

Malgré un consensus apparent, le terme suscite des tensions sur le terrain :

| Type de résistance | Argumentation observée | | --- | --- | | Sentiment de jugement | Certains enseignants perçoivent l'injonction à la bienveillance comme une critique de leurs pratiques passées, sous-entendant qu'ils ne l'auraient pas été auparavant. | | Opposition à l'exigence | Une crainte que l'attention portée au bien-être des élèves ne se fasse au détriment de l'effort nécessaire à la réussite académique. | | Crise de l'autorité | La bienveillance est parfois vue comme une entrave à l'autorité face à des manquements disciplinaires chroniques. | | Complexité systémique | La multiplication des élèves à besoins éducatifs particuliers (EBEP) met les équipes sous pression, rendant la posture bienveillante difficile à maintenir sans formation adéquate. |

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3. Les Gestes Professionnels de la Bienveillance

La bienveillance se traduit par des actes concrets et une posture éthique dans la relation pédagogique :

La puissance du Feedback

Le levier le plus efficace pour la réussite des élèves est le feedback positif. Il doit être :

• Centré sur l'activité et la méthodologie de l'élève.

• Formulé chaleureusement.

• Porteur de confiance et d'espoir dans les capacités de l'élève.

L'attention aux signaux de vulnérabilité

Le professionnel bienveillant doit être attentif aux signes de fragilité qui peuvent mener au décrochage :

• Signes de découragement ou discours négatif sur l'école.

• Absentéisme et arythmies.

• Sentiments d'insécurité (peur de prendre la parole, honte).

• Mutisme, isolement ou passages fréquents à l'infirmerie/vie scolaire.

Une communication renouvelée

L'horizontalité et l'authenticité sont cruciales pour les nouvelles générations :

• Passer d'un rôle purement académique à une relation de personne à personne.

• Pratiquer l'écoute active (savoir se taire pour laisser l'élève s'exprimer).

• Faire preuve de transparence et de prévisibilité.

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4. Domaines d'Application et Leviers d'Action

L'Évaluation comme espace de sécurisation

L'évaluation est une source majeure de stress (environ 60 % des élèves se disent angoissés par les évaluations). Une évaluation bienveillante implique :

• La suppression de l'implicite.

• Le droit à l'erreur et à la remédiation (possibilité de recommencer).

• Un cadre rassurant qui ne sacrifie pas l'exigence intellectuelle.

La lutte contre le harcèlement (Programme PHARE)

La bienveillance s'incarne dans la création d'une « communauté protectrice » :

• Utilisation de la méthode de la « préoccupation partagée ».

• Recherche d'alternatives à la sanction punitive immédiate pour l'intimidateur, en visant le développement de compétences psychosociales.

La transformation des espaces

La bienveillance passe par une réflexion sur le cadre de vie :

• Création de « jardins zen » ou de salles de calme.

• Réinvention des salles d'étude (espaces de coworking, possibilité de travailler debout ou dans des fauteuils).

• Mise en place de dispositifs permettant le mouvement (ballons, vélos-bureaux) pour favoriser la concentration, notamment des élèves hyperactifs.

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5. Éthique et Pilotage : La Bienveillance Collective

La bienveillance ne doit pas être une initiative isolée mais une stratégie d'établissement.

• Le rôle du diagnostic : Utiliser l'auto-évaluation (domaine du climat scolaire et du bien-être) pour identifier les besoins réels des élèves et des familles, en évitant les solutions préconçues.

• La Qualité de Vie au Travail (QVT) : Il existe un lien direct entre le bien-être des personnels et celui des élèves. Un encadrement bienveillant (feedback positif du chef d'établissement, convivialité, confiance déléguée) favorise l'engagement des équipes.

• L'éthique de la rencontre : S'intéresser à la singularité de ce que vit l'élève, au-delà de ses difficultés scolaires. Comme le souligne la sociologie, l'éducation par la rencontre est un levier de raccrochage puissant.

• Le partage et la convivialité : Des actions simples, comme le partage de nourriture (fruits en libre-service, repas de Noël partagé entre agents, élèves et chefs étoilés), peuvent transformer radicalement la relation sociale au sein d'un établissement.

Conclusion sur l'autorité bienveillante

L'autorité et la bienveillance sont compatibles. L'autorité s'exerce de manière éthique lorsqu'elle respecte l'intégrité morale de l'élève.

La sincérité de l'adulte, y compris dans l'expression de ses propres limites ou l'admission d'une erreur, renforce paradoxalement sa légitimité auprès des jeunes.

DOI: 10.1016/j.molcel.2026.01.022

Resource: cryoSPARC (RRID:SCR_016501)

Curator: @scibot

SciCrunch record: RRID:SCR_016501

What is this?

DOI: 10.1016/j.immuni.2026.01.011

Resource: RStudio (RRID:SCR_000432)

Curator: @scibot

SciCrunch record: RRID:SCR_000432

What is this?

L’Évolution du Rôle Paternel : Fondements Biologiques, Neurobiologiques et Sociaux

Résumé Exécutif

Ce document analyse la transformation du rôle des pères à travers le prisme de l'anthropologie évolutionniste et des neurosciences.

Contrairement aux autres grands singes africains où le soin paternel est quasi inexistant, l'être humain a développé une capacité unique de "coopérative breeding" (élevage coopératif).

Cette évolution, dictée par les contraintes climatiques du Pléistocène et l'augmentation massive du volume cérébral des nourrissons, a nécessité l'implication des pères et d'autres membres du groupe pour assurer la survie de l'espèce.

Les recherches récentes démontrent que les hommes disposent de circuits neuronaux et hormonaux ancestraux, partagés avec d'autres vertébrés, qui s'activent lors d'un contact prolongé et intime avec le nouveau-né.

L'éloignement historique des pères du soin direct est identifié non pas comme une barrière biologique, mais comme une construction sociale datant de la révolution néolithique.

La réengagement des pères aujourd'hui présente des bénéfices psychologiques et physiologiques majeurs, tant pour l'enfant que pour le parent.

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I. Le Paradoxe Évolutionnaire de la Paternité Humaine

L'exception humaine chez les primates

Dans la lignée des grands singes africains dont l'humain descend, le soin des nourrissons est traditionnellement l'apanage exclusif des mères.

Chez la plupart des 5 500 espèces de mammifères, l'investissement mâle est rare, limité souvent aux espèces monogames où la certitude de paternité est élevée.

La rupture du Pléistocène

L'émergence du soin paternel humain s'enracine dans les conditions extrêmes du Pléistocène en Afrique :

• Contraintes environnementales : Un climat devenant plus frais et sec, marqué par des précipitations instables.

• Le coût du cerveau : Le cerveau humain a triplé de volume, passant de 900 cm³ chez Homo erectus à 1 350 cm³ chez Homo sapiens.

• La dépendance du nourrisson : Les bébés humains sont les plus "coûteux" de la planète en termes de ressources et de temps de maturation.

Une mère seule ne pouvait assurer leur survie sans aide extérieure (soins alloparentaux et paternels).

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II. Mécanismes Neurobiologiques et Hormonaux

Les pères ne sont pas biologiquement "désarmés" face aux soins des nourrissons ; ils possèdent un équipement physiologique latent qui s'active sous certaines conditions.

Transformations Hormonales

Lorsqu'un père s'occupe activement d'un bébé, son profil hormonal se modifie de manière significative :

• Prolactine : Les niveaux augmentent, favorisant les réponses de soin (une hormone originellement liée à la lactation chez la mère).

• Ocytocine : Des poussées de cette "hormone du lien" sont observées chez les pères impliqués.

• Testostérone : On observe une diminution du taux de testostérone, facilitant une attitude plus douce et attentive.

Activation Cérébrale

Une étude de 2014 (Ruth Feldman et son équipe) a révélé des données cruciales sur les pères agissant comme soignants principaux :

• Activation de l'amygdale : Chez les pères en contact prolongé et intime avec leur bébé dès la naissance, l'activation de l'amygdale (zone liée à la vigilance et au soin maternel) est quatre fois supérieure à celle des pères n'apportant qu'une aide secondaire.

• Anciens circuits : Ces circuits neuronaux sont hautement conservés et se trouvent dans des zones primitives du cerveau (hypothalamus), similaires à celles activées chez les mères.

| Hormone / Zone | Effet chez le père impliqué | | --- | --- | | Prolactine | Augmentation des réponses de soin et d'attention. | | Ocytocine | Renforcement du lien affectif et de l'affiliation. | | Testostérone | Diminution des niveaux circulants. | | Amygdale | Vigilance accrue et réaction immédiate aux besoins du bébé. |

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III. Les Racines Ancestrales : De l'Eau à la Terre

L'anthropologie évolutionniste suggère que les capacités de soin mâle sont bien plus anciennes que les mammifères eux-mêmes.

• L'héritage des poissons : Il y a 400 millions d'années, certains poissons mâles pratiquaient déjà le soin des œufs (ex: le cichlidé du Tanganyika ou le poisson-mâchoire qui porte sa progéniture dans sa bouche).

• La plasticité de la Prolactine : À l'origine, cette hormone servait à réguler l'équilibre hydrique chez les organismes aquatiques avant d'être "réutilisée" par l'évolution pour les fonctions de reproduction et de soin.

• Conservation génétique : Les gènes responsables de la production de molécules comme l'isotocine chez les poissons sont les précurseurs directs de l'ocytocine chez les mammifères.

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IV. La Construction Sociale de la Masculinité

Si la biologie prédispose les hommes au soin, l'histoire récente a créé une rupture.

Le tournant du Néolithique

L'adoption de l'agriculture et de l'élevage a transformé les structures sociales :

• Apparition de la propriété : La nécessité de protéger les terres et les troupeaux a favorisé l'émergence de sociétés patriarcales.

• Redéfinition de la masculinité : L'identité masculine s'est déplacée vers la protection des institutions, du statut et de la propriété, éloignant physiquement les hommes de la sphère domestique et des nourrissons.

• Institutions patrilinéaires : Ces structures ont perduré jusqu'à l'époque moderne, excluant souvent les femmes et confinant les hommes à des rôles de pourvoyeurs distants.

La "Sélection Sociale" par le bébé

Le nourrisson humain a lui-même évolué pour encourager ce soin. Dès son plus jeune âge, le bébé utilise son cortex préfrontal médial pour :

• Monitorer son entourage.

• Apprendre à s'ingratiatier et attirer l'attention.

• Compétiter pour obtenir des soins alloparentaux par son attrait physique et comportemental.

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V. Implications et Bénéfices Modernes

Le réengagement des pères dans les soins primaires n'est pas seulement un retour à une nécessité biologique ancienne, mais un levier de santé publique.

• Santé mentale et longévité : Les relations de soin augmentent l'espérance de vie et réduisent les risques de dépression.

• Lutte contre les addictions : Les circuits de la récompense (dopamine) activés par l'amour parental sont les mêmes que ceux sollicités par les drogues.

Un engagement profond envers un enfant pourrait agir comme un protecteur contre les "décès par désespoir" et les addictions.

• Sens et finalité : Le soin direct apporte un sentiment immédiat d'utilité et de but, contrecarrant la solitude et l'aliénation sociale.

"Les mâles d'aujourd'hui, lorsqu'ils sont en contact suffisant et en proximité intime prolongée avec les bébés, possèdent l'équipement nécessaire. Ils sont aussi équipés pour s'occuper des bébés que les mères le sont."

[Paper-level Aggregated] PMCID: PMC4675689

Evidence Type(s): Oncogenic, Predictive, Functional

Justification: Oncogenic: The D835 mutations are reported to cause FLT3 TKI resistance in patients, indicating their role in tumorigenesis and cancer progression. Predictive: The study suggests that specific D835 mutations can predict the level of resistance to type II FLT3 inhibitors, which can inform treatment decisions. Functional: The text discusses the functional implications of D835 mutations on the stability of the DFG-out conformation and their impact on inhibitor binding, demonstrating the functional consequences of these mutations.

Gene→Variant (gene-first): FLT3(2322):D835 FLT3(2322):D835A/E FLT3(2322):D835H FLT3(2322):D835V/Y FLT3(2322):D835E/N FLT3(2322):D835N/E FLT3(2322):D835Y/V

Genes: FLT3(2322)

Variants: D835 D835A/E D835H D835V/Y D835E/N D835N/E D835Y/V

[Paragraph-level] PMCID: PMC4675689 Section: RESULTS PassageIndex: 2

Evidence Type(s): Predictive, Oncogenic

Justification: Predictive: The passage discusses how various D835 substitutions correlate with resistance to FLT3 inhibitors, indicating their predictive value regarding treatment response. Oncogenic: The D835 mutations are reported to cause resistance in patients, suggesting that these somatic variants contribute to tumor development or progression.

Gene→Variant (gene-first): 2322:D835 2322:D835A/E 2322:D835H 2322:D835V/Y

Genes: 2322

Variants: D835 D835A/E D835H D835V/Y

[Paragraph-level] PMCID: PMC4675689 Section: RESULTS PassageIndex: 2

Evidence Type(s): Predictive, Oncogenic

Justification: Predictive: The passage discusses how various D835 substitutions correlate with resistance to FLT3 inhibitors, indicating their predictive value regarding treatment response. Oncogenic: The D835 mutations are reported to cause resistance in patients, suggesting that these somatic variants contribute to tumor development or progression.

Gene→Variant (gene-first): 2322:D835 2322:D835A/E 2322:D835H 2322:D835V/Y

Genes: 2322

Variants: D835 D835A/E D835H D835V/Y

Salen de personaje y se reclaman cosas

Representar, actuar como juego - Teatro y metateatro

Document de Synthèse : Le Programme EVARS – Enjeux, Histoire et Mise en Application

Résumé Exécutif

L’adoption à l’unanimité du programme EVARS (Éducation à la Vie Affective, Relationnelle et Sexuelle) par le Conseil supérieur de l’éducation le 3 février 2025 marque un tournant historique dans le système éducatif français.

Fruit de plus de 50 ans de luttes et d'évolutions législatives, ce programme vise à institutionnaliser une éducation complète à la sexualité, de la maternelle à la terminale.

L'objectif central est de transformer une obligation légale souvent négligée — la loi Aubri de 2001 prévoyant trois séances annuelles — en une réalité pédagogique concrète.

Les enjeux sont multiples : prévention des violences sexuelles (touchant statistiquement trois enfants par classe), lutte contre les stéréotypes de genre, promotion du consentement et déconstruction des représentations toxiques issues notamment de la pornographie.

Malgré cette victoire institutionnelle, la mise en œuvre se heurte à des défis persistants : une désinformation active de mouvements traditionalistes, un manque de formation des personnels et des contraintes de financement.

La réussite du programme repose désormais sur une synergie entre l'institution scolaire, les associations expertes et l'implication des familles.

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1. Perspective Historique et Évolution Légale

L'éducation à la sexualité n'est pas un concept récent, mais son approche a radicalement évolué, passant d'une logique de contrôle à une logique d'émancipation.

1.1. Les prémices (XIXe - milieu XXe siècle)

• Fin du XIXe siècle : Apparition des premiers textes, oscillant entre la préservation de l'innocence enfantine et des impératifs de santé publique (lutte contre la syphilis et enjeux démographiques).

• 1947-1948 : Le rapport de l'inspecteur général François marque la première prise en compte institutionnelle de la nécessité d'une éducation à la sexualité.

1.2. De l'information à l'éducation (1973 - 2001)

• 1973 : Une circulaire fondamentale distingue l'information sexuelle (reproduction, assurée par les SVT) de l'éducation à la sexualité (dimension affective et sociale).

• 1998 : Sous l'impulsion de Jack Lang, la circulaire "Toutmonde" met l'accent sur la prévention du sida.

• 4 juillet 2001 (Loi Aubri/Péri) : La loi rend obligatoires trois séances d'éducation à la sexualité par an à chaque niveau de classe.

Cependant, dans les faits, seuls 15 à 20 % des élèves en bénéficient réellement.

1.3. Vers le programme EVARS de 2025

• Le programme adopté en 2025 remplace des initiatives plus fragiles ou contestées comme les "ABCD de l'égalité" (2013).

• Il s'inscrit dans un cadre européen standardisé, nommant l'enseignement "Éducation à la vie affective et relationnelle" (EVAR) pour le premier degré et y ajoutant le terme "Sexuelle" (EVARS) pour le second degré afin d'apaiser les craintes parentales.

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2. Les Enjeux Majeurs de l'EVARS

Le programme repose sur trois piliers de compétences : se connaître et vivre avec son corps, construire des relations épanouies, et trouver sa place dans la société en tant que citoyen libre et responsable.

2.1. Prévention des violences sexuelles

• Constat alarmant : Selon la CIIVISE, 160 000 enfants sont victimes de violences sexuelles chaque année, soit environ trois enfants par classe.

• Rôle de l'école : L'éducation permet de nommer les parties du corps (brisant le tabou de la "zette" ou du sexe), d'identifier l'intimité et d'apprendre à dénoncer les attouchements.

• Protection : L'absence de mots et une pudeur excessive favorisent les agresseurs. Le programme EVARS apprend aux enfants qu'ils ont le droit de dire "non".

2.2. Lutte contre les stéréotypes et la masculinité toxique

• Impact du numérique : 73 % des adolescents garçons sont exposés en ligne à des stéréotypes de domination masculine (données d'octobre 2025).

• Déconstruction : Le programme vise à libérer les garçons de l'injonction à la violence ou à la répression émotionnelle ("apprendre à pleurer avant d'apprendre les armes") et les filles de l'intériorisation de la soumission.

2.3. Accès à une information fiable

• En l'absence d'éducation formelle, la pornographie devient la source principale d'information, véhiculant des modèles relationnels faussés et violents dès le CM1.

• L'EVARS offre un cadre clinique et serein pour aborder des sujets complexes sans jugement.

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3. Modalités d'Application et Défis de Terrain

3.1. Les "Ateliers de l'égalité" : Un modèle pédagogique

Des associations comme En avant Toute(s) déploient des interventions concrètes (du CE2 à la 5e) :

• Méthodologie : Utilisation de l'éducation populaire (débats, théâtre-forum, jeux de cartes) pour partir de la parole de l'élève.

• Non-mixité : Des temps séparés entre filles et garçons sont parfois utilisés pour favoriser la libération de la parole sur les violences vécues avant une mise en commun.

• Outils pratiques : Création de "réseaux de soutien" où l'enfant identifie les adultes ressources en cas de problème.

3.2. Obstacles institutionnels et financiers

• Formation : Il existe un besoin impérieux de former les enseignants via les INSPÉ pour leur donner la confiance nécessaire face aux sujets "sensibles".

• Statut des heures : Si les séances sont obligatoires, elles ne sont pas toujours intégrées aux programmes évalués, ce qui complexifie leur financement (nécessité de dotations horaires pour les heures supplémentaires dans le secondaire).

• Restriction des intervenants : Une circulaire limite l'intervention des associations dans les écoles primaires, laissant la charge aux seuls enseignants, ce qui peut freiner la mise en œuvre faute d'expertise externe.

3.3. La résistance idéologique

• L'école fait face à une "hystérie collective" ou des rumeurs persistantes (accusations infondées d'apprendre la masturbation aux jeunes enfants).

• Des groupes traditionalistes et des mouvements d'extrême droite s'organisent pour délégitimer le programme, utilisant des plateformes médiatiques pour diffuser de la désinformation.

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4. Recommandations pour une Mise en Œuvre Réussie

| Axe d'effort | Actions préconisées | | --- | --- | | Transparence | Rendre les programmes consultables par tous les parents sur Éduscol pour désamorcer les fantasmes. | | Implication parentale | Organiser des "cafés des parents" et les inciter à porter la demande d'EVARS dans les conseils d'école. | | Soutien aux enseignants | Assurer la protection institutionnelle des professeurs face aux menaces de groupes radicaux. | | Synergie associative | Maintenir le rôle des associations agréées qui apportent une expertise complémentaire et une posture d'adulte neutre. | | Élargissement | Étendre ces formations au secteur périscolaire et aux établissements spécialisés (IME, CFA). |

Conclusion

Le programme EVARS n'est pas une menace pour les familles, mais un "cadeau pour les générations futures".

En enseignant le respect, le consentement et l'empathie au même titre que la grammaire ou les mathématiques, l'école remplit sa mission fondamentale : former des citoyens lucides, capables d'aimer sans posséder et de s'affirmer sans écraser.

La réussite de ce projet repose sur le passage définitif de la "pudeur à la pédagogie".

Briefing Doc : "Le parcours de l'élève au périscope" Source : Excerpts from the radio show "Le parcours de l'élève au périscope"

Date de diffusion : 11 mars 2025

Participants :

• Jean-Marc Moulet : Inspecteur général de l'éducation, du sport et de la recherche
• Philippe Montoya : IEN en scolarisation des élèves en situation de handicap, conseiller technique école inclusive du recteur de l'académie de Toulouse
• Patrick Avogadro : Personnel de direction, Lycée professionnel Les Grippeaux (académie de Poitiers)
• Noémie Olympio : Enseignante-chercheuse sur les trajectoires des élèves (LEST CNRS, Université Aix-Marseille)

• Raphaël Mata Duvignot : Présentateur de la minute juris

• Thème principal : Le parcours de l'élève, ses enjeux, les dispositifs d'accompagnement, les inégalités et les perspectives.

Structure de l'émission (d'après l'extrait) :

• Enjeux autour du parcours de l'élève : Définition, étapes clés, accompagnement du système éducatif, objectifs au-delà de l'insertion professionnelle, influence du territoire et position de la recherche.

• Minute Juris : Présentation des cadres légaux et administratifs du parcours de l'élève (socle commun, redoublement, orientation, classes et groupes spécifiques).

• Témoignages de terrain (Table ronde) : Expériences dans le premier et second degré, forces du système, enjeux territoriaux, discriminations, découverte des métiers, inclusion des élèves en situation de handicap, formation des enseignants et partenaires.

• Minute Bibli : Présentation de ressources bibliographiques.

Principaux thèmes et idées clés :

1. Définition et complexité du parcours de l'élève :

• Le parcours de l'élève englobe "tout ce qu'un élève va vivre à l'intérieur de l'école à l'extérieur de l'école pour se construire réussir son orientation et arriver à une insertion professionnelle la meilleure possible." (Jean-Marc Moulet)

• Il existe une distinction avec les "parcours éducatifs" (réforme de 2008) qui sont plus axés sur les éducations transversales, au sein desquels figure le "parcours avenir", central pour l'orientation au collège.

• Le parcours est différencié selon les niveaux (primaire, collège, lycée), avec des dispositifs spécifiques pour accompagner les difficultés (plans personnalisés au primaire, SEGPA au collège, spécialisations au lycée).

• Au lycée (surtout professionnel), l'éventail des parcours s'élargit avec des secondes thématiques et des possibilités d'approfondissement ou d'immersion professionnelle en terminale.

Le lycée général et technologique offre une multiplication des choix de disciplines et de couplages.

• Le système éducatif accompagne via des heures dédiées à l'orientation dès la 4ème, l'accompagnement personnalisé au lycée et le rôle des équipes éducatives et des psychologues de l'Éducation nationale.

2. Objectifs multiples du parcours :

• L'objectif n'est pas uniquement l'insertion professionnelle, mais aussi la "fabrication de citoyens qui soient heureux" et la "diversification des possibles". (Jean-Marc Moulet)

• Il s'agit de lutter contre le déterminisme social et les pressions de genre en élargissant le "panel des possibles" pour que les élèves se révèlent dans ce qui est le meilleur pour eux.

• Le socle commun assure l'acquisition des compétences nécessaires à l'orientation pour tous les citoyens à la fin de la scolarité obligatoire.

3. Personnalisation et choix :

• L'idée est d'avoir un parcours "le plus personnalisé proche des envies possibles des jeunes". (Jean-Marc Moulet)
• L'offre de choix est aujourd'hui beaucoup plus large qu'auparavant, correspondant à une plus grande diversité de profils.
• La valorisation du lycée professionnel est un enjeu éducatif et économique fort, en lien avec les besoins du marché du travail.

4. Influence du territoire et mobilité :

• La proximité du secteur économique influence l'orientation.

L'information sur l'orientation est déléguée aux régions (loi de 2018) pour tenir compte des enjeux économiques locaux et favoriser la mobilité régionale.

• La mobilité des élèves est centrale, et informer sur les opportunités régionales peut engager certains élèves à s'y orienter.

• Les projets éducatifs de territoire (PEDT) sont des leviers importants pour lutter contre les inégalités culturelles et favoriser la mobilité dès le primaire.

• Des initiatives comme les cordées de la réussite et les internats d'excellence visent à pallier les inégalités territoriales et à élever les ambitions des élèves.

5. Le regard de la recherche : Inégalités et déterminismes :

• La notion de parcours renvoie aux "périodes charnières" (aménagements précoces, premiers paliers d'orientation en 3ème et seconde). (Noémie Olympio)

• Malgré la volonté d'uniformité (tronc commun), le système est marqué par des "éléments d'inégalité" et un fort "déterminisme scolaire et social des trajectoires".

• La performance scolaire en fin de primaire est un bon prédicteur des possibilités futures. L'orientation est socialement marquée (à performance égale, un enfant de parents diplômés du supérieur a plus de chances de faire un bac général).

• Les données de la DEP (panel d'élèves) montrent l'importance du "capital informationnel des familles", du "niveau d'aspiration des familles" et du "maintien des aspirations" (phénomène de "refroidissement des aspirations" parfois non lié à la performance scolaire).

• La "représentation de l'utilité des diplômes" est également inégalement répartie et corrélée à la résilience scolaire.

• Le système actuel, avec des aménagements précoces (comme la SEGPA), peut rendre les trajectoires "peu réversibles" et socialement marquées.

• Le "capital informationnel" se constitue par la catégorie socio-professionnelle, la représentation du monde, le rapport à la mobilité et les "stratégies éducatives des parents" (plus ou moins "opérantes").

6. Cadre légal et administratif (Minute Juris) :

• L'article L 111-1 du code de l'éducation garantit l'organisation des parcours en fonction des élèves.

• Le socle commun de connaissances, de compétences et de culture (défini par la loi de 2005 et refondé en 2013) est au cœur du système et évalué à la fin de chaque cycle.

• Le redoublement est un dispositif rare et exceptionnel (codifié à l'article L 31-7), privilégiant des stratégies de prévention et d'accompagnement.

Il est interdit en maternelle. La décision fait l'objet d'un dialogue avec les familles et peut être contestée.

• L'orientation scolaire (articles L331-7 et D331-31) est encadrée par des voies définies par arrêté ministériel et implique un dialogue entre familles et équipes pédagogiques. En cas de désaccord, une procédure de recours existe.

• Des classes et groupes spécifiques (article D 332-5), comme les SEGPA (article D 332-7) et les ULIS (article L12-1), permettent un parcours différencié pour répondre aux besoins des élèves, y compris en situation de handicap.

La différence de traitement basée sur les besoins n'est pas considérée comme une rupture d'égalité.

• Le principe de mutabilité du service public d'éducation implique une innovation et un ajustement continu des pratiques pédagogiques.

7. Témoignages de terrain et solutions :

• Les parcours diffèrent déjà au primaire en fonction du territoire et des projets menés (y compris le temps périscolaire). La distance au collège et au lycée impacte également les parcours.

• Les projets éducatifs de territoire (PEDT) et le regroupement de collectivités sont essentiels pour offrir des opportunités culturelles et de mobilité.

• Les cordées de la réussite lient les collèges à des grandes écoles pour susciter l'ambition. Les internats d'excellence lèvent l'obstacle de la distance.

• Les campus des métiers d'excellence (CMQ) favorisent la mobilité et le lien avec l'économie des territoires, en produisant des ressources pour les collèges (jeux, plateformes numériques, accueil).

• Il est crucial de travailler sur l'ouverture du champ des possibles et le capital informationnel sans paternalisme, en s'appuyant sur des données fiables (taux d'employabilité, mobilité professionnelle).

• La découverte des métiers dès la 5ème (voire plus tôt, comme dans les pays anglo-saxons) est essentielle pour contrer les déterminismes.

La rencontre avec des professionnels a un impact déterminant (l'exemple d'une heure d'intervention d'une scientifique sur l'orientation des filles).

• Des actions locales (forums des métiers, visites d'entreprises, mini-stages, stages de seconde) permettent aux élèves de découvrir la diversité des professions.

• Le soutien au parcours dans les lycées professionnels (ateliers CV, rencontres avec des professionnels et anciens élèves) vise à faciliter l'insertion et la poursuite d'études.

Le parcours différencié en fin d'année permet des stages de professionnalisation ou des ateliers de préparation à la vie étudiante.

• La formation d'initiatives locales (FIL) rapproche les enseignants des différents niveaux pour harmoniser les attentes.

8. Inclusion des élèves en situation de handicap :

• La mobilité est un enjeu crucial, nécessitant des outils spécifiques (applications d'aide au déplacement).

• L'ambition pour ces élèves doit être élevée (faible taux en lycée général et technologique). Il existe un "plafond de verre" à faire sauter.

• Les universités et grandes écoles développent une forte dynamique inclusive (référents handicap, aménagements). La convention "A tout pour tous" à Toulouse et les initiatives pour les étudiants avec TSA sont des exemples.

• Des plateformes d'accompagnement à l'inclusion professionnelle sont mises en place.

• La connaissance des dispositifs par les enseignants est fondamentale. L'action "Enseignement supérieur et handicap, c'est possible" vise à informer.

9. Formation des enseignants et partenaires :

• Les psychologues de l'Éducation nationale et les CIO ont un rôle central.

• Il est important d'associer les médecins de l'Éducation nationale pour anticiper les contre-indications dans certaines filières professionnelles.

• La région (information, orientation mobile), l'ONISEP (compétences à s'orienter, plateforme "Avenir"), les professeurs principaux et les DDFPT (en lycée professionnel) sont des partenaires clés.

• Le travail en équipe et en réseau (campus des métiers et des qualifications) est essentiel.

• Il faut renforcer les partenariats entre le collège et le lycée, ainsi qu'avec l'enseignement supérieur (continuum Bac-3 / Bac+3).

• La gestion algorithmique de l'orientation peut alimenter l'autocensure, nécessitant une meilleure explicitation des stratégies et des accompagnements pour les élèves les moins favorisés.

• Le bureau des entreprises dans les lycées professionnels renforce le lien avec le monde du travail. Le réseau associatif peut apporter une expertise complémentaire.

• Il est important de lier le stage de seconde aux expériences vécues au collège.

Conclusion et perspectives (Jean-Marc Moulet) :

• Le système éducatif évolue pour faciliter et mieux accompagner les parcours, en aidant les familles les plus fragiles.

• L'objectif ne doit pas être uniquement l'insertion professionnelle, mais aussi la formation à la mobilité professionnelle et à la plasticité face aux évolutions du marché du travail.

• La question du décrochage scolaire, souvent lié à des difficultés d'orientation, pourrait faire l'objet d'une prochaine table ronde.

Ressources bibliographiques (Minute Biblie) :

Rapport de l'Inspection générale sur la découverte des métiers au collège (mai 2024).

Articles de Noémie Olympio sur l'orientation en lycée professionnel, les aspirations et le capital social et culturel.

Site de l'ONISEP et plateforme "Avenir".

Author response:

The following is the authors’ response to the previous reviews

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

The authors have adequately addressed all of my concerns. I have no further questions or concerns.

We thank the Reviewer #1. 

Reviewer #2 (Recommendations for the authors):

We thank the Reviewer #2 for thoughtful recommendations.

(1) Figure 1A, 1B, 2B, 2C, etc.: The Y-axis label is confusing. I assume the intention was to make big numbers small by dividing by 1000. The comma makes the label confusing. Perhaps, make the label more "mathematical" as in "Avp density ((transcript/µm2) * 10-3)" or rearrange the math to be clearer as in "Avp density (transcript/1000 per µm2)".

Great suggestion and done exactly as suggested in Figures 1, 2 and 4.

(2) Figure 1B and 1C: The figure and legend do not match up. Either switch the figures or the legends. Currently, legend 1B describes image 1C.

Agreed and done as suggested.

(3) Figure 2A is broken up into separate pages/panels. It could be integrated better or separated to make A and B, then shift B and C to C and D.

Great suggestion and we have done exactly as suggested.

(4) Figure 2 legend: I recommend putting the scale bar info with (A) rather than at the end. The stars used in the figure are not explained in the legend.

Good points. We have made all necessary changes as suggested.

(5) Supplementary Figure 1B: The legend states that the data are the number of transcript-containing cells, but the figure states transcript number.

We thank the Reviewer for pointing out this typo. We corrected all graph legends in the Supplementary Figure 1.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

Review of the manuscript titled " Mycobacterial Metallophosphatase MmpE acts as a nucleomodulin to regulate host gene expression and promotes intracellular survival".

The study provides an insightful characterization of the mycobacterial secreted effector protein MmpE, which translocates to the host nucleus and exhibits phosphatase activity. The study characterizes the nuclear localization signal sequences and residues critical for the phosphatase activity, both of which are required for intracellular survival.

Strengths:

(1) The study addresses the role of nucleomodulins, an understudied aspect in mycobacterial infections.

(2) The authors employ a combination of biochemical and computational analyses along with in vitro and in vivo validations to characterize the role of MmpE.

Weaknesses:

(1) While the study establishes that the phosphatase activity of MmpE operates independently of its NLS, there is a clear gap in understanding how this phosphatase activity supports mycobacterial infection. The investigation lacks experimental data on specific substrates of MmpE or pathways influenced by this virulence factor.

We thank the reviewer for this insightful comment and agree that identification of the substrates of MmpE is important to fully understand its role in mycobacterial infection. MmpE is a putative purple acid phosphatase (PAP) and a member of the metallophosphoesterase (MPE) superfamily. Enzymes in this family are known for their catalytic promiscuity and broad substrate specificity, acting on phosphomonoesters, phosphodiesters, and phosphotriesters (Matange et al., Biochem J, 2015). In bacteria, several characterized MPEs have been shown to hydrolyze substrates such as cyclic nucleotides (e.g., cAMP) (Keppetipola et al., J Biol Chem, 2008; Shenoy et al., J Mol Biol, 2007), nucleotide derivatives (e.g., AMP, UDP-glucose) (Innokentev et al., mBio, 2025), and pyrophosphate-containing compounds (e.g., Ap4A, UDP-DAGn) (Matange et al., Biochem J., 2015). Although the binding motif of MmpE has been identified, determining its physiological substrates remains challenging due to the low abundance and instability of potential metabolites, as well as the limited sensitivity and coverage of current metabolomic technologies in mycobacteria.

(2) The study does not explore whether the phosphatase activity of MmpE is dependent on the NLS within macrophages, which would provide critical insights into its biological relevance in host cells. Conducting experiments with double knockout/mutant strains and comparing their intracellular survival with single mutants could elucidate these dependencies and further validate the significance of MmpE's dual functions.

We thank the reviewer for the comment. Deletion of the NLS motifs did not impair MmpE’s phosphatase activity in vitro (Figure 2F), indicating that MmpE's enzymatic function operates independently of its nuclear localization. Indeed, we confirmed that Fe³⁺-binding ability via the residues H348 and N359 is required for enzymatic activity of MmpE. We have expanded on this point in the Discussion section “MmpE is a bifunctional virulence factor in Mtb”.

(3) The study does not provide direct experimental validation of the MmpE deletion on lysosomal trafficking of the bacteria.

We thank the reviewer for the comment. To validate the role of MmpE in lysosome maturation during infection, we conducted fluorescence colocalization assays in THP-1 macrophages infected with BCG strains, including WT, ∆MmpE, Comp-MmpE, Comp-MmpE^ΔNLS1, Comp-MmpE^ΔNLS2, Comp-MmpE^ΔNLS1-2. These strains were stained with the lipophilic membrane dye DiD, while macrophages were treated with the acidotropic probe LysoTracker^TM Green (Martins et al., Autophagy, 2019). The result indicated that ΔMmpE and MmpE^NLS1-2 mutants exhibited significantly higher co-localization with LysoTracker compared to WT and Comp-MmpE strains (New Figure 5G), suggesting that MmpE deletion leads to enhanced lysosomal maturation during infection.

(4) The role of MmpE as a mycobacterial effector would be more relevant using virulent mycobacterial strains such as H37Rv.

We thank the reviewer for the comment. Previously, the role of Rv2577/MmpE as a virulence factor has been demonstrated in M. tuberculosis CDC 1551, where its deletion significantly reduced bacterial replication in mouse lungs at 30 days post-infection (Forrellad et al., Front Microbiol, 2020). However, that study did not explore the underlying mechanism of MmpE function. In our study, we found that MmpE enhances M. bovis BCG survival in macrophages (THP-1 and RAW264.7 both) and in mice (Figure 3, Figure 7A), consistent with its proposed role in virulence. To investigate the molecular mechanism by which MmpE promotes intracellular survival, we used M. bovis BCG as a biosafe surrogate and this model is widely accepted for studying mycobacterial pathogenesis (Wang et al., Nat Immunol, 2015; Wang et al., Nat Commun, 2017; Péan et al., Nat Commun, 2017).

Reviewer #2 (Public review):

Summary:

In this paper, the authors have characterized Rv2577 as a Fe3+/Zn2+ -dependent metallophosphatase and a nucleomodulin protein. The authors have also identified His348 and Asn359 as critical residues for Fe3+ coordination. The authors show that the proteins encode for two nuclease localization signals. Using C-terminal Flag expression constructs, the authors have shown that the MmpE protein is secretory. The authors have prepared genetic deletion strains and show that MmpE is essential for intracellular survival of M. bovis BCG in THP-1 macrophages, RAW264.7 macrophages, and a mouse model of infection. The authors have also performed RNA-seq analysis to compare the transcriptional profiles of macrophages infected with wild-type and MmpE mutant strains. The relative levels of ~ 175 transcripts were altered in MmpE mutant-infected macrophages and the majority of these were associated with various immune and inflammatory signalling pathways. Using these deletion strains, the authors proposed that MmpE inhibits inflammatory gene expression by binding to the promoter region of a vitamin D receptor. The authors also showed that MmpE arrests phagosome maturation by regulating the expression of several lysosome-associated genes such as TFEB, LAMP1, LAMP2, etc. These findings reveal a sophisticated mechanism by which a bacterial effector protein manipulates gene transcription and promotes intracellular survival.

Strength:

The authors have used a combination of cell biology, microbiology, and transcriptomics to elucidate the mechanisms by which Rv2577 contributes to intracellular survival.

Weakness:

The authors should thoroughly check the mice data and show individual replicate values in bar graphs.

We kindly appreciate the reviewer for the advice. We have now updated the relevant mice data in the revised manuscript.

Reviewer #3 (Public review):

Summary:

In this manuscript titled "Mycobacterial Metallophosphatase MmpE Acts as a Nucleomodulin to Regulate Host Gene Expression and Promote Intracellular Survival", Chen et al describe biochemical characterisation, localisation and potential functions of the gene using a genetic approach in M. bovis BCG and perform macrophage and mice infections to understand the roles of this potentially secreted protein in the host cell nucleus. The findings demonstrate the role of a secreted phosphatase of M. bovis BCG in shaping the transcriptional profile of infected macrophages, potentially through nuclear localisation and direct binding to transcriptional start sites, thereby regulating the inflammatory response to infection.

Strengths:

The authors demonstrate using a transient transfection method that MmpE when expressed as a GFP-tagged protein in HEK293T cells, exhibits nuclear localisation. The authors identify two NLS motifs that together are required for nuclear localisation of the protein. A deletion of the gene in M. bovis BCG results in poorer survival compared to the wild-type parent strain, which is also killed by macrophages. Relative to the WT strain-infected macrophages, macrophages infected with the ∆mmpE strain exhibited differential gene expression. Overexpression of the gene in HEK293T led to occupancy of the transcription start site of several genes, including the Vitamin D Receptor. Expression of VDR in THP1 macrophages was lower in the case of ∆mmpE infection compared to WT infection. This data supports the utility of the overexpression system in identifying potential target loci of MmpE using the HEK293T transfection model. The authors also demonstrate that the protein is a phosphatase, and the phosphatase activity of the protein is partially required for bacterial survival but not for the regulation of the VDR gene expression.

Weaknesses:

(1) While the motifs can most certainly behave as NLSs, the overexpression of a mycobacterial protein in HEK293T cells can also result in artefacts of nuclear localisation. This is not unprecedented. Therefore, to prove that the protein is indeed secreted from BCG, and is able to elicit transcriptional changes during infection, I recommend that the authors (i) establish that the protein is indeed secreted into the host cell nucleus, and (ii) the NLS mutation prevents its localisation to the nucleus without disrupting its secretion.

We kindly appreciate the reviewer for this insightful comment. To confirm the translocation of MmpE into the host nucleus during BCG infection, we first detected the secretion of MmpE by M. bovis BCG, using Ag85B as a positive control and GlpX as a negative control (Zhang et al., Nat commun, 2022). Our results showed that MmpE- Flag was present in the culture supernatant, indicating that MmpE is secreted by BCG indeed (new Figure S1C).

Next, we performed immunoblot analysis of the nuclear fractions from infected THP-1 macrophages expressing FLAG-tagged wild-type MmpE and NLS mutants. The results revealed that only wild-type MmpE was detected in the nucleus, while MmpE^ΔNLS1, MmpE^ΔNLS2 and MmpE^ΔNLS1-2 were not detectable in the nucleus (New Figure S1D). Taken together, these findings demonstrated that MmpE is a secreted protein and that its nuclear translocation during infection requires both NLS motifs.

Demonstration that the protein is secreted: Supplementary Figure 3 - Immunoblotting should be performed for a cytosolic protein, also to rule out detection of proteins from lysis of dead cells. Also, for detecting proteins in the secreted fraction, it would be better to use Sauton's media without detergent, and grow the cultures without agitation or with gentle agitation. The method used by the authors is not a recommended protocol for obtaining the secreted fraction of mycobacteria.

We kindly appreciate the reviewer for the advice. To avoid the effects of bacterial lysis, we cultured the BCG strains expressing MmpE-Flag in Middlebrook 7H9 broth with 0.5% glycerol, 0.02% Tyloxapol, and 50 µg/mL kanamycin at 37 °C with gentle agitation (80 rpm) until an OD₆₀₀ of approximately 0.6 (Zhang et al., Nat Commun, 2022). Subsequently, we assessed the secretion of MmpE-Flag in the culture supernatant, using Ag85B as a positive control and GlpX as a negative control (New Figure S1C). The results showed that GlpX was not detected in the supernatant, while MmpE and Ag85B were detected, indicating that MmpE is indeed a secreted protein in BCG.

Demonstration that the protein localises to the host cell nucleus upon infection: Perform an infection followed by immunofluorescence to demonstrate that the endogenous protein of BCG can translocate to the host cell nucleus. This should be done for an NLS1-2 mutant expressing cell also.

We thank the reviewer for the suggestion. We agree that this experiment would be helpful to further verify the ability of MmpE for nuclear import. However, MmpE specific antibody is not available for us for immunofluorescence experiment. Alternatively, we performed nuclear-cytoplasmic fractionation for the THP-1 cells infected with the M. bovis BCG strains expressing FLAG-tagged wild-type MmpE, as well as NLS deletion mutants (MmpE^ΔNLS1, MmpE^ΔNLS2, and MmpE^ΔNLS1-2). The WT MmpE is detectable in both cytoplasmic and nuclear compartments, while MmpE^ΔNLS1, MmpE^ΔNLS2 or MmpE^ΔNLS1-2 were almost undetectable in nuclear fractions (New Figure S1D), suggesting that both NLS motifs are necessary for nuclear import.

(2) In the RNA-seq analysis, the directionality of change of each of the reported pathways is not apparent in the way the data have been presented. For example, are genes in the cytokine-cytokine receptor interaction or TNF signalling pathway expressed more, or less in the ∆mmpE strain?

We thank the reviewer for the comment. The KEGG pathway enrichment diagrams in our RNA-seq analysis primarily reflect the statistical significance of pathway enrichment based on differentially expressed genes, but do not indicate the directionality of genes expression changes. To address this concern, we conducted qRT-PCR on genes associated with the cytokine-cytokine receptor interaction pathway, specifically IL23A, CSF2, and IL12B. The results showed that, compared to the WT strain, infection with the ΔMmpE strain resulted in significantly increased expression levels of these genes in THP-1 cells (Figure 4F, Figure S4B), consistent with the RNA-seq data. Furthermore, we have submitted the complete RNA-seq dataset to the NCBI GEO repository [GSE312039], which includes normalized expression values and differential expression results for all detected genes.

(3) Several of these pathways are affected as a result of infection, while others are not induced by BCG infection. For example, BCG infection does not, on its own, produce changes in IL1β levels. As the author s did not compare the uninfected macrophages as a control, it is difficult to interpret whether ∆mmpE induced higher expression than the WT strain, or simply did not induce a gene while the WT strain suppressed expression of a gene. This is particularly important because the strain is attenuated. Does the attenuation have anything to do with the ability of the protein to induce lysosomal pathway genes? Does induction of this pathway lead to attenuation of the strain? Similarly, for pathways that seem to be downregulated in the ∆mmpE strain compared to the WT strain, these might have been induced upon infection with the WT strain but not sufficiently by the ∆mmpE strain due to its attenuation/ lower bacterial burden.

We thank the reviewer for the comment. Previous studies have shown that wild-type BCG induces relatively low levels of IL-1β, while retaining partial capacity to activate the inflammasome (Qu et al., Sci Adv, 2020). Our data (Figures 3G) show that infection with the ΔMmpE strain results in enhanced IL-1β expression, consistent with findings by Master et al. (Cell Host Microbe, 2008), in which deletion of zmp1 in BCG or M. tuberculosis led to increased IL-1β levels due to reduced inhibition of inflammasome activation.

In the revised manuscript, we have provided additional qRT-PCR data using uninfected macrophages as a baseline control. These results demonstrate that the WT strain suppresses lysosome-associated gene expression, whereas the ΔMmpE strain upregulates these genes, indicating that MmpE inhibits lysosome-related genes expression (Figure 4G). Furthermore, bacterial burden analysis revealed that ∆mmpE exhibited ~3-fold lower intracellular survival than the WT strain in THP-1 cells. However, when lysosomal maturation was inhibited, the difference in bacterial load between the two strains was reduced to ~1-fold (New Figures S6B and C). These findings indicate that MmpE promotes intracellular survival primarily by inhibiting lysosomal maturation, which is consistent with a previous study (Chandra et al., Sci Rep, 2015).

(4) CHIP-seq should be performed in THP1 macrophages, and not in HEK293T. Overexpression of a nuclear-localised protein in a non-relevant line is likely to lead to several transcriptional changes that do not inform us of the role of the gene as a transcriptional regulator during infection.

We thank the reviewer for the comment. We performed ChIP-seq in HEK293T cells based on their high transfection efficiency, robust nuclear protein expression, and well-annotated genome (Lampe et al., Nat Biotechnol, 2024; Marasco et al., Cell, 2022). These characteristics make HEK293T an ideal system for the initial identification of genome-wide chromatin binding profiles by MmpE.

Further, we performed comprehensive validation of the ChIP-seq findings in THP-1 macrophages. First, CUT&Tag and RNA-seq analyses in THP-1 cells revealed that MmpE modulates genes involved in the PI3K–AKT signaling and lysosomal maturation pathways (Figure 4C; Figure S5A-B). Correspondingly, we found that infection with the ΔMmpE strain led to reduced phosphorylation of AKT (S473), mTOR (S2448), and p70S6K (T389) (New Figure 5E-F), and upregulation of lysosomal genes such as TFEB, LAMP1, and LAMP2 (Figure 4G), compared to infection with the WT strain, and lysosomal maturation in cells infected with the ΔMmpE strain more obviously (New Figure 5G). Additionally, CUT&Tag profiling identified MmpE binding at the promoter region of the VDR gene, which was further validated by EMSA and ChIP-qPCR. Also, qRT-PCR demonstrated that MmpE suppresses VDR transcription, supporting its role as a transcriptional regulator (Figure 6). Collectively, these data confirm the biological relevance and functional significance of the ChIP-seq findings obtained in HEK293T cells.

(5) I would not expect to see such large inflammatory reactions persisting 56 days post-infection with M. bovis BCG. Is this something peculiar for an intratracheal infection with 1x107 bacilli? For images of animal tissue, the authors should provide images of the entire lung lobe with the zoomed-in image indicated as an inset.

We thank the reviewer for the comment. The lung inflammation peaked at days 21–28 and had clearly subsided by day 56 across all groups (New Figure 7B), consistent with the expected resolution of immune responses to an attenuated strain like M. bovis BCG. This temporal pattern is in line with previous studies using intravenous or intratracheal BCG vaccination in mice and macaques, which also demonstrated robust early immune activation followed by resolution over time (Smith et al., Nat Microbiol, 2025; Darrah et al., Nature, 2020).

In this study, the infectious dose (1×10⁷ CFU intratracheal) was selected based on previous studies in which intratracheal delivery of 1×10⁷ CFU produced consistent and measurable lung immune responses and pathology without causing overt illness or mortality (Xu et al., Sci Rep, 2017; Niroula et al., Sci Rep, 2025). We have provided whole-lung lobe images with zoomed-in insets in the source dataset.

(6) For the qRT-PCR based validation, infections should be performed with the MmpE-complemented strain in the same experiments as those for the WT and ∆mmpE strain so that they can be on the same graph, in the main manuscript file. Supplementary Figure 4 has three complementary strains. Again, the absence of the uninfected, WT, and ∆mmpE infected condition makes interpretation of these data very difficult.

We thank the reviewer for the comment. As suggested, we have conducted the qRT-PCR experiment including the uninfected, WT, ∆mmpE, Comp-MmpE, and the three complementary strains infecting THP-1 cells (Figure 4F and G; New Figure S4B–D).

(7) The abstract mentions that MmpE represses the PI3K-Akt-mTOR pathway, which arrests phagosome maturation. There is not enough data in this manuscript in support of this claim. Supplementary Figure 5 does provide qRT-PCR validation of genes of this pathway, but the data do not indicate that higher expression of these pathways, whether by VDR repression or otherwise, is driving the growth restriction of the ∆mmpE strain.

We thank the reviewer for the comment. In the updated manuscript, we have provided more evidence. First, the RNA-seq analysis indicated that MmpE affects the PI3K-AKT signaling pathway (Figure 4C). Second, CUT&Tag analysis suggested that MmpE binds to the promoter regions of key pathway components, including PRKCB, PLCG2, and PIK3CB (Figure S5A). Third, confocal microscopy showed that ΔMmpE strain promotes significantly increased lysosomal maturation compared to the WT, a process downstream of the PI3K-AKT-mTOR axis (New Figure 5G).

Further, we measured protein phosphorylation for validating activation of the pathway (Zhang et al., Stem Cell Reports, 2017). Our results showed that cells infected with WT strains exhibited significantly higher phosphorylation of Akt, mTOR, and p70S6K compared to those infected with ΔMmpE strains (New Figures 5E and F). Moreover, the dual PI3K/mTOR inhibitor BEZ235 abolished the survival advantage of WT strains over ΔMmpE mutants in THP-1 macrophages (New Figure S6B and C). Collectively, these results support that MmpE activates the PI3K–Akt–mTOR signaling pathway to enhance bacterial survival within the host.

(8) The relevance of the NLS and the phosphatase activity is not completely clear in the CFU assays and in the gene expression data. Firstly, there needs to be immunoblot data provided for the expression and secretion of the NLS-deficient and phosphatase mutants. Secondly, CFU data in Figure 3A, C, and E must consistently include both the WT and ∆mmpE strain.

We thank the reviewer for the comment. We have now added immunoblot analysis for expression and secretion of MmpE mutants. The result show that NLS-deficient and phosphatase mutants can detected in supernatant (New Figure S1C). Additionally, we have revised Figures 3A, 3C, and 3E to consistently include both the WT and ΔMmpE strains in the CFU assays (Figures 3A, 3C, and 3E).

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

The authors should attempt to address the following comments:

(1) Please perform densitometric analysis for the western blot shown in Figure 1E.

We sincerely thank the reviewer for the suggestion. In the updated manuscript, we have performed densitometric analysis of the western blot shown in New Figure 1F and G.

(2) Is it possible to measure the protein levels for MmpE in lysates prepared from infected macrophages.

We thank the reviewer for the comment. In the revised manuscript, we performed immunoblot analysis to measure MmpE levels in lysates from infected macrophages. The results demonstrated that wild-type MmpE was present in both the cytoplasmic and nuclear fractions during infection in THP-1 cells (New Figure S1D).

(3) The authors should perform circular dichroism studies to compare the secondary structure of wild type and mutant proteins (in particular MmpEHis348 and MmpEAsn359.

We thank the reviewer for this valuable suggestion. We agree that circular dichroism spectroscopy could provide useful information in comparison of the differences on the secondary structures. However, due to the technical limitations, we instead compared the structures of wild-type MmpE and the His348 and Asn359 mutant proteins predicted by AlphaFold. These structural models showed almost no differences in secondary structures between the wild-type and mutants (Figure S1B).

(4) The authors should perform more experiments to determine the binding motif for MmpE in the promoter region of VDR.

We thank the reviewer for this suggestion. In the current study, we have identified the MmpE-binding motif within the promoter region of VDR using CUT&Tag sequencing. This prediction was further validated by ChIP-qPCR and EMSA (Figure 6). These complementary approaches collectively support the identification of a specific MmpE-binding motif and demonstrate its functional relevance. Such approach was acceptable in many publications (Wen et al., Commun Biol, 2020; Li et al., Nat Commun, 2022).

(5) Were the transcript levels of VDR also measured in the lung tissues of infected animals?

We thank the reviewer for this suggestion. In the revised manuscript, we have performed qRT-PCR to assess VDR transcript levels in the lung tissues of infected mice (New Figure S8B).

(6) How does MmpE regulate the expression of lysosome-associated genes?

We thank the reviewer for this question. Our experiments suggested that MmpE suppresses lysosomal maturation probably by activating the host PI3K–AKT–mTOR signaling pathway (New Figure 5E–I). This pathway is well established as a negative regulator of lysosome biogenesis and function (Yang et al., Signal Transduct Target Ther, 2020; Cui et al., Nature, 2023; Cui et al., Nature, 2025). During infection, THP-1 cells infected with the WT showed increased phosphorylation of Akt, mTOR, and p70S6K compared to those infected with ΔMmpE (New Figure S5C, New Figure 5E and F), and concurrently downregulated key lysosomal maturation markers, including TFEB, LAMP1, LAMP2, and multiple V-ATPase subunits (Figure 4G). Given that PI3K–AKT–mTOR signaling suppresses TFEB activity and lysosomal gene transcription (Palmieri et al., Nat Commun, 2017), we propose that MmpE modulates lysosome-associated gene expression and lysosomal function probably by PI3K–AKT–mTOR signaling pathway.

(7) Mice experiment:

(a) The methods section states that mice were infected intranasally, but the legend for Figure 6 states intratracheally. Kindly check?

(b) Supplementary Figure 7 - this is not clear. The legend says bacterial loads in spleens (CFU/g) instead of DNA expression, as shown in the figure.

(c) The data in Figure 6 and Figure S7 seem to be derived from the same experiment, but the number of animals is different. In Figure 6, it is n = 6, and in Figure S7, it is n=3.

We thank the reviewer for the comments.

(a) The infection was performed intranasally, and the figure legend for New Figure 7 has now been corrected.

(b) We adopted quantitative PCR method to measure bacterial DNA levels in the spleens of infected mice. We have now revised the legend.

(c) We have conducted new experiments where each experiment now includes six mice. The results are showed in Figure 7B and C, as well as in the new Figure S8.

(8) The authors should show individual values for various replicates in bar graphs (for all figures).

We thank the reviewer for this helpful suggestion. We have now updated all relevant bar graphs to include individual data points for each biological replicate.

(9) The authors should validate the relative levels of a few DEGs shown in Figure 3F, Figure 3G, and Figure S4C, in the lung tissues of mice infected with wild-type, mutant, and complemented strains.

We thank the reviewer for this suggestion. In the revised manuscript, we have performed qRT-PCR to validate the expression levels of selected DEGs, including inflammation-related and lysosome-associated genes, in lung tissues from mice infected with wild-type, mutant, and complemented strains (New Figure S8C-H).

(10) Did the authors perform an animal experiment using a mutant strain complemented with the phosphatase-deficient MmpE (Comp-MmpE-H348AN359H)?

We appreciate the reviewer's comment. We agree that an additional animal experiment would be useful to assess the effects of the phosphatase. However, our study mainly focused on interpreting the function of the nuclear localization of MmpE during BCG infection. Additionally, we have assessed the role of the phosphatase of MmpE during infection with cell model (Figure 3E).

Minor comment:

The mutant strain should be verified by either Southern blot or whole genome sequencing.

We thank the reviewer for this comment. We verified deletion of mmpE gene by PCR method (Figure S3A-D) which was acceptable in many publications (Zhang et al., PLoS Pathog, 2020; Zhang et al., Nat Commun, 2022).

Reviewer #3 (Recommendations for the authors):

(1) Line 195: cytokine.

We thank the reviewer for the comments. We have now corrected it.

(2) Line 225: rewording required.

Corrected.

(3) Figure 4A. "No difference" instead of "No different".

Corrected.

(4) "KommpE" should be replaced with "∆mmpE strain" (∆=delta symbol).

Corrected.

(5) Supplementary Figure 7. The figure legend states CFU assays, but the y-axis and the graph seem to depict IS1081 quantification.

We thank the reviewer for the comment. The figure is based on IS1081 quantification using qRT-PCR, not CFU assays. We have now revised the legend for New Figure S8A.

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Briefing : Prévention des Addictions et Accompagnement des Jeunes (3-25 ans)

Synthèse

Ce document synthétise les enjeux actuels de la lutte contre les addictions chez les jeunes, tels que présentés par la Mission interministérielle de lutte contre les drogues et les conduites addictives (MILDECA).

Le point central de cette analyse est la vulnérabilité biologique du cerveau des jeunes, qui ne finit sa maturation qu'aux alentours de 25 ans.

Toute consommation prématurée altère le système nerveux et impacte directement la réussite scolaire et l'insertion sociale.

La stratégie de prévention préconisée repose sur un changement de paradigme : s'éloigner des interventions ponctuelles pour privilégier le développement des compétences psychosociales (CPS) à travers des programmes probants évalués par la recherche.

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I. État des Lieux et Réalité des Addictions en France

L'addiction est définie comme une dépendance psychique et comportementale liée à l'utilisation de substances psychoactives qui perturbent le système nerveux central.

Contrairement aux idées reçues, le profil de l'addict n'est pas marginalisé ; il concerne l'ensemble de la population.

Données de santé publique et coûts sociaux

Les chiffres soulignent une problématique majeure de santé publique, souvent banalisée par rapport à d'autres crises sanitaires :

• Tabac : 75 000 décès par an.

• Alcool : 41 000 décès par an (soit un "demi-Covid" annuel récurrent).

• Coût social : L'alcool et le tabac coûtent chacun 120 milliards d'euros par an à la société, contre 10 milliards pour les autres drogues.

• Violences : L'alcool est impliqué dans plus d'un tiers des violences en général, et jusqu'à 80 % des violences faites aux femmes selon certains territoires.

La banalisation culturelle

La France présente des taux de consommation excessivement élevés. Un adulte sur quatre dépasse les repères de consommation à moindre risque (plus de 2 verres par jour ou 10 verres par semaine).

Cette culture de l'alcool s'installe dès l'enfance, souvent au sein de la famille (initiation lors de fêtes familiales, usage de boissons type "Champomy" qui préparent au marketing de l'alcool).

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II. Les Jeunes : Une Population à Haute Vulnérabilité

L'adolescence est une période à risque marqué par le besoin de découverte de sensations et l'influence du groupe de pairs.

Le cerveau en construction

Le cerveau humain n'achève sa formation qu'à 25 ans.

Toute consommation de substances psychoactives avant cet âge provoque des altérations cognitives durables, affectant directement les capacités d'apprentissage.

Lien avec le décrochage scolaire

Les addictions alimentent différentes formes de décrochage :

1. Le décrochage discret : L'élève est présent physiquement mais désengagé, ses facultés étant altérées par les produits (ex: consommation de cannabis avant les cours).

2. Le décrochage par l'échec : Malgré un travail réel, l'élève ne parvient plus à suivre en raison des effets cognitifs des substances.

3. L'influence de l'environnement : Le manque de cadre protecteur familial et l'accessibilité trop aisée aux produits (vente interdite aux mineurs mal respectée) aggravent ces risques.

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III. Analyse des Substances et Nouveaux Comportements

| Substance / Comportement | État de la situation chez les jeunes | Risques et caractéristiques | | --- | --- | --- | | Alcool | 44 % d'expérimentation en 6ème ; 85 % à 17 ans. | Développement du binge drinking (API) ; consommation banalisée en famille. | | Tabac | En baisse constante (perçu comme cher, "odorant" et sans effet immédiat). | Le risque n'est pas proportionnel à la quantité : l'arrêt total est la seule protection réelle. | | Cannabis | 600 000 jeunes de 17 ans en situation de dépendance. | Teneur en THC beaucoup plus élevée qu'il y a 20 ans ; risques de psychose et mal-être accrus. | | Cocaïne | Diffusion croissante dans tous les milieux professionnels. | Risques cardiovasculaires graves (AVC) avant 50 ans ; absence de traitement médical de substitution. | | Protoxyde d'azote | Usage de plus en plus fréquent via de grandes bonbonnes industrielles. | Risques immédiats : brûlures, chutes, paralysies neurologiques graves. | | Jeux d'argent | Croissance de 30 à 40 % (paris sportifs, poker). | Marketing agressif ciblant les milieux défavorisés ; risque financier et isolement. | | Écrans / Jeux vidéo | Usage intensif (plus de 4h/jour pour les 15-24 ans). | Impact sur le sommeil et l'activité physique ; pas de lien direct systématique avec l'échec scolaire. |

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IV. La Prévention par les Compétences Psychosociales (CPS)

La MILDECA préconise de délaisser les "coups médiatiques" ou les interventions policières ponctuelles au profit du développement des CPS.

Ce sont les capacités d'une personne à répondre aux épreuves de la vie et à maintenir un état de bien-être.

Les trois piliers des CPS

• Cognitives : Prise de décision, auto-contrôle, pensée critique face au marketing.

• Émotionnelles : Régulation du stress, gestion des émotions, confiance en soi.

• Sociales : Empathie, communication, résistance à la pression des pairs.

Programmes probants et évalués

Plusieurs programmes ont démontré leur efficacité par des suivis longitudinaux de chercheurs :

• Tina et Tony (4-6 ans) : Activités ludiques en maternelle.

• Good Behavior Game (Élémentaire) : Travail sur le comportement en groupe.

• Unplug (12-14 ans) : 12 séances interactives en collège pour apprendre à dire non et décrypter les influences.

• Primavera : Programme de transition école-collège.

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V. Recommandations pour les Professionnels et les Familles

Posture éducative

• Changement de comportement des adultes : Le développement des CPS nécessite que les adultes incarnent eux-mêmes ces compétences (coopération, gestion non violente des conflits).

• Valorisation positive : La "prédiction de l'échec" par un enseignant peut enfermer l'élève dans un cercle vicieux. À l'inverse, une vision positive favorise la résilience.

• Lutte contre les contrevérités : Il est crucial de déconstruire l'idée que le cannabis est une "drogue douce" ou que l'alcool est inoffensif en milieu familial.

Dispositifs d'aide

• CJC (Consultations Jeunes Consommateurs) : Accueil anonyme et gratuit pour les jeunes et leurs parents.

• Plateformes numériques :

◦ Faminum : Pour réguler l'usage des écrans en famille.    ◦ Maad Digital : Média d'information scientifique sur les addictions adapté aux jeunes.

• Programmes de soutien à la parentalité : Travailler la relation jeune-famille pour renforcer l'environnement protecteur.

En conclusion, la prévention efficace ne consiste plus à parler uniquement des produits, mais à armer les jeunes de capacités relationnelles et émotionnelles leur permettant de faire des choix responsables face à un environnement de plus en plus incitatif.

Author response:

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

eLife Assessment

This valuable study examines the role of E2 ubiquitin enzyme, Uev1a in tissue resistance to oncogenic RasV12 in Drosophila melanogaster polyploid germline cells and human cancer cell lines. The incomplete evidence suggests that Uev1a works with the E3 ligase APC/C to degrade Cyclin A, and the strength of evidence could be increased by addressing the expression of CycA in the ovaries and the uev1a loss of function in human cancer cells. This work would be of interest to researchers in germline biology and cancer.

Thank you for your valuable assessment. The requested data on CycA expression (Figure 4E-G) and uev1a loss-of-function in human cancer cells (Figure 8 and Figure 8-figure supplement 2) have been added to the revised manuscript.

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study uncovers a protective role of the ubiquitin-conjugating enzyme variant Uev1A in mitigating cell death caused by over-expressed oncogenic Ras in polyploid Drosophila nurse cells and by RasK12 in diploid human tumor cell lines. The authors previously showed that overexpression of oncogenic Ras induces death in nurse cells, and now they perform a deficiency screen for modifiers. They identified Uev1A as a suppressor of this Ras-induced cell death. Using genetics and biochemistry, the authors found that Uev1A collaborates with the APC/C E3 ubiquitin ligase complex to promote proteasomal degradation of Cyclin A. This function of Uev1A appears to extend to diploid cells, where its human homologs UBE2V1 and UBE2V2 suppress oncogenic Ras-dependent phenotypes in human colorectal cancer cells in vitro and in xenografts in mice.

Strengths:

(1) Most of the data is supported by a sufficient sample size and appropriate statistics.

(2) Good mix of genetics and biochemistry.

(3) Generation of new transgenes and Drosophila alleles that will be beneficial for the community.

We greatly appreciate your comments.

Weaknesses:

(1) Phenotypes are based on artificial overexpression. It is not clear whether these results are relevant to normal physiology.

Downregulation of Uev1A, Ben, and Cdc27 together significantly increased the incidence of dying nurse cells in normal ovaries (Figure 5-figure supplement 2), indicating that the mechanism we uncovered also protects nurse cells from death during normal oogenesis.

(2) The phenotype of "degenerating ovaries" is very broad, and the study is not focused on phenotypes at the cellular level. Furthermore, no information is provided in the Materials and Methods on how degenerating ovaries are scored, despite this being the most important assay in the study.

Thank you for pointing out this issue. We quantified the phenotype of nurse cell death using “degrading/total egg chambers per ovary”, not “degenerating ovaries”. Normal nurse cell nuclei exhibit a large, round morphology in DAPI staining (see the first panel in Figure 1D). During early death, they become disorganized and begin to condense and fragment (see the second panel in Figure 1D). In late-stage death, they are completely fragmented into small, spherical structures (see the third panel in Figure 1D), making cellular-level phenotypic quantification impossible. Since all nurse cells within the same egg chamber are interconnected, their death process is synchronous. Thus, quantifying the phenotype at the egg-chamber level is more practical than at the cellular level. We have added the description of this death phenotype and its quantification to the main text (Lines 104-108).

(3) In Figure 5, the authors want to conclude that uev1a is a tumor-suppressor, and so they over-express ubev1/2 in human cancer cell lines that have RasK12 and find reduced proliferation, colony formation, and xenograft size. However, genes that act as tumor suppressors have loss-of-function phenotypes that allow for increased cell division. The Drosophila uev1a mutant is viable and fertile, suggesting that it is not a tumor suppressor in flies. Additionally, they do not deplete human ubev1/2 from human cancer cell lines and assess whether this increases cell division, colony formation, and xenograph growth.

We apologize for any misleading description. We aimed to demonstrate that UBE2V1/2, like Uev1A in Drosophila “nos>Ras^G12V+bam-RNAi” germline tumors, suppress oncogenic KRAS-driven overgrowth in diploid human cancer cells. Importantly, this function of Uev1A and UBE2V1/2 is dependent on Ras-driven tumors; there is no evidence that they act as broad tumor suppressors in the absence of oncogenic Ras. Drosophila uev1a mutants were lethal, not viable (see Lines 135-137), and germline-specific knockdown of uev1a (nos>uev1a-RNAi) caused female sterility without inducing tumors. These findings suggest that Uev1A lacks tumor-suppressive activity in the Drosophila female germline in the absence of Ras-driven tumors. We have revised the manuscript to prevent misinterpretation. Furthermore, we have added data demonstrating that the combined knockdown of UBE2V1 and UBE2V2 significantly promotes the growth of KRAS-mutant human cancer cells, as suggested (Figure 8 and Figure 8-figure supplement 2).

(4) A critical part of the model does not make sense. CycA is a key part of their model, but they do not show CycA protein expression in WT egg chambers or in their over-expression models (nos.RasV12 or bam>RasV12). Based on Lilly and Spradling 1996, Cyclin A is not expressed in germ cells in region 2-3 of the germarium; whether CycA is expressed in nurse cells in later egg chambers is not shown but is critical to document comprehensively.

We appreciate your critical comment. CycA is a key cyclin that partners with Cdk1 to promote cell division (Edgar and Lehner, 1996). Notably, nurse cells are post-mitotic endocycling cells (Hammond and Laird, 1985) and typically do not express CycA (Lilly and Spradling, 1996) (see the last sentence, page 2518, paragraph 3 in this 1996 paper). However, their death induced by oncogenic Ras^G12V is significantly suppressed by monoallelic deletion of either cycA or cdk1 (Zhang et al., 2024). Conversely, ectopic CycA expression in nurse cells triggers their death (Figure 4C, D). These findings suggest that polyploid nurse cells exhibit high sensitivity to aberrant division-promoting stress, which may represent a distinct form of cellular stress unique to polyploid cells. In the revised manuscript, we have provided the CycA-staining data, comparing its expression in normal nurse cells versus cells undergoing oncogenic Ras^G12V-induced death (Figure 4E-G).

(5) The authors should provide more information about the knowledge base of uev1a and its homologs in the introduction.

Thank you for your suggestion. In the revised introduction, we have provided a more detailed description of Uev1A (Lines 72-79). Additionally, we have introduced its human homologs, UBE2V1 and UBE2V2, in the main text (Lines 143-145).

Reviewer #2 (Public review):

Summary:

The authors performed a genetic screen using deficiency lines and identified Uev1a as a factor that protects nurse cells from RasG12V-induced cell death. According to a previous study from the same lab, this cell death is caused by aberrant mitotic stress due to CycA upregulation (Zhang et al.). This paper further reveals that Uev1a forms a complex with APC/C to promote proteasome-mediated degradation of CycA.

In addition to polyploid nurse cells, the authors also examined the effect of RasG12V-overexpression in diploid germline cells, where RasG12V-overexpression triggers active proliferation, not cell death. Uev1a was found to suppress its overgrowth as well.

Finally, the authors show that the overexpression of the human homologs, UBE2V1 and UBE2V2, suppresses tumor growth in human colorectal cancer xenografts and cell lines. Notably, the expression of these genes correlates with the survival of colorectal cancer patients carrying the Ras mutation.

Strength:

This paper presents a significant finding that UBE2V1/2 may serve as a potential therapy for cancers harboring Ras mutations. The authors propose a fascinating mechanism in which Uev1a forms a complex with APC/C to inhibit aberrant cell cycle progression.

We greatly appreciate your comments.

Weakness:

The quantification of some crucial experiments lacks sufficient clarity.

Thank you for highlighting this issue. We have provided more details regarding the quantification data in the revised manuscript.

References

Edgar, B.A., and Lehner, C.F. (1996). Developmental control of cell cycle regulators: a fly's perspective. Science 274, 1646-1652.

Hammond, M.P., and Laird, C.D. (1985). Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster. Chromosoma 91, 267-278.

Lilly, M.A., and Spradling, A.C. (1996). The Drosophila endocycle is controlled by Cyclin E and lacks a checkpoint ensuring S-phase completion. Genes Dev 10, 2514-2526.

Zhang, Q., Wang, Y., Bu, Z., Zhang, Y., Zhang, Q., Li, L., Yan, L., Wang, Y., and Zhao, S. (2024). Ras promotes germline stem cell division in Drosophila ovaries. Stem Cell Reports 19, 1205-1216.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) The figure legends insufficiently describe the figures. One example is Figure 3, where there are no details in the figure legend about what conditions apply to each panel and each lane of the gels.

For clarity and brevity, detailed experimental conditions are described in the Materials and Methods section. Figure legends therefore focus on summarizing the key findings. Thank you for your understanding!

(2) The font size on the figure is too small.

Thank you for your constructive suggestion. In response, we have enlarged all font sizes to improve readability.

(3) There are places where the authors overstate their results, and there are issues with the clarity of the text:

(3a) Lines 170: "excessive" is not appropriate. Their prior study showed a mild increase in proliferation.

“Excessive” has been removed in the revised manuscript (Lines 215-216).

(3b) Line 187-8: The authors should restate this sentence. Here's a possibility. Over-expression of Uev1a suppressed the phenotypes caused by CycA over-expression.

This sentence has been restated as “Notably, this cell death was suppressed by co-overexpression of CycA and Uev1A, indicating a genetic interaction between them”. (Lines 229-231).

(3c) Lines 266-7: The properties of Uev1a (ie, lacking a conserved Cys) should be in the introduction.

This information has been added to the revised introduction (Lines 74-76).

(3d) Line 318: "markedly" is an overstatement of the prior results.

Our quantification data revealed that “nos>Ras^G12V; bam^-/-” ovaries are three times larger than “nos>GFP; bam^-/-” control ovaries (see Figure 4A-C in Zhang et al., Stem Cell Reports 19, 1205-1216). Given this substantial difference, we think that using "markedly" is not an overstatement.

(4) Data not shown occurs in a few places in the text. Given the ability to supply supplemental information in eLife preprints, these data should be shown.

Thanks for your suggestion. All “not shown” data have been added to the revised manuscript.

Reviewer #2 (Recommendations for the authors):

Major Comments

(1) Cyclin A (CycA) is a key player in this study, but the authors do not provide evidence showing the upregulation of CycA following Ras overexpression in either polyploid or diploid cells. Data on CycA expression should be included.

Thank you for your constructive suggestion. These data have been added to the revised manuscript (Figure 4E-G).

(2) DNA replication stress, cellular senescence, and cell death should be assessed under Ras overexpression (RasOE) and RasOE + Uev1A RNAi conditions to support the model proposed in Figure 4F.

We apologize for any confusion caused by our initial model. We do not have evidence that DNA replication stress and cellular senescence occur under these conditions. Cell death can be readily detected through the presence of fragmented nuclei and condensed DNA (see Figure 1D). The model has been updated accordingly (Figure 9E).

(3) Appropriate controls should be performed alongside the experimental sets. The same nos>Ras+GFPi data set was repeatedly used in Figures 1I, 2B, 2H, and Figures 2, S2B, which is not ideal.

All these experiments were performed under identical conditions. Therefore, we deem it appropriate to use the same control data across these analyses.

(4) Overall, the microscopic images are too small and hard to see.

Thank you for raising this important point. In the revised manuscript, all images and the font size on figures have been enlarged for improved clarity.

(5) Figure 1H

Why is the frequency of egg chamber degradation quite less in nos>RasG12V+GFP-RNAi (about 40%) than nos > RasG12V (about 80%)? And the authors do not show that there is a significant difference between those two conditions, although it should be there. We will need the explanation from the authors on why there is a difference here.

These overexpression experiments were conducted using the GAL4/UAS system. While both “nos>Ras^G12V+GFP-RNAi” and “nos>Ras^G12V” contain a single nos-GAL4 driver, they differ in UAS copy number: the former incorporates two UAS elements compared to only one in the latter (see the detailed genotypes in Source data 2). These results demonstrate that UAS copy number impacts experimental outcomes in our system.

In the previous paper (Zhang et al. (2024), Figure 7H shows that the frequency of egg chambers in nos>RasG12V is 33%, although this paper shows it as about 80%. There seems to be a difference in flies' age (previous paper: 7d, this paper: 3d), but this data raises the question of why nos>RasG12V shows more egg chamber degradation this time.

We greatly appreciate your careful observation. The nurse-cell-death phenotype exhibits a spectrum from mild to severe manifestations [see Figure 1D and our response to weekness (2) in Reviewer #1’s public reviews]. While our 2024 paper exclusively quantified egg chambers with severe phenotypes as degrading, the current study included both mild and severe cases in this classification. We do not think fly age could account for this substantial phenotypic difference. A detailed description of the nurse-cell-death phenotype and its quantification have been added to the revised manuscript (Lines 104-108).

In the following experiments, only nos>RasG12V+GFP-RNAi is used as a control (Figures 2B, H, S2B). I wonder if these results would give us a different conclusion if nos>RasG12V were used as a control.

As explained above, the UAS copy number does matter in our analyses, so it is important to keep them identical for comparison.

(6) In the abstract, the authors mention that uev1a is an intrinsic factor to protect cells from RasG12V-induced cell death. RasG12V does not induce much cell death of cystocytes with bam-gal4, whereas it induces a lot of nurse cells' death. Does it mean the intrinsic expression level of uev1a is low in nurse cells (or polyploid cells) compared to cystocytes (or diploid cells)?

Overexpression of Ras^G12V driven by bam-GAL4 exhibited only minimal nurse cell death (Figure 1D, E). Additionally, Uev1A exhibited low intrinsic expression levels in both cystocytes and nurse cells (Figure 3E and Figure 5-figure supplement 1).

(7) Is uev1a-RNAi alone sufficient to induce egg chamber degradation? Or does it have any effect on ovarian development? (Related to question #1 in minor comments)

While nos>uev1a-RNAi resulted in female sterility, it alone was insufficient to induce egg chamber degradation. However, simultaneous downregulation of Uev1A, Ben, and Cdc27 triggered significant egg chamber degradation (Figure 5-figure supplement 2).

(8) Which stages of egg chambers get degraded with RasG12V induction?

This is a good question. In our analyses, we noted that degrading egg chambers exhibited considerable size variability (Figure 1D). Because degradation disrupts normal morphological cues, precise staging of these egg chambers is nearly impossible.

(9) I suggest testing the cellular senescence marker as well if the authors mention that CycA-degradation by Uev1a-APC/C complex prevents cellular senescence induced by RasG12V in a schematic image of Figure 4 (e.g., Dap/p21, SA-β-gal).

As addressed in our response to your Major Comment (2), we lacked experimental evidence to support cellular senescence in this context. We have therefore revised the model accordingly (Figure 9E). While this study focuses specifically on cell death, investigating potential roles of cellular senescence remains an important direction for future research. Thank you for your suggestion!

Minor Comments

(1) Figure 1D: Df#7584

It seems that the late-stage egg chamber is missing in this condition. Why does this occur without egg chamber degradation? Is there a possibility that we do not see egg chamber degradation because this deficiency line does not have a properly developed egg chamber that can have a degradation?

While this image represents only a single sample, we have confirmed the presence of late-stage egg chambers in other samples. If “Df#7584/+” females were unable to support late-stage egg chamber development, complete sterility would be expected due to the lack of mature eggs. However, as shown in this image (Figure 1D), the ovary contains mature eggs, and the “Df#7584/+” fly strain remains fertile.

(2) Based on the results that DDR signaling functions as keeping egg chambers from degradation, the authors may be better to check the DNA-damage markers in nos>RasG12V, nos>RasG12V +uev1a. (e.g. γ-H2AX)

Thank you for your constructive recommendation. These data have been added to the revised manuscript (Figure 3C).

Traumas, Criminalité et Judiciarisation : Analyse des Trajectoires de Rétablissement des Jeunes Hommes

Résumé Exécutif

Ce document synthétise les recherches menées par l'Institut universitaire Jeunes en difficulté sur les liens profonds entre les expériences traumatiques vécues durant l'enfance (ACE) et les parcours criminels des garçons et jeunes hommes au Québec.

L'analyse révèle que la population judiciaire masculine présente une surreprésentation massive de traumas complexes, souvent négligés par rapport à ceux des femmes.

Ces traumas altèrent le développement neurologique et créent une « mentalité de zone de guerre » où la déviance devient une stratégie de survie logique.

Le processus de « désistement » (l'abandon de la criminalité) ne se limite pas à l'arrêt des délits, mais nécessite une transformation identitaire profonde, souvent entravée par un système carcéral qui génère de nouveaux traumatismes.

L'intervention doit impérativement évoluer vers des approches sensibles aux traumas pour briser le cycle de la violence et de la réincarcération.

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1. Cadre Conceptuel des Expériences Potentiellement Traumatisantes (EPT)

Définition et Prévalence

Les expériences potentiellement traumatisantes vécues durant l’enfance (souvent appelées ACE - Adverse Childhood Experiences) sont des événements de sévérité variable, souvent chroniques, survenant dans l'environnement familial ou social. Elles perturbent le développement physique et psychologique.

Les dix catégories principales identifiées sont :

• 1. Abus émotionnel
• 2. Abus physique
• 3. Abus sexuel
• 4. Négligence émotionnelle
• 5. Négligence physique
• 6. Violence familiale
• 7. Usage de substances chez un parent
• 8. Incarcération d'un parent
• 9. Séparation ou divorce des parents
• 10. Placement hors de la famille d'origine

Impacts Statistiques sur la Santé et le Comportement

L'exposition à ces expériences multiplie de manière exponentielle les risques à l'âge adulte :

• Santé mentale : Une personne exposée à sept traumas durant l'enfance a 980 % de risques supplémentaires de développer un trouble de santé mentale.

• Suicide : Le risque de tentative de suicide est 30 fois plus élevé chez les personnes ayant vécu plusieurs ACE.

• Dépendances : Risque 5 fois plus élevé pour l'alcoolisme et 10 fois plus élevé pour la toxicomanie (drogues illicites).

• Victimisation : Risque 7 fois plus élevé d'être victime de violence à l'âge adulte.

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2. Mécanismes de Liaison : Du Trauma à la Délinquance

Impacts Neurobiologiques

Les traumas affectent des zones critiques du cerveau, expliquant certains comportements dits « criminels » :

• Hippocampe : Atrophie ou dysfonctionnement impactant la régulation des émotions.

• Lobe préfrontal : Altération de la gestion des émotions, des communications interpersonnelles et du raisonnement moral.

• Fonctions exécutives : Difficulté à contrôler les impulsions, à planifier l'avenir et à réagir aux renforcements (positifs ou négatifs).

Cela rend les approches classiques cognitivo-comportementales moins efficaces si le trauma n'est pas traité.

Le Trauma Complexe et la Masculinité

Le trauma complexe, bien que non encore intégré au DSM-5, est reconnu internationalement. Chez les garçons, il se manifeste souvent par :

• La « Mentalité de zone de guerre » : Le jeune perçoit le monde comme hostile et traite tout étranger comme un ennemi potentiel. La déviance est alors perçue comme une réponse logique et justifiée.

• Insensibilité et retrait : Sous l'influence d'une vision hégémonique de la masculinité (stoïcisme, force), les jeunes hommes peuvent refuser l'aide, se replier sur eux-mêmes ou paraître dénués d'empathie, ce qui est en réalité un symptôme traumatique.

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3. Le Cycle de la Violence et de l'Incarcération

Le système actuel tend à nourrir un cercle vicieux plutôt qu'à le briser :

1. Trauma initial : Exposition aux ACE.

2. Stratégies d'adaptation : Usage de drogues, criminalité pour survie ou appartenance.

3. Incarcération : Souvent vécue comme un nouveau traumatisme. Les mesures de coercition, l'isolement et la violence entre détenus exacerbent les symptômes de stress post-traumatique.

4. Conséquences carcérales : Les personnes ayant vécu au moins quatre ACE ont 15 fois plus de risques de s'automutiler et 8 fois plus de risques de tenter de se suicider en prison.

« Je ne me sens pas en sécurité en ce moment, ni dehors, ni en dedans. Si je rentre en dedans... je n'aurai pas le choix de me crisser la corde autour du cou, sinon il y en a d'autres qui vont le faire. » — Témoignage d'un jeune judiciarisé.

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4. Les Trajectoires de Désistement du Crime

Le désistement n'est pas simplement l'absence de récidive, mais un processus identitaire décliné en trois niveaux :

• Primaire : Une simple pause ou accalmie dans les activités criminelles.

• Secondaire : Changement d'identité (ne plus se percevoir comme un contrevenant).

• Tertiaire : Reconnaissance sociale et intégration pleine dans la communauté.

Typologies des parcours de désistement

| Type | Caractéristiques | Besoins | | --- | --- | --- | | Convertis | Faible statut socio-économique, besoin d'appartenance comblé par le crime. | Soutien communautaire massif pour adopter une identité prosociale. | | Repentants | Statut social favorable, délits rationalisés, peu d'ACE. | L'arrestation suffit souvent à provoquer la prise de conscience. | | Rescapés | Grand isolement, troubles de santé mentale sévères, multiples ACE. | Équipes multidisciplinaires spécialisées (santé, logement, pharmacologie). |

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5. Le Cas Particulier des Gangs de Rue : Blessures Morales

Pour les jeunes affiliés aux gangs, le trauma prend la forme de blessures morales :

• Trahison : Le gang, initialement perçu comme une famille de substitution face à la négligence parentale, finit par exploiter la vulnérabilité du jeune.

• Dissonance cognitive : Sentiment de honte et de culpabilité lié aux actes violents commis sous pression.

• Syndrome de Stockholm : Développement d'un lien affectif fondé sur le trauma envers ceux qui les mettent en danger.

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6. Pistes d'Intervention et Recommandations

L'analyse conclut à l'urgence de transformer les pratiques judiciaires et cliniques :

1. Intervention sensible aux traumas : Tester des modèles (comme le Special Housing Unit aux États-Unis) qui forment le personnel et les détenus.

Résultats observés : diminution de l'anxiété, de la dépression et des agressions physiques.

2. Dépistage systématique des ACE : Comprendre le passé pour ne pas voir le jeune comme un « déchet » (terme cité par les répondants) mais comme un individu en réaction à son milieu.

3. Humanisation des services correctionnels : Réduire l'utilisation de la force et de l'isolement, particulièrement pour ceux ayant des troubles de santé mentale.

4. Rétablir l'espoir : Le désistement est possible pour la majorité si l'on agit sur la santé mentale, les dépendances et la création de nouvelles relations sociales valorisantes.

« On n'est pas des déchets... on est des êtres vivants pareils. » — Appel à la reconnaissance de la dignité humaine par un jeune incarcéré.

Author response:

We thank the reviewers for their thorough and constructive evaluation of our manuscript titled “PSD-95 drives binocular vision maturation critical for predation”. The reviewers raised several important conceptual and technical points. Here, we address and provide additional context on the major themes and outline our planned revisions.

We acknowledge that the current prey capture task cannot directly adjudicate between PSD-95 binocular vision impairments or sensorimotor processing deficits. However, we did not observe any major impairment supporting a sensorimotor processing deficit, in contrast to a major impairment in line with binocular vision impairment. Evidence from Huang et al. (2015) [1], Favaro et al. (2018) [2] and our data with the visual water task (VWT) — thus requiring identical sensorimotor but differential visual processing—clearly demonstrated intact visual acuity but impaired orientation discrimination in PSD-95 KO mice. Therefore, we believe that a binocular integration deficit is the most likely explanation of PSD-95 KO binocular impairments. In line with this, it is unlikely that aberrations in binocular eye movements account for the observations. We appreciate that alternative explanations remain possible and merit explicit discussion. Accordingly, we intend to expand the discussion of these alternatives.

Importantly, we will provide additional experimental data demonstrating that knock-down of PSD-95 in V1 but not in superior colliculus, significantly decreases orientation discrimination analyzed with the VWT, as we had shown for PSD-95 KO mice (while control knock-down does not have this effect). We believe that this new evidence better delineates the potential neuroanatomical locus of the PSD-95-associated deficits.

Furthermore, we will provide additional head movement analyses, as suggested by Reviewer 1. Specifically, we will investigate the head angle in relation to the cricket (azimuth) in time (±1 second) around prey contact under light and dark conditions.

We will also address the potential impact of PSD-95 KO learning deficits. We agree that there are more impairments in the PSD-95 KO brain, as has been published previously. But strikingly, the binocular impairment was dominating the sensory processing. This cannot be convincingly explained by learning deficits. In fact, we have observed improved learning of PSD-95 KO mice with some tasks (e.g. cocaine conditioned place preference) [3], but no significant differences in the VWT [1,2]. Learning differences were described for another PSD-95 mouse line, expressing the N-terminus with two PDZ domains [4]. To avoid potential learning dependent confounds, we have chosen salient stimuli, like water aversion, and prey capture to reduce impacts of potential learning defects.

We agree on the strength of the random dot stereograms to isolate stereoscopic computations. However, it requires special filters in front of either eye, which renders it unsuitable for the VWT. The lengthy training with less silent stimuli of water reward, could potentially add additional confounds of PSD-95 KO deficits. Thus, we think that this would be something for future experiments to allow for integration of different visual inputs. However, the combined improved performance of WT mice with binocular vision for prey capture (depth percept) and orientation discrimination (summation) is already supporting the importance of binocular vision in mice and the dominant defect in PSD-95 KO mice.

Finally, we will address the other points raised by the reviewers through clearer exposition and reorganization of the manuscript.

Once again, we would like to thank the reviewers for their thoughtful and constructive feedback, which we believe will substantially strengthen the manuscript.

(1) Huang, X., Stodieck, S. K., Goetze, B., Cui, L., Wong, M. H., Wenzel, C., Hosang, L., Dong, Y., Löwel, S., and Schlüter, O. M. (2015). Progressive maturation of silent synapses governs the duration of a critical period. Proc. Natl. Acad. Sci. 112, E3131–E3140. https://doi.org/10.1073/pnas.1506488112.

(2) Favaro, P.D., Huang, X., Hosang, L., Stodieck, S., Cui, L., Liu, Y., Engelhardt, K.-A., Schmitz, F., Dong, Y., Löwel, S., et al. (2018). An opposing function of paralogs in balancing developmental synapse maturation. PLOS Biol. 16, e2006838. https://doi.org/10.1371/journal.pbio.2006838.

(3) Shukla, A., Beroun, A., Panopoulou, M., Neumann, P.A., Grant, S.G., Olive, M.F., Dong, Y., and Schlüter, O.M. (2017). Calcium‐permeable AMPA receptors and silent synapses in cocaine‐conditioned place preference. EMBO J. 36, 458–474. https://doi.org/10.15252/embj.201695465.

(4) Migaud, M., Charlesworth, P., Dempster, M., Webster, L.C., Watabe, A.M., Makhinson, M., He, Y., Ramsay, M.F., Morris, R.G.M., Morrison, J.H., et al. (1998). Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein. Nature 396, 433–439. https://doi.org/10.1038/24790.

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DOI: 10.1038/s41556-026-01879-y

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DOI: 10.1016/j.immuni.2026.01.028

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DOI: 10.1007/s12011-025-04961-y

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DOI: 10.1007/s12011-025-04961-y

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DOI: 10.1101/pdb.rec10907

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Curator: @areedewitt04

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What is this?

Hay muchos campos actualmente donde se aplica la ciencia de la información tal como menciona este apartado que abarca la mayoría sino todos los aspectos de la vida actualmente, tanto a nivel personal como a nivel organizacional o de estado/gobierno, en calidad de usuario y proveedor o comunidad para comunidad. Al tener una presencia tan indiscutible hace que la profundización de sus procesos e investgaciones generen valor dentro de la misma disciplina tanto como para los que se benefician de los resultados.

Este apartado tiene un poder y peso bastante grandes. El impacto de las redes sociales en las personas y en la industria es algo de doble filo, si bien se mencionan cosas tales cómo que "el impacto de la opinión pública puede extenderse de maneras inimaginables" y que las personas están interesadas en las nuevas formas en cómo se recibe la información, hay que tener en cuenta que esta misma información queda expuesta en la red (a veces hasta sin consentimiento ni conocimiento de las personas), Facebook y su creador se han vuelto inmersos en varias polémicas cómo ya sabemos, pero es este el riesgo de manejar datos sensibles, los algoritmos moldean a las personas, las redes sociales vuelven dependientes a estas personas, si bien la forma en cómo nos comunicamos y consumimos información ha crecido a pasos gigantes cabe recalcar que vamos perdiendo humanidad e identidad propia a base del consumismo provisto por la industria

Si bien, se define la comunicación de la información cómo algo "unilateral" -que está muy bien, dependiendo desde que arista se vea- cambiaría esto (Incluso con la mención "desde antes de la llegada del Internet") al hecho de que esto puede llegar a modificarse cómo algo BILATERAL en algunos o en la gran mayoría de casos.

Un caso puntual sería, donde un individuo difunde información y esta llega a un receptor o un espacio receptivo que está a la espera de este conocimiento para seguir difundiéndolo interactúa con este primer individuo y su conocimiento compartido creando y generando el famoso "intercambio de saberes".

Esto nace de que no es que haya un solo creador de conocimiento que simplemente se encarga de difundirlo y ya, sino que en su lugar, aparecería un agente externo que lo recibe e intercambia conocimiento con este

Me gusta cómo se hace un pequeño desglose (de la GRAN variedad de carreras vinculadas) sus características y funciones, ya que brinda una perspectiva llamativa sobre en donde y en qué se puede desempeñar un individuo, sin embargo, para hacer un poco más llamativo el apartado sobre las carreras, recomendaría asociar más de estas mismas

Si bien es una concepción general muy contundente y clara, desde un punto subjetivo no deja de ser susceptible a cambios y redefiniciones dependiendo de la persona y el campo desde el que se explique, ya que esto es un campo de acción tan amplio que no se puede limitar a una simple (o única) definición

Esto es algo completamente cierto que muchos de nosotros podemos llegar a olvidar o no tener en cuenta y es que se vive esa cooperación o integración de varios campos de acción, conocimiento o profesiones que permiten buscar soluciones más integrales en cooperativo

Si bien podría considerarse algo "básico" no puedo dejar pasar que es un bien necesario el hecho que es bueno eso de aclararle de forma inicial, a la audiencia y/o lectores que no hay que confundir o tergiversar conceptos por más parecidos o afines que sean.

A demás de lo que se menciona sobre la ciencia de la informacion, agregaria que es una necesidad del ser humano para su desarrollo integral.

La gestión de estas áreas en conjunto, permiten que la información sea accesible, confiable y útil para la sociedad.

Las nuevas tecnologías aportan en gran medida a la investigación científica porque permite que los académicos lleven a cabo proyectos de forma remota y, así, culminarlos en un periodo de tiempo más corto que antes.

Podríamos considerar que se está hablando de comunicación científica, en específico, R&D collaboration (investigación y desarrollo). Ya que, se habla de varias instituciones trabajando juntas para lograr el avance de la ciencia.

Sea en el contexto social u organizacional existe un factor relevante que abordar en esta ciencia: la ética. Es la capacidad de lograr la integridad personal y laboral en nuestro oficio.

Esta ciencia es trasversal, los científicos de la información se adaptan a los entornos de estudio para integrarse y promover el conocimiento.

Agregaría a esta afirmación que la información deber ser también legitimizada, llegar hasta su fuente y complementarla con su contexto para así acercarnos desde un perspectiva óptima.

Este debate me trae recuerdos de mi primer semestre, ya que fue uno de los primeros temas que vi en la asignatura de ciencia de la información, en ese entonces una de las conclusiones que fue nuestro objeto de estudio es la información y todo lo relacionado a esta.

Aquí se encapsula lo que había mencionado sobre la importancia de la interacción con los usuarios y como somos una disciplina del servicio, sin embargo debo admitir que me sorprende algunas de las áreas incluidas, como es el caso de la psicología cognitiva, que a primera instancia me la imaginaria más en otros campos.

Efectivamente, el componente técnico y computacional es muy importante, y no solo es nuestro campo, el avance tecnológico nos obliga adaptarnos a esas nuevas tendencias o correr el riesgo de ser obsoletos, sin embargo no se la da la importancia que se le debe en la carrera.

Considero esta definición como correcta, sin embargo en considero que también es necesario agregar que somos una disciplina del servicio a los usuarios y por lo tanto nuestras interacciones con el publico fuera de la unidades de información es de igual forma importante.

Reviewer #1 (Public review):

Summary:

Abdelmageed et al. investigate age-related changes in the subcellular localization of DNA polymerase kappa (POLK) in the brains of mice. POLK has been actively investigated for its role in translesion DNA synthesis and involvement in other DNA repair pathways in proliferating cells, very little is known about POLK in a tissue-specific context or let alone in post-mitotic cells. The authors investigated POLK subcellular distribution in the brains of young, middle-aged, and old mice via immunoblotting of fractioned tissue extracts and immunofluorescence (IF). Immunoblotting revealed a progressive decrease in the abundance of nuclear POLK, while cytoplasmic POLK levels concomitantly increased. Similar findings were present when IF was performed on brain sections. Further IF studies of cingulate cortex (Cg1), motor cortex (M1, M2), and somatosensory (S1) cortical regions all showed an age-related decline in nuclear POLK. Nuclear speckles of POLK decrease in each region, meanwhile the number of cytoplasmic POLK granules decreases in all four regions, but granule size is increasing. The authors report similar findings for REV1, another Y-family DNA polymerase.

The authors then investigate the colocalization of POLK with other DNA damage response (DDR) proteins in either pyramidal neurons or inhibitory interneurons. At 18 months of age, DNA damage marker gH2AX demonstrated colocalization with nuclear POLK, while strong colocalization of POLK and 8-oxo-dG was present in geriatric mice. The authors find that cytoplasmic POLK granules colocalize with stress granule marker G3BP1, suggesting that the accumulated POLK ends up in the lysosome.

Brain regions were further stained to identify POLK patterns in NeuN+ neurons, GABAergic neurons, and other non-neuronal cell types present in the cortex. Microglia associated with pyramidal neurons or inhibitory interneurons were found to have higher abundance of cytoplasmic POLK. The authors also report that POLK localization can be regulated by neuronal activity induced by Kainic acid treatment. Lastly, the authors suggest that POLK could serve as an aging clock for brain tissue, but POLK deserves further characterization and correlation to functional changes before being considered for a biomarker.

Strengths:

Investigation of TLS polymerases in specific tissues and in post-mitotic cells is largely understudied. The potential changes in sub cellular localization of POLK and potentially other TLS polymerases opens up many questions about DNA repair and damage tolerance in the brain and how it can change with age.

Weaknesses:

The work is quite novel and interesting, and the authors do suggest some potentially interesting roles for POLK in the brain, but these are in of themselves a bit speculative. The majority of the findings of this paper draw upon findings from POLK antibody and its presumed specificity for POLK. However, this antibody has not been fully validated and would benefit from further validation of the different band sizes. More mechanistic investigation is needed before POLK could be considered as a brain aging clock but does not preclude the potential for using POLK as a biological "dating" system for the brain.

Comments on revisions:

The revised manuscript is suitably improved and addresses reviewer comments.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

Abdelmageed et al. investigate age-related changes in the subcellular localization of DNA polymerase kappa (POLK) in the brains of mice. POLK has been actively investigated for its role in translesion DNA synthesis and involvement in other DNA repair pathways in proliferating cells, very little is known about POLK in a tissue-specific context, let alone in post-mitotic cells. The authors investigated POLK subcellular distribution in the brains of young, middle-aged, and old mice via immunoblotting of fractioned tissue extracts and immunofluorescence (IF). Immunoblotting revealed a progressive decrease in the abundance of nuclear POLK, while cytoplasmic POLK levels concomitantly increased. Similar findings were present when IF was performed on brain sections. Further, IF studies of the cingulate cortex (Cg1), the motor cortex (M1, M2), and the somatosensory (S1) cortical regions all showed an age-related decline in nuclear POLK. Nuclear speckles of POLK decrease in each region, meanwhile, the number of cytoplasmic POLK granules decreases in all four regions, but granule size is increasing. The authors report similar findings for REV1, another Y-family DNA polymerase.

The authors then investigate the colocalization of POLK with other DNA damage response (DDR) proteins in either pyramidal neurons or inhibitory interneurons. At 18 months of age, DNA damage marker gH2AX demonstrated colocalization with nuclear POLK, while strong colocalization of POLK and 8-oxo-dG was present in geriatric mice. The authors find that cytoplasmic POLK granules colocalize with stress granule marker G3BP1, suggesting that the accumulated POLK ends up in the lysosome.

Brain regions were further stained to identify POLK patterns in NeuN+ neurons, GABAergic neurons, and other non-neuronal cell types present in the cortex. Microglia associated with pyramidal neurons or inhibitory interneurons were found to have a higher abundance of cytoplasmic POLK. The authors also report that POLK localization can be regulated by neuronal activity induced by Kainic acid treatment. Lastly, the authors suggest that POLK could serve as an aging clock for brain tissue, but POLK deserves further characterization and correlation to functional changes before being considered as a biomarker.

Strengths:

Investigation of TLS polymerases in specific tissues and in post-mitotic cells is largely understudied. The potential changes in sub-cellular localization of POLK and potentially other TLS polymerases open up many questions about DNA repair and damage tolerance in the brain and how it can change with age.

Weaknesses:

The work is quite novel and interesting, and the authors do suggest some potentially interesting roles for POLK in the brain, but these are in and of themselves a bit speculative. The majority of the findings of this paper draw upon findings from POLK antibody and its presumed specificity for POLK. However, this antibody has not been fully validated and needs further work. Further validation experiments using Polk-deficient or knocked-down cells to investigate antibody specificity for both immunoblotting and immunofluorescence should be performed. More mechanistic investigation is needed before POLK could be considered as a brain aging clock.

We are thankful for the overall enthusiasm and positive comments.

(a) Concern over POLK antibody characterization in mouse:

We performed siRNA and shRNA knock downs in mouse primary cortical neurons as well as efficiently transfectable murine lines like 4T1 and Neuro-2A showing knock down of 99kDa and 120kDa bands recognized by sc-166667 anti-POLK antibody (exact figure number Figure 1 and S1). We show that in IF sc-166667 and A12052 (Figure S1G) shows similar immunostaining patterns and we used sc-166667 in all reported figures and western blots.

(b) More mechanistic investigation is needed before POLK could be considered as a brain aging clock:

We sincerely appreciate the valuable suggestion. We agree as a terminal assay POLK nucleo-cytoplasmic status is not practical for longitudinal studies. However, we believe it may serve an investigative/correlative endogenous signal for determining tissue age, that may be useful to "date" brain sections, since not many such cell biological markers exist. We have added clarification texts to address this.

Reviewer #2 (Public review):

Summary:

Abdelmageed et al., demonstrate POLK expression in nervous tissue and focus mainly on neurons. Here they describe an exciting age-dependent change in POLK subcellular localization, from the nucleus in young tissue to the cytoplasm in old tissue. They argue that the cytosolic POLK is associated with stress granules. They also investigate the cell-type specific expression of POLK, and quantitate expression changes induced by cell-autonomous (activity) and cell nonautonomous (microglia) factors.

I think it is an interesting report but requires a few more experiments to support their findings in the latter half of the paper. Additionally, a more mechanistic understanding of the pathways regulating POLK dynamics between the nucleus and cytosol, what is POLK doing in the cytosol, and what is it interacting with; would greatly increase the impact of this report. However, additional mechanistic experiments are mostly not needed to support much of the currently presented results, again, it would simply increase the impact.

(a) Concern on more mechanistic understanding of the pathways regulating POLK dynamics between the nucleus and cytosol:

We sincerely appreciate the reviewer’s enthusiasm and valuable guidance in helping us better understand the mechanism of nuclear-cytoplasmic POLK dynamics. Previously, we developed a modified aniPOND (accelerated native isolation of proteins on nascent DNA) protocol, which we termed iPoKD-MS (isolation of proteins on Pol kappa synthesized DNA followed by mass spectrometry), to capture proteins bound to nascent DNA synthesized by POLK in human cell lines (bioRxiv https://www.biorxiv.org/content/10.1101/2022.10.27.513845v3). In this dataset, we identified potential candidates that may regulate nuclear/cytoplasmic POLK dynamics. These candidates are currently undergoing validation in human cell lines, and we are preparing a manuscript on these findings. Among these, some candidates, including previously identified proteins such as exportin and importin (Temprine et al., 2020, PMID: 32345725), are being explored further as potential POLK nuclear/cytoplasmic shuttles. We are also conducting tests on these candidates in mouse cortical primary neurons to assess their role in POLK dynamics. In the revised version of the manuscript, we have included a discussion of our current understanding.

(b) Question on “… what is POLK doing in the cytosol, and what is it interacting with …”: Our data so far indicate that POLK accumulates in stress granules and lysosomes. We are very grateful for the reviewer’s insightful suggestions and will make every effort to incorporate them in the revised manuscript. We characterized POLK accumulation in the cytoplasm using six additional endo-lysosomal markers, as recommended by the reviewer. This data is now part of entirely new Figure 3.

Reviewer #3 (Public review):

Summary:

In this study, the authors show that DNA polymerase kappa POLK relocalizes in the cytoplasm as granules with age in mice. The reduction of nuclear POLK in old brains is congruent with an increase in DNA damage markers. The cytoplasmic granules colocalize with stress granules and endo-lysosome. The study proposes that protein localization of POLK could be used to determine the biological age of brain tissue sections.

Strengths:

Very few studies focus on the POLK protein in the peripheral nervous system (PNS). The microscopy approach used here is also very relevant: it allows the authors to highlight a radical change in POLK localization (nuclear versus cytoplasmic) depending on the age of the neurons. 

The conclusions of the study are strong. Several types of neurons are compared, the colocalization with several proteins from the NHEJ and BER repair pathways is tested, and microscopy images are systematically quantified.

Weaknesses:

The authors do not discuss the physical nature of POLK granules. There is a large field of research dedicated to the nature and function of condensates: in particular numerous studies have shown that some condensates but not all exhibit liquid-like properties (https://www.nature.com/articles/nrm.2017.7, https://pubmed.ncbi.nlm.nih.gov/33510441/ https://www.mdpi.com/2073-4425/13/10/1846). The change of physical properties of condensates is particularly important in cells undergoing stress and during aging. The authors should discuss this literature.

We highly appreciate the reviewer bringing up the context of biomolecular condensates. Our iPoKD-MS data referenced above suggests candidates from various biomolecular condensates that we are currently investigating. We appreciate the reviewer providing important literature cited these articles in text and potential biomolecular condensates are discussed in the revised version. 

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

The work is quite novel and interesting, and the authors do suggest some potentially interesting roles for POLK in the brain, but these are in of themselves a bit speculative. The majority of the findings of this paper rely upon the POLK antibody and its specificity for POLK, which is not fully characterized and needs further work (validation of antibodies using immunoblots of Polk KO cells or siRNA KD of POLK in murine cells) to provide confidence in the authors' findings. 

Points

siRNA knockdown of Polk in primary neurons showed a dramatic reduction in signal by IF even though qPCR analysis showed a reduction of only ~35% at the transcript level. Typically many DNA repair genes need to be knocked down by 80% or more to see discernable differences at the protein level. siRNA knockdown in a murine cell line (MEFs, neurons, or some other easily transfectable cell type) needs to be performed with immunoblotting with whole cell and fractionated (nuclear/cytoplasmic) lysates in order to better validate the anti-POLK antibodies and which bands that are visualized during immunoblotting are specific to POLK.

We performed siRNA and shRNA knock downs in mouse primary cortical neurons as well as efficiently transfectable murine lines like 4T1 and Neuro-2A showing knock down of 99kDa and 120kDa bands recognized by sc-166667 anti-POLK antibody (exact figure number Figure 1 and S1). We show that in IF sc-166667 and A12052 (Figure S1G) shows similar immunostaining patterns and we used sc-166667 in all reported figures and western blots.

Figure 1B and C, it is not clear which antibody(ies) are used for the immunoblotting of nuclear and cytoplasmic fractions and for a blot with whole tissue lysates. Please place the antibody vendor or clone next to the corresponding blot or describe it in the figure legend. Bands of varying sizes are present in 1B (and Figure S1) but only a band at 99 kDa was shown in 1C. Because there are no bands of equivalent size present in the nuclear and cytoplasmic fractions in Figure 1B, please describe or denote which bands were used for quantification purposes for nuclear and cytoplasmic POLK.

This has been clarified by using only one antibody throughout the manuscript sc-166667. We observed in whole cell lysate an intense ~99kDa and a faint ~120kDa band, which gets intense in nuclear fraction and is absent in cytoplasmic fraction. We have noted this in multiple human cell lines and hiPSC-derived neurons, which is our ongoing work. We do not know yet if the ~120kDa is a modification or isoform of POLK. We have hints from our proteomics data that it may be SUMOylated or ubiquitinylated or other post translational modifications. We added this in the discussion section.

Figure 1I, is there a quantification beyond just the representative image? There is no green staining pattern outside the cytoplasm in the 1-month-old M1 images that is present in all the other images in the panel.

Fig 1I is now Fig S1G in the revised manuscript. Since REV1 and POLH were not central to the study that focused on POLK, they were meant to be exploratory data panels and as such we did not quantify beyond the qualitative evaluation, which broadly resembled POLK’s disposition with age. We have noted there are some sample to sample variability in the background signal. In general, outside the cytoplasm as subcellularly segmented by fluorescent nissl expression, tends to be variable by brain areas but also higher in older brains

"Association with PRKDC further suggests POLK's role in the "gap-filling" step in the NHEJ repair pathway in neurons." There is no strong evidence in the literature for mammalian POLK playing a role in NHEJ. Some description of a role in HR has been described, however. The reference regarding the iPoKD-MS data set that provides evidence of POLK associating with BER and NHEJ factors is listed as Paul, 2022 but is in the reference list as Shilpi Paul 2022.

We removed this speculative statement and citation fixed.

Figure 4A, what is the age of the mouse for the representative images?

19 months and now mentioned in the figure legend

Figure 4C, Could the data from the different ages be plotted side by side to better evaluate the differences for each cell type/region?

Data is plotted side by side

Why was the one-month time point chosen as this could still represent the developing and not mature murine brain? 

Reviewer correctly noted that a 1 month brain is still developing, but mostly from the behavioral and circuit maturation standpoint. However, from cell division and neurogenesis perspective, that is considered to be complete by first postnatal month, with neuron production thereafter largely restricted to specialized adult niches in the dentate gyrus and subventricular zone–olfactory bulb pathway; these adult neurogenic stem cells are embryonically derived and are regulated in ways that are distinct from the early, expansionary developmental waves of neurogenesis. In our study we performed our measurements in the cortical areas only. (Caviness et al., 1995, PMID: 7482802; Ansorg et al., 2012, PMID: 22564330; Ming & Song, 2011, PMID: 21609825; Bond et al., 2015, PMID: 26431181; Bond et al., 2021, PMID: 33706926; Bartkowska et al., 2022, PMID: 36078144). Also, in Figure 6A it was incorrectly mentioned to be just 1month, we rechecked our metadata and noted that young brains were comprised of 1 and 2 month old brains and now it has been corrected.

Furthermore, can the authors describe which sex of mice was used in these experiments and the justification if a single sex was used? If both sexes were used, were there any dimorphic differences in POLK localization patterns?

This is an important aspect, but in the beginning to keep mice numbers within manageable limits, we were focusing more on the age component. While both males and female brains were assayed but due to uneven sample distribution between sexes, we could not estimate if there were any statistically significant sexual dimorphic differences in IN, PN and NNs. Future studies will investigate the sex component as a function of age.

The suggestion of POLK as a brain aging clock may be a bit premature as the functional and behavioral consequences of cytoplasmic POLK sequestration are not fully known. Furthermore, investigation of POLK levels in other genetic models of neurodegeneration or with gerotherapeutics would be needed to establish if the POLK brain clock is responsive to changes that shift brain aging. Lastly, this clock may be impractical and not useful for longitudinal studies due to the terminal nature of assessing POLK levels.

We agree as a terminal assay POLK nucleo-cytoplasmic status is not practical for longitudinal studies. However, we believe it may serve an investigative/correlative endogenous signal for determining tissue age, that may be useful to "date" brain sections, since not many such cell biological markers exist. We have added clarification text.

Some discussion of the Polk-null mice is warranted, as they only have a slightly shortened lifespan, and any disease phenotypes were not reported. This stands in contrast to other DNA repair-deficient mice that mimic premature aging and show behavioral and motor deficits. This calls into question the role of POLK in brain aging.

Discussion statements on Polk-null mice has been added.

Please correct the catalog number for the SCBT anti-POLK antibody to sc-166667

Typographical error has been corrected

Reviewer #2 (Recommendations for the authors):

Results:

Figure by figure 

(1) A progressive age-associated shift in subcellular localization of POLK The authors state that POLK has not been studied in nervous tissue before and they want to see if it is expressed, and if it changes subcellular location as a function of age. The authors argue age = stress like that seen in previous models using genotoxic agents and cancer cells. Indeed, POLK seems to convincingly change subcellular location from the nucleus to larger cytosolic puncta. 

(2) Nuclear POLK co-localizes with DNA damage response and repair proteins This was a difficult dataset for me to decipher. To me, it appears as though POLK colocalizes with these examined proteins in the CYTOSOL, not the nucleus. Especially, in the oldest mice.

We added in the discussion that DNA repair proteins were observed to be present in the cytoplasm and biomolecular condensates citing relevant reviews and primary references.

(3) POLK in the cytoplasm is associated with stress granules and lysosomes in old brains LAMP1 has some issues as a lysosome marker. The authors even state it can be on endosomes. It would be nice to use a marker for mature lysosomes, some fluorescent reporter that is activated only by lysosomal proteases or pH. It is also of interest if POLK is localized to the membrane or the inside of these structures. The authors have access to an airyscan which is sufficient to examine luminal vs membrane localization on larger organelles like lysosomes.

We thank the reviewer for pushing us to investigate the nature of cytoplasmic POLK in endo-lysosomal compartments. We have now added a full-page figure on the cell biological results from six different markers, subset (Cathepsin B and D) are known to present in the lumens of endo-lysosomes, in Figure 3. Further high-resolution membrane vs lumen was not pursued, which is perhaps better suited in cultured neurons rather than thick fixed tissues.

(4) Differentially altered POLK subcellular expression amongst excitatory, inhibitory, and nonneuronal cells in the cortex.

This seems fine. I don't see anything wrong with the author's statement that there is more POLK in neurons vs non-neuronal cells. 

(5) Microglia associated with IN and PN have significantly higher levels of cytoplasmic POLK I don't see really any convincing evidence of the author's claim here. They find a difference at early-old age, but not at old-old, or other ages. This is explained by "However, this effect is lost in late-old age (Figure 5D), likely due to the MG-mediated removal of the INs.". But no trend being observed, no experiment to show sufficiency, and no experiment to uncover a directional relationship; this is a tough claim to stand by.

Changes made in text to reflect speculative nature of this observation

(6) Subcellular localization of POLK is regulated by neuronal activity

Interesting and fairly difficult experiment. Can the authors talk more about what these values mean? I am confused as to why there is a decline in nuclear puncta at 80 min. Also, why are POLK counts in 6c similar at baseline between young and early-old? In Figures 5 and 6 I also worry about statistical analysis. Are all assumptions checked to use t-tests? Why not always use a test that has fewer assumptions?

We have explained in the text the artificial nature of few hour long acute slice preparations is very different and inherently a stressful environment, especially for the old brains, compared to the vascular perfused PFA fixed brain tissues tested between young and old ages.

We don’t have a proper explanation for the initial dip in nuclear puncta in both young and old brains at 80min of very similar magnitude. It could be a separate biological phenomenon that occurs at much shorter time scales that would not otherwise be captured in a fixed tissue assay and needs careful investigation using live tissue fluorescence imaging that is beyond the scope of this manuscript.

We apologize for the typographical error in the figure legend. We rechecked our R code and the tests were all Wilcoxon rank-sum (Mann–Whitney U) two-sided nonparametric.

Figure 6B & E had absurdly small p values due to large sample numbers. So, we implemented random sampling of 100 cells repeating for 200 times and presented the distribution of p values and Cohen’s d in the supplement and reported the median p value and Cohen’s in the main plot.

(7) POLK as an endogenous "aging clock" for brain tissue

Trainable model. What are the criteria for the model, and how does it work? The cutoffs it uses to classify each age group might be interesting in that the model may have identified a trait the researchers were unaware of. Otherwise, it is not especially useful. Maybe as an independent 'blind' analysis of the data?

We have added a better description of the models, assumptions and how two different unsupervised approaches converge on the same set of features with high AUROCs.

Minor questions:

The cartoons (1a, 2a-b, 5a, 6a) help a lot. However, I still had to work a bit to understand some of the graphs (e.g., 5d, 6b-e, fig 7). Is there a simpler way to present them? Maybe simply additional labelling? I'm not sure.

A more thorough discussion of statistical tests is warranted I think. I am not very clear why some were chosen (t-test vs nonparametric with fewer assumptions). Infinitesimally small p values also make me think maybe incorrect tests were done or no power analysis was performed beforehand. A fix for this is just discussing what went into the testing methods and why they were chosen.

Statistical analysis for Fig2 (using Generalized Estimating Equations), and Fig6 (with random repeated subsampling; method explained in text, figure legend updated and supplementary data on the distribution of p values and cohen’s d are added) to address the very small p values. Descriptions rewritten in relevant text.

In the absence of further mechanistic experiments, it would still be interesting to hear what the authors think is going on and what the significance of this altered subcellular location means. How do the authors think this is occurring? I think they are arguing that cytosolic localization of POLK is 100% detrimental to the neuron. ("The reduction of nuclear POLK in old brains is congruent with an increase in DNA damage markers") Do they have any idea what the 'bug' is in the POLK system then?

Statements in the discussion has been added.

Reviewer #3 (Recommendations for the authors):

POLK is detected as small " as small "speckles" inside the nucleus at a young age (1-2 months) and larger "granules" can be seen in the cytoplasm at progressively older time points (>9 months). In the nucleus, is POLK bound to DNA? In the cytoplasm, how are the POLK molecules organized: are they bound to a substrate or are they just organized as a proteins condensate without DNA?

In human U2OS cell line Dnase1 treatment leads to loss of POLK from the nucleus as well as its activity as reported in Fig5 of Paul, S. et. al. 2023 bioRxiv. While we haven’t reproduced these results in mouse primary neurons, we anticipate a similar situation which will be tested in the future. We have addressed limited aspects of the POLK in the cytoplasm in all new Fig3 with six endo-lysosomal markers, and added text.

When POLK proteins accumulate in the cytoplasm in aging cells, do they also repair condensates in the cytoplasm? What is the function of cytoplasmic POLK granules? More generally, is it known if other granules or foci, such as repair foci are found in the cytoplasms in aging cells, or in cells under stress?

Six markers for endo-lysosomes were tested to characterize the cytoplasmic granules now shown in Fig3.

While the authors quantify the number and sizes of the POLK signal, they don't discuss their physical nature. Some membrane-less condensates exhibit liquid-like properties, such as stress granules, P-bodies, or in the nucleus some repair condensates. In some diseased tissues, some condensates lose their liquid properties and become solid-like. Is it known if POLK condensates behave like liquid condensates or they are simply formed by bound molecules on DNA? Since they are larger and fewer in the cytoplasm, is it because several small puncta fused together to form a larger one? It would be worthwhile to discuss these points.

Discussion statements on the nature of condensates in context of the POLK cytoplasmic signal has been added.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

The manuscript titled, "Sleep-Wake Transitions Are Impaired in the AppNL-G-F Mouse Model of Early Onset Alzheimer's Disease", is about a study of sleep/wake phenomena in a knockin mouse strain carrying "three mutations in the human App gene associated with elevated risk for early onset AD". Traditional, in-depth characterization of sleep/wake states, EEG parameters, and response to sleep loss are employed to provide evidence, "supporting the use of this strain as a model to investigate interventions that mitigate AD burden during early disease stages". The sleep/wake findings of earlier studies (especially Maezono et al., 2020, as noted by the authors) were extended by several important, genotype-related observations, including age-related hyperactivity onset that is typically associated with increased arousal, a normal response to loss of sleep and to multiple sleep latency testing, and a stronger AD-like phenotype in females. The authors conclude that the AppNL-G-F mice demonstrate many of the human AD prodromal symptoms and suggest that this strain may serve as a model for prodromal AD in humans, confirming the earlier results and conclusions of Maezono et al. Finally, based on state bout frequency and duration analyses, it is suggested that the AppNL-G-F mice may develop disruptions in mechanism(s) involved in state transition.

Strengths:

The study appears to have been, technically, rigorously conducted with high quality, in-depth traditional assessment of both state and EEG characteristics, with the concordant addition of activity and temperature. The major strengths of this study derive from observations that the AppNL-G-F mice: (1) are more hyperactive in association with decreased transitions between states; (2) maintain a normal response to sleep deprivation and have normal MSLT results; and (3) display a sex specific, "stronger" insomnia-like effect of the knockin in females.

Weaknesses:

The weaknesses stem from the study's impact being limited due to its being largely confirmatory of the Maezono et al. study, with advances of importance to a potentially more focused field. Further, the authors conclude that AppNL-G-F mice have disrupted mechanism(s) responsible for state transition; however, these were not directly examined. The rationale for this conclusion is stated by the authors as based on the observations that bouts of both W and NREM tend to be longer in duration and decreased in frequency in AppNL-G-F mice. Although altered mechanism(s) of state transition (it is not clear what mechanisms are referenced here) cannot be ruled out, other explanations might be considered. For example, increased arousal in association with hyperactivity would be expected to result in increased duration of W bouts during the active phase. This would also predictably result in greater sleep pressure that is typically associated with more consolidated NREM bouts, consistent with the observations of bout duration and frequency.

Reviewer 1 succinctly summarizes the advances of this study beyond the ground-breaking Maezono et al (2020) study of this “humanized” mouse model exhibiting amyloid deposition. Whereas Maezono et al. conducted sleep/wake studies on male App^NL-G-F mice at 6 and 12 months of age, we had the unusual opportunity to study both sexes of homozygous App^NL-G-F mice and WT littermates at 14-18 months of age and to conduct a longitudinal assessment of many of the same individuals at 18-22 months. In addition to baseline sleep/wake and EEG spectral analyses, we (1) measured subcutaneous body temperature and activity to obtain a broader picture of the physiology and behavior of this strain at advanced ages; (2) assessed baseline sleepiness in this strain using the murine version of the clinically-relevant Multiple Sleep Latency Test (MSLT); (3) evaluated the response of App^NL-G-F mice and WT littermates to a perturbation of the sleep homeostat; (4) compared the sleep/wake characteristics of male vs. female App^NL-G-F mice at 18-22 months and, (5) to assess the stability of the phenotypes, analyzed these data over a continuous 14-d recording rather than the conventional 24h recordings typical of most sleep/wake studies including Maezono et al. We found that a long wake/short sleep phenotype was characteristic of homozygous App^NL-G-F mice at these advanced ages which is also evident in the Maezono et al. (2020) study at 12 months of age (but not at 6 months), although the authors do not comment on this phenotype and instead focus on the reduced REM sleep which is particularly evident in female App^NL-G-F mice in our study. Remarkably, despite being awake ~20% longer per day, we find that App^NL-G-F mice are no sleepier than WT mice as determined by the MSLT and that their sleep homeostat is intact when challenged by 6-h sleep deprivation. At both advanced ages, the long wake/short sleep phenotype is due primarily to longer Wake bouts and shorter bouts of both NREM and REM sleep during the dark phase. Moreover, hyperactivity develops in older in App^NL-G-F mice, particularly females, which contributes to this phenotype. We agree with Reviewer 1 that “hyperactivity would be expected to result in increased duration of W bouts during the active phase” and that this could result in more consolidated NREM bouts and we will modify the manuscript to discuss this alternative. However, the suggestion of greater sleep pressure is not borne out by the MSLT studies as we did not observe the shorter sleep latencies and increased sleep during the nap opportunities on the MSLT that we have observed in other mouse strains. Moreover, due to their short sleep phenotype, App^NL-G-F mice would be entering the sleep deprivation study with a greater sleep debt than WT mice, yet we did not observe greater EEG Slow Wave Activity in this strain during recovery from sleep deprivation. Thus, we have suggested that App^NL-G-F mice are unable to transition from Wake to sleep as readily as their WT littermates. Our observations summarized above set the stage for subsequent mechanistic studies in aged App^NL-G-F mice, although realistically, mice of this age and genotype are a rare commodity.

Reviewer #2 (Public review):

Summary:

The authors have used a knock-in mouse model to explore late-in-life amyloid effects on sleep. This is an excellent model as the mutated genes are regulated by the endogenous promoter system. The sleep study techniques and statistical analyses are also first-rate.

The group finds an age-dependent increase in motor activity in advanced age in the NLGF homozygous knock-in mice (NLGF), with a parallel age-dependent increase in body temperature, both effects predominate in the dark period. Interestingly, the sleep patterns do not quite follow the sleep changes. Wake time is increased in NLGF mice, and there is no progression in increased wake over time. NREMS and REM sleep are both reduced, and there is no progression. Sleep-wake effects, however, show a robust light:dark effect with larger effects in the dark period. These findings support distinct effects of this mutation on activity and temperature and on sleep. This is the first description of the temporal pattern of these effects. NLGF mice show wake stability (longer bout durations in the dark period (their active period) and fewer brief arousals from sleep. Sleep homeostasis across the lights-on period is normal. Wake power spectral density is unaffected in NLGF mice at either age. Only REM power spectra are affected, with NLGF mice showing less theta and more delta. There are interesting sex differences, with females showing no gene difference in wake bout number, while males show a gene effect. Similarly, gene effects on NREM bout number seem larger in males than in females. Although there was no difference in homeostatic response, there was normalization of sleep-wake activity after sleep deprivation.

Strengths:

Approach (model extent of sleep phenotyping), analysis.

Weaknesses:

The weaknesses are summarized below and are viewed as "addressable".

(1) The term insomnia. Insomnia is defined as a subjective dissatisfaction with sleep, which cannot be ascertained in a mouse model. The findings across baseline sleep in NLGF mice support increased wake consolidation in the active period. The predominant sleep period (lights on) is largely unaffected, and the active period (lights off) shows increased activity and increased wake with longer bouts. There is a fantastic clue where NLGF effects are consistent with increased hypocretinergic (orexinergic) neuron activity in the dark period, and/or increased drive to hypocretin neurons from PVH.

(2) Sleep-wake transitions are impaired: This should not be termed an impairment. It could actually be beneficial to have greater state stability, especially wake stability in the dark or active period. There is reduced sleep in the model that can be normalized by short-term sleep loss. It is fascinating that recovery sleep normalized sleep in the NLGF in the immediate lights-on and light-off period. This is a key finding.

Reviewer 2 suggests a provocative hypothesis to test. Curiously, although a recent Science paper suggests that hyperexcitable hypocretin/orexin neurons in aging mice results in greater sleep/wake fragmentation, hyperexcitability of this system could result in hyperactivity and longer wake bouts in aged App^NL-G-F mice.

Reviewer #3 (Public review):

Summary:

In this study, Tisdale et al. studied the sleep/wake patterns in the biological mouse model of Alzheimer's disease. The results in this study, together with the established literature on the relationship of sleep and Alzheimer's disease progression, guided the authors to propose this mouse model for the mechanistic understanding of sleep states that translates to Alzheimer's disease patients. However, the manuscript currently suffers from a disconnect between the physiological data and the mechanistic interpretations. Specifically, the claim of "impaired transitions" is logically at odds with the observed increase in wake-state stability or possible hyperactivity. Additionally, the description of the methods, the quantification, and the figure presentation could be substantially improved. I detail some of my concerns below.

Strengths:

The selection of the knock-in model is a notable strength as it avoids the artifacts associated with APP overexpression and more closely mimics human pathology. The study utilizes continuous 14-day EEG recordings, providing a unique dataset for assessing chronic changes in arousal states. The assessment of sex as a biological variable identifies a more severe "insomniac-like" phenotype in females, which aligns with the higher prevalence and severity of Alzheimer's disease in women.

Weaknesses:

The study seems to lack a clear hypothesis-driven approach and relies mostly on explorative investigations. Moreover, lack of quantitative analytical methods as well as shaky logical conclusions, possibly not supported by data in its current form, leaves room for major improvement.

Since this paper studied sleep states, the "Methods" section is quite unclear on what specific criteria were used to classify sleep states. There is no quantitative description of classifying sleep based on clear, reproducible procedures. There are many reasonably well-characterized sleep scoring systems used in rat electrophysiological literature, which could be useful here. The authors are generally expected to describe movement speed and/or EMG and/or EEG (theta/delta/gamma) criteria used to classify these epochs. The subjective (manual) nature of this procedure provides no verifiable validation of the accuracy and interpretability of the results.

One of the bigger claims is that "state transition mechanism(s)" are impaired. However, Figure 7 shows that model mice exhibit significantly more long wake bouts (>260s) and fewer short wake bouts (<60s). Logically, an "impaired switch" (the flip-flop model, Saper et al., 2010) results in state fragmentation. The data here show the opposite: the wake state has become too stable. This suggests the primary defect is not in the transition mechanism itself, but possibly in a pathological increase in arousal drive (hyper-arousal), likely linked to the dark-phase hyperactivity shown in Figures 4 and 5. Also, a point to note is that this finding is not new.

Figure 3 heatmaps lack color bars and units. Spectral power must be quantitatively defined and methods well-explained in the Methods section. Without these, the reader cannot discern if the "reduced power" in females is a global suppression of signal or a frequency-specific shift. Additionally, the representative example used to claim shorter sleep bouts lacks the statistical weight required for a major physiological conclusion. How does a cooler color (not clear what range and what the interpretation is) mean shorter sleep bout in female mice? The authors should clearly mark the frequency ranges that support their claims. In this figure, there is a question mark following the theta/delta range. The authors should avoid speculation and state their claims based on facts. They should also add the theta and delta ranges in the plot, such that readers can draw their own conclusions.

Figure 8 and the MSLT results show that model mice are "no sleepier than WT mice" and have a functional homeostatic rebound. This presents a logical flaw in the "insomnia" narrative. True insomnia in AD patients typically involves a failure of the homeostatic process or a debilitating accumulation of sleep debt. If these mice do not show increased sleepiness (shorter latency) despite ~19% less sleep, the authors might be describing a "reduced need" for sleep or a "hyper-aroused" state, possibly not a clinical insomnia phenotype.

In Figure 9, LFP power shown and compared in percentages is problematic, as LFP power distribution is known to be skewed (follows power law). This is particularly problematic here because all the frequencies above ~20 Hz seem to be totally flattened or nonexistent, which makes this comparison of power severely limited and biased towards the relative frequency in the highly skewed portion of the LFP power spectrum, i.e., very low frequency ranges like delta, theta, and possibly beta. This ignores low, mid, and high gamma as well as ripple band frequencies. NREM sleep is known to have relatively greater ripple band (100-250 Hz) power bursts in hippocampal regions, and REM sleep is known to have synchronous theta-gamma relationships.

We agree with the reviewer that the “Classification of arousal states” section was missing the key description of how we scored the recordings into arousal states based on EEG, EMG and locomotor activity; this was an oversight as the corresponding text exists in all our previous sleep/wake studies published over several decades. Reviewer 1 also points out the alternative interpretation that “the wake state has become too stable.” However, I think we are using different words to say the same thing: that the transition from wake to sleep is impaired whether it is due to hyperarousal or to a defect in the flip/flop switch that results in greater Wake stability. We will revise Fig 3 (Reviewer 2 suggests combining with Fig 14) but note that the X-axis is labelled 0-25 Hz and that this figure was intended to be descriptive -- illustrating how unusual the female App^NL-G-F mice are relative to WT -- rather than a quantitative analysis of spectral power as in Fig. 14. Both Reviewer 2 and 3 suggest that we are using “insomnia” incorrectly, which we have simply used to describe less sleep per 24h period. Reviewer 2 states that “Insomnia is defined as a subjective dissatisfaction with sleep” and Reviewer 3 suggests a narrow definition of insomnia as due only to “a failure of the homeostatic process or a debilitating accumulation of sleep debt.” In a revised manuscript, we will define “insomnia” as an operational term to succinctly mean “less sleep”. Regarding the problem of presenting spectral power in percentages, we completely agree with the reviewer. However, we intentionally presented spectral power density, a measure of relative power, as in Figure 3A and 3B of Maezono et al. (2020). At the risk of making Fig. 9 even more busy, we will revise Fig. 9 to add labels for all Y-axes.

In addition to a revised Fig. 9, in the revised manuscript, we will reformat Tables 1-3, Figs. S1 and S2 for legibility and correct an error in Fig. 7.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study addresses an important clinical challenge by proposing muscle network analysis as a tool to evaluate rehabilitation outcomes. The research direction is relevant, and the findings suggest further research. The strength of evidence supporting the claims is, however, limited: the improvements in function are not directly demonstrated, the robustness of the method is not benchmarked against already published approaches, and key terminology is not clearly defined, which reduces the clarity and impact of the work.

Comments:

There are several aspects of the current work that require clarification and improvement, both from a methodological and a conceptual standpoint.

First, the actual improvements associated with the rehabilitation protocol remain unclear. While the authors report certain quantitative metrics, the study lacks more direct evidence of functional gains. Typically, rehabilitation interventions are strengthened by complementary material (e.g., videos or case examples) that clearly demonstrate improvements in activities of daily living. Including such evidence would make the findings more compelling.

We thank the reviewer for their careful consideration of our work. We agree that direct evidence for the functional gains achieved by patients is important for establishing the efficacy of a clinical intervention and that this evidence should provide comprehensive insights for clinicians, from videos to case examples as suggested. Our aim here was apply a novel computational framework to a cohort of patients undergoing rehabilitation, and in doing so, provide empirical support for its utility in standardised motor assessments. We have shown that our novel approach can identify distinct physiological responses to VR vs PT conditions across the post-stroke cohort (see Fig.2B and associated text). Hence, although the data contains virtual reality vs. conventional physical therapy experimental conditions which likely holds important insights into the clinical use case of virtual reality interventions, we did not focus on such complementary evidence in this study. In future work, research groups (including our own) investigating the important question of clinical intervention efficacy will likely gain unique and useful mechanistic insights using our approach.

Moreover, a threshold of 5 points at the FMA-UE was considered as MCID, to distinguish between responder and non-responder patients, which represents an acknowledged and applicable measure in the clinical field. The use of single cases represents low evidence of change from the perspective of expert clinicians, raising concerns on the clinical meaningful of reported results. All this given, we chose to provide stronger evidence of clinical effect (i.e. comparison between responders and non-responders) interpreted from the perspective of muscle synergies, than to support our results in single selected cases, representing a bias in terms of translation to population of people survived to a stroke.

Second, the claim that the proposed muscle network analysis is robust is not sufficiently substantiated. The method is introduced without adequate reference to, or comparison with, the extensive literature that has proposed alternative metrics. It is also not evident whether a simpler analysis (e.g., EMG amplitude) might produce similar results. To highlight the added value of the proposed method, it would be important to benchmark it against established approaches. This would help clarify its specific advantages and potential applications. Moreover, several studies have shown very good outcomes when using AI and latent manifold analyses in patients with neural lesions. Interpreting the latent space appears even easier than interpreting muscle networks, as the manifolds provide a simple encoding-decoding representation of what the patient can still perform and what they can no longer do.

To address the reviewers concerns regarding adequate evidence for the claims made about the presented framework, we have now included an application of the conventional muscle synergy analysis approach based on non-negative matrix factorisation to the post-stroke cohort (see Supplementary materials Fig.5 and associated text). We made efforts to make this comparison as fair as possible by applying the conventional approach at the population level also and clustering the activation coefficients using a similar yet more conventional approach, agglomerative clustering. Accompanying the output of this application, we have included several points of where our framework improves significantly upon conventional muscle synergy analysis:

“Comparison with conventional approaches

To more directly illustrate the advantages of the proposed framework, we carried out a standardised pre-processing of the EMG data in line with conventional muscle synergy analysis. This included rectification, low-pass filtration (cut-off: 20Hz) and smooth resampling of EMG waveforms to 50 timepoints. All data for each participant at each session was separately normalised by channel-wise variance, concatenated together and input into non-negative matrix factorisation (NMF) ('nnmf' Matlab function, 10 replications) to extract 11 muscle synergies (W1-11 of Supplementary Materials Fig.5(Left)) and their time-varying activations. The number of components to extract was determined in a conventional way as the number of components required to explain >75% of the data variance. The extracted muscle synergies included distinct shoulder- (e.g. W2), elbow (e.g. W8) and forearm-level (e.g. W1) muscle covariation patterns along with more isolated muscle contributions (e.g. UT in W3, TL in W10).

Regarding the clustering results of our framework and how they compare to conventional approaches, to facilitate this comparison we applied agglomerative clustering to the time-varying activation coefficients of all participants, trials, tasks separately for pre- and post-sessions and employed the 'evalclusters' Matlab function (Ward linkage clustering, Calinski Harabasz criterion, Klist search = 2:21) for each session. We identified two clusters both at pre-session (Criterion = 1.69) and post-session (Criterion = 1.81) as optimal fits to the population data (see Supplementary Materials Fig.5(Right)). We found no associations between pre- or post-session cluster partitions and participants FMA-UE scores. Nevertheless, we did identify significant associations between the pre-session clustering’s and S_Pre (X² = 7.08, p = 0.008) and between post-session clustering’s and conventionally-defined treatment responders (X² = 4.2, p = 0.04). These findings, along with the similar two-way clustering structure found using the NIF, highlights important commonalities between these approaches.

To summarise the main advantages of our framework over this conventional approach:

- Lower dimensionality and enhanced interpretability of extracted components.

Our framework yields a lower number of population-level components that correspond more consistently to meaningful biomechanical and physiological functions.

- Integration of pairwise muscle relationships.

By incorporating muscle-pair level analysis, our framework captures coordinated interactions between primary and stabilising muscles—relationships that conventional NMF approaches overlook.

- Separation of task-relevant and task-irrelevant activity.

The NIF isolates task-relevant coordination patterns, distinguishing them from task-irrelevant interactions driven by biomechanical or task constraints. On the other hand, task-relevant and -irrelevant muscle contributions are intermixed in conventional muscle synergy analysis.

- Ability to identify complementary functional roles.

The NIF characterises whether muscle pairs act in similar or complementary ways, providing richer insight into motor control strategies.

- Reduced dependence on variance-based optimisation.

Unlike conventional methods that rely on maximising variance explained, our framework allows detection of subtle but functionally significant interactions that contribute less to total variance.

- Improved detection of clinically relevant population structure.

The clustering component of our framework revealed distinct post-stroke subgroups with important clinical relevance, distinguishing moderately and severely impaired cohorts and treatment responders and non-responders from pre-treatment data.”

This supplementary analysis is referred to in the Methods section of the main text with reference to previous similar comparisons between our framework and conventional approaches:

“Towards finding an effective approach to clustering participants in this data based on differences in impairment severity and therapeutic (non-)responsiveness, we found that conventional clustering algorithms (e.g. agglomerative, k-means etc.) could not provide substantive outputs (see Supplementary Materials Fig.5 and associated text for a direct comparison with conventional approaches), perhaps resulting from the complex interdependencies between the modular activations.”

“To facilitate comparisons with existing approaches, we performed a conventional muscle synergy analysis on the post-stroke cohort (see Supplementary Materials Fig.5 and associated text). Further comparisons with conventional approaches can be found in our previous work (O’Reilly & Delis, 2022).”

Further, we have also referred to a previous analysis of this post-stroke dataset using the conventional approach in the discussion section, where we point out how our approach can identify salient features of post-stroke physiological responses that conventional approaches cannot:

“Further, the NIF demonstrated here an enhanced capability over traditional approaches to identify these crucial patterns, as earlier work on related versions of this dataset could not identify any differentiable fractionation events across the cohort (Pregnolato et al., 2025).”

Overall, the utility of conventional muscle synergy analysis is well recognised across the field (Hong et al 2021). Our proposed approach builds on this conventional method by addressing key limitations to further enhance this clinical utility. We also agree that manifold learning approaches are an exciting area of research that we aim to incorporate into our framework in future research. Specifically, manifold learning methods like Laplacian eigenmaps can readily be applied to the co-membership matrix produced by our clustering algorithm, exploiting the geometry of this matrix to provide a continuous rather than discrete representation of population structure. We have highlighted this possibility in the discussion section:

“Indeed, in future work, we aim to apply manifold learning approaches to the co-membership matrix derived from this clustering algorithm, providing a continuous representation of the population structure.”

Third, the terminology used throughout the manuscript is sometimes ambiguous. A key example is the distinction made between "functional" and "redundant" synergies. The abstract states: "Notably, we identified a shift from redundancy to synergy in muscle coordination as a hallmark of effective rehabilitation-a transformation supported by a more precise quantification of treatment outcomes."

However, in motor control research, redundancy is not typically seen as maladaptive. Rather, it is a fundamental property of the CNS, allowing the same motor task to be achieved through different patterns of muscle activity (e.g., alternative motor unit recruitment strategies). This redundancy provides flexibility and robustness, particularly under fatiguing conditions, where new synergies often emerge. Several studies have emphasized this adaptive role of redundancy. Thus, if the authors intend to use "redundancy" differently, it is essential to define the term explicitly and justify its use to avoid misinterpretation.

We appreciate the reviewers concerns regarding the terminology employed in this study. Indeed, we agree that redundancy is seen in the motor control literature as a positive feature of biological systems, appearing to contradict the interpretations of the redundancy-to-synergy information conversion result we have presented. We also wish to highlight that across the motor control literature and beyond, the idea of redundancy is often conflated with the related but distinct notion of degeneracy. Traditional motor control research has also recognised this difference, for example, Latash has outlined this difference in the seminal work on motor abundance (https://doi.org/10.1007/s00221-012-3000-4). A key reference discussing this conflation and these two concepts in an information-theoretic way is found here: https://doi.org/10.1093/cercor/bhaa148. To summarise what their arguments mean for our work:

- System degeneracy relates to the ability of different system components to contribute towards the same task in a context-specific way.

- System redundancy corresponds to the degree of functional overlap among system components.

Hence, conceptually speaking, informational redundancy as employed in our study (i.e. functionally-similar muscle interactions) links with system redundancy in that it quantifies the functional overlap of system components. This definition of system redundancy implies that it is an unavoidable by-product of degenerate systems (inefficient use of degrees of freedom) which should be minimised where possible. As a result of stroke, in our study and related previous work patients displayed increased informational redundancy, linking with the abnormal co-activations they typically experience for example and with previous results from traditional muscle synergy analysis showing fewer components extracted as a function of motor impairment post-stroke (i.e. higher informational redundancy) (Clark et al. 2010). Our novel contribution here is to convey how effective rehabilitation is underpinned by a redundancy-to-synergy information conversion across the muscle networks, relating in a loose sense conceptually to a reduction in system redundancy and enhancement of system degeneracy (i.e. functionally differentiated system components contributing towards task performance).

Together, and alongside the mathematical descriptions of redundant (functionally-similar) and synergistic (functionally-complementary) information in what types of functional relationships they capture, we believe the intuition behind this finding has clear links with previous research showing a) the merging of muscle synergies in response to post-stroke impairment (i.e. functional de-differentiation), b) reduction in abnormal couplings with effective rehabilitation (i.e. functional re-differentiation). To communicate this more clearly to readers, we have included the following in the corresponding discussion section:

“Previous research has shown that functional redundancy increases post-stroke (Cheung et al., 2012; Clark et al., 2010), reflecting the characteristic loss of functional specificity (i.e. functional de-differentiation) of muscle interactions post-stroke. Enhanced synergy with treatment here thus reflects the functional re-differentiation of predominantly flexor-driven muscle networks towards different, complementary task-objectives across the seven upper-limb motor tasks performed (Kim et al., 2024b), leading to improved motor function among responders.”

Finally, we have screened the updated manuscript for consistent use of terminology including functional/redundant/synergistic.

References

Clark DJ, Ting LH, Zajac FE, Neptune RR, Kautz SA. Merging of healthy motor modules predicts reduced locomotor performance and muscle coordination complexity post-stroke. Journal of neurophysiology. 2010 Feb;103(2):844-57.

Hong YN, Ballekere AN, Fregly BJ, Roh J. Are muscle synergies useful for stroke rehabilitation?. Current Opinion in Biomedical Engineering. 2021 Sep 1;19:100315.

Latash ML. The bliss (not the problem) of motor abundance (not redundancy). Experimental brain research. 2012 Mar;217(1):1-5.

O'Reilly D, Delis I. Dissecting muscle synergies in the task space. Elife. 2024 Feb 26;12:RP87651.

Sajid N, Parr T, Hope TM, Price CJ, Friston KJ. Degeneracy and redundancy in active inference. Cerebral Cortex. 2020 Nov;30(11):5750-66.

Reviewer #2 (Public review):

Summary:

This study analyzes muscle interactions in post-stroke patients undergoing rehabilitation, using information-theoretic and network analysis tools applied to sEMG signals with task performance measurements. The authors identified patterns of muscle interaction that correlate well with therapeutic measures and could potentially be used to stratify patients and better evaluate the effectiveness of rehabilitation.

However, I found that the Methods and Materials section, as it stands, lacks sufficient detail and clarity for me to fully understand and evaluate the quality of the method. Below, I outline my main points of concern, which I hope the authors will address in a revision to improve the quality of the Methods section. I would also like to note that the methods appear to be largely based on a previous paper by the authors (O'Reilly & Delis, 2024), but I was unable to resolve my questions after consulting that work.

I understand the general procedure of the method to be: (1) defining a connectivity matrix, (2) refining that matrix using network analysis methods, and (3) applying a lower-dimensional decomposition to the refined matrix, which defines the sub-component of muscle interaction. However, there are a few steps not fully explained in the text.

(1) The muscle network is defined as the connectivity matrix A. Is each entry in A defined by the co-information? Is this quantity estimated for each time point of the sEMG signal and task variable? Given that there are only 10 repetitions of the measurement for each task, I do not fully understand how this is sufficient for estimating a quantity involving mutual information.

We acknowledge the confusion caused here in how many datapoints were incorporated into the estimation of II. The number of datapoints included in each variable involved was in fact no. of timepoints x 10 repetitions. Hence for the EMGs employed in this analysis with a sampling rate of 2000Hz, the length of variables involved in this analysis could easily extend beyond 20,000 datapoints each. We have clarified this more specifically in the corresponding section of the methods:

“We carried out this application in the spatial domain (i.e. interactions between muscles across time (Ó’Reilly & Delis, 2022)) by concatenating the 10 repetitions of each task executed on a particular side (i.e. variables of length no. of timepoints x 10 trials) and quantifying II with respect to this discrete task parameter codified to describe the motor task performed at each timepoint for each trial included.”

In the previous paper (O'Reilly & Delis, 2024), the authors initially defined the co-information (Equation 1.3) but then referred to mutual information (MI) in the subsequent text, which I found confusing. In addition, while the matrix A is symmetrical, it should not be orthogonal (the authors wrote A^TA = I) unless some additional constraint was imposed?

We thank the reviewer for spotting this typo in the previous paper describing a symmetric matrix as A^TA = I which is in fact related to orthogonality instead. To clarify this error, in the current study we have correctly described the symmetric matrix as A = A^T here:

“We carried out this application in the spatial domain (i.e. interactions between muscles across time (Ó’Reilly & Delis, 2022)) by concatenating the 10 repetitions of each task executed on a particular side (i.e. variables of length no. of timepoints x 10 trials) and quantifying II with respect to this discrete task parameter codified to describe the motor task performed at each timepoint for each trial included. This computation was performed on all unique m_x and m_y pairings, generating symmetric matrices (A) (i.e. A = A^T) composed separately of non-negative redundant and synergistic values (Fig.5).”

Regarding the reviewers point about the reference to MI after equation 1.3 of the previous paper where co-Information is defined, we were referring both to the task-relevant and task-irrelevant estimates analysed there collectively in a general sense as ‘MI estimates’ as they both are derived from mutual information, task-irrelevant being the MI between two muscles conditioned on a task variable (conditional mutual information) and task-relevant being the difference between two MI values (co-I is a higher-order MI estimate). This removed the need to continuously refer to each separately throughout the paper which may in its own way cause some confusion. For clarity, in the results of that paper we also provided context for each MI estimate on how they were estimated (see beginning of “Task-irrelevant muscle couplings” and “Task-redundant muscle couplings” and “Task-synergistic muscle couplings” results sections), referring throughout the Venn diagrams depicting them (see Fig.1 of previous paper). In the present study however, for brevity and focus we did not perform an analysis on task-irrelevant muscle interactions and so decided to focus our terminology on co-I (II), a higher-order MI estimate. We acknowledge that this may have caused some confusion but highlight the efforts made to communicate each measure throughout the previous and present study. We have explicitly pointed out this specific focus on task-dependent muscle couplings in this paper at the end of the introduction of the updated manuscript:

“To do so, here we focussed our analysis on quantifying task-dependent muscle couplings (collectively referred to as II), extracting functionally-similar (i.e. redundant) and -complementary (i.e. synergistic) modules…”

(2) The authors should clarify what the following statement means: "Where a muscle interaction was determined to be net redundant/synergistic, their corresponding network edge in the other muscle network was set to zero."

We acknowledge this sentence was unclear/misleading and have now clarified this statement in the following way:

“This computation was performed on all unique m_x and m_y pairings, generating sparse symmetric matrices (A) (i.e. A = A^T) composed separately of non-negative redundant and synergistic values (Fig.5).” Additionally, we have now included an additional figure (fig.5) describing this text graphically.

(3) It should be clarified what the 'm' values are in Equation 1.1. Are these the co-information values after the sparsification and applying the Louvain algorithm to the matrix 'A'? Furthermore, since each task will yield a different co-information value, how is the information from different tasks (r) being combined here?

We thank the reviewer for their attention to detail. For clarity, at the related section of Equation 1.1, we have clarified that the input matrix is composed of co-I estimates:

“The input matrix for PNMF consisted of the sparsified A on both affected and unaffected sides from all participants at both pre- and post-sessions concatenated in their vectorised forms. More specifically, the input matrix composed of redundant or synergistic values was configured such that the set of unique muscle pairings (1 … K) on affected and unaffected sides (m_aff and m_unaff respectively)…”.

The co-I estimates in this input matrix are indeed those that survived sparsification in previous steps, however, for determining the number of modules to extract using the Louvain algorithm, this step has no direct impact or transformation on the co-I estimates and is simply employed to derive an empirical input parameter for dimensionality reduction. We refer the reviewer to the following part of this paragraph where this is described:

“The number of muscle network modules identified in this final consensus partition was used as the input parameter for dimensionality reduction, namely projective non-negative matrix factorisation (PNMF) (Fig.1(D)) (Yang & Oja, 2010). The input matrix for PNMF consisted of the sparsified A on both affected and unaffected sides from all participants at both pre- and post-sessions concatenated together in their vectorised form.”

Finally, as the reviewer has mentioned, the co-I estimates from the same muscles pairings but for different tasks, experimental sessions and participants are indeed different, reflecting their task-specific tuning, changes with rehabilitation and individual differences. To combine these representations into low-dimensional components, we employed projective non-negative matrix factorisation (PNMF). As outlined in the previous paper and earlier work on this framework (O’ Reilly & Delis, 2022), application of dimensionality reduction here can generate highly generalisable motor components, highlighting their ability to effectively represent large populations of participants, tasks and sessions, while allowing interesting individual differences mentioned by the reviewer to be buffered into the corresponding activation coefficients. These activation coefficients are for this reason the focus of the cluster analyses in the present study to characterise the post-stroke cohort. We have explicitly provided this reason in the methods section of the updated manuscript:

“We focussed on $a$ here as the extraction of population-level functional modules enabled the buffering of individual differences into the space of modular activations, making them an ideal target for identifying population structure.”

(4) In general, I recommend improving the clarity of the Methods section, particularly by being more precise in defining the quantities that are being calculated. For example, the adjacency matrix should be defined clearly using co-information at the beginning, and explain how it is changed/used throughout the rest of the section.

We thank the reviewer for their constructive advice and have gone to lengths to improve the clarity of the methods section. Firstly, we have addressed all the reviewers comments on various specific sections of the methods, including more clearly the ‘why’ and ‘how’ of what was performed. Secondly, we have now included an additional figure illustrating how co-information was quantified at the network level and separated into redundant and synergistic values (see Fig.5 of updated manuscript). Finally, we have re-structured several paragraphs of the methods section to enhance flow with additional subheadings for clarity.

(5) In the previous paper (O'Reilly & Delis, 2024), the authors applied a tensor decomposition to the interaction matrix and extracted both the spatial and temporal factors. In the current work, the authors simply concatenated the temporal signals and only chose to extract the spatial mode instead. The authors should clarify this choice.

The reviewer is correct in that a different dimensionality reduction approach was employed in the previous paper. In the present study, we instead chose to employ projective non-negative matrix factorisation, as was employed in a preliminary paper on this framework (O’Reilly & Delis, 2022). This decision was made simply based on aiming to maintain brevity and simplicity in the analysis and presentation of results as we introduce other tools to the framework (i.e. the clustering algorithm). Indeed, we could have just as easily employed the tensor decomposition to extract both spatial and temporal components, however we believed the main take away points for this paper could be more easily communicated using spatial networks only. To clarify this difference for readers we have included the following in the methods section:

“The choice of PNMF here, in contrast to the space-time tensor decomposition employed in the parent study (O’Reilly & Delis, 2024), was chosen simply to maintain brevity by focussing subsequent analyses on the spatial domain.”

References

Ó’Reilly D, Delis I. A network information theoretic framework to characterise muscle synergies in space and time. Journal of Neural Engineering. 2022 Feb 18;19(1):016031.

O'Reilly D, Delis I. Dissecting muscle synergies in the task space. Elife. 2024 Feb 26;12:RP87651.

Recommendations for the authors:

Reviewing Editor Comments:

Both reviewers are concerned with the manuscript in its current form. They questioned the relevance of the current approach in providing functional or mechanistic explanations about the rehabilitation process of post-stroke patients. Our eLife Assessment would change if you include comparisons between your current method and classical ones, in addition to improving the description of your method to strengthen the evidence of its robustness.

Reviewer #1 (Recommendations for the authors):

There is a minor typographical error in Figure 2 ("compononents" should be corrected).

This error has been rectified.

Reviewer #2 (Recommendations for the authors):

The authors should be able to address most of my concerns by providing a substantially improved version of the Methods section.

See above responses to the reviewers comments regarding the methods section.

However, I would like the authors to explain in full detail (potentially including a simulation or power analysis) the procedure for estimating the co-information quantity, and to clarify whether it is robust given the sample size used in this paper.

We refer the reviewer to our previous responses outlining with greater clarity the number of samples included in the estimation of co-I. We would also like to mention here that our framework does not make inferences on the statistical significance of individual muscle couplings (i.e. co-I estimates). Instead, these estimates are employed collectively for the sole purpose of pattern recognition. Nevertheless, to generate reliable estimates of the muscle couplings, we have employed a substantial number of samples for each co-I estimate (>20k samples in each variable) addressing the reviewers main concern her.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

The study by Wu et al. uses endogenous bruchpilot expression in a cell-type-specific manner to assess synaptic heterogeneity in adult Drosophila melanogaster mushroom body output neurons. The authors performed genomic on locus tagging of the presynaptic scaffold protein bruchpilot (BRP) with one part of splitGFP (GFP11) using the CRISPR/Cas9 methodology and co-expressed the other part of splitGFP (GFP1-10) using the GAL4/UAS system. Upon expression of both parts of splitGFP, fluorescent GFP is assembled at the N-terminus of BRP, exactly where BRP is endogenously expressed in active zones. For manageable analysis, a high-throughput pipeline was developed. This analysis evaluated parameters like location of BRP clusters, volume of clusters, and cluster intensity as a direct measure of the relative amount of BRP expression levels on site, using publicly available 3D analysis tools that are integrated in Fiji. Analysis was conducted for different mushroom body cell types in different mushroom body lobes using various specific GAL4 drivers. To test this new method of synapse assessment, Wu et al. performed an associative learning experiment in which an odor was paired with an aversive stimulus and found that, in a specific time frame after conditioning, the new analysis solidly revealed changes in BRP levels at specific synapses that are associated with aversive learning.

Strengths:

Expression of splitGFP bound to BRP enables intensity analysis of BRP expression levels as exactly one GFP molecule is expressed per BRP. This is a great tool for synapse assessment. This tool can be widely used for any synapse as long as driver lines are available to co-express the other part of splitGFP in a cell-type-specific manner. As neuropils and thus the BRP label can be extremely dense, the analysis pipeline developed here is very useful and important. The authors have chosen an exceptionally dense neuropil - the mushroom bodies - for their analysis and convincingly show that BRP assessment can be achieved with such densely packed active zones. The result that BRP levels change upon associative learning in an experiment with odor presentation paired with punishment is likewise convincing, and strongly suggests that the tool and pipeline developed here can be used in an in vivo context.

Weaknesses:

Although BRP is an important scaffold protein and its expression levels were associated with function and plasticity, I am still somewhat reluctant to accept that synapse structure profiling can be inferred from only assessing BRP expression levels and BRP cluster volume. Also, is it guaranteed that synaptic plasticity is not impaired by the large GFP fluorophore? Could the GFP10 construct that is tagged to BRP in all BRP-expressing cells, independent of GAL4, possibly hamper neuronal function? Is it certain that only active zones are labeled? I do see that plastic changes are made visible in this study after an associative learning experiment with BRP intensity and cluster volume as read-out, but I would be reassured by direct measurement of synaptic plasticity with splitGFP directly connected to BRP, maybe at a different synapse that is more accessible.

We appreciate the reviewer’s comments. In the revised manuscript, we have clarified that Brp is an important, but not the only player in the active zone. We have included new data to demonstrate that split-GFP tagging does not severely affect the localization and plasticity of Brp and the function of synapses by showing: (1) nanoscopic localization of Brp::rGFP using STED imaging; (2) colocalization between Brp::rGFP and anti-Brp signals/VGCCs; (3) activity-dependent Brp remodeling in R8 photoreceptors; (4) no defect in memory performance when labeling Brp::rGFP in KCs; These four lines of additional evidence further corroborate our approach to characterize endogenous Brp as a proxy of active zone structure.

Reviewer #2 (Public review):

Summary:

The authors developed a cell-type specific fluorescence-tagging approach using a CRISPR/Cas9 induced spilt-GFP reconstitution system to visualize endogenous Bruchpilot (BRP) clusters as presynaptic active zones (AZ) in specific cell types of the mushroom body (MB) in the adult Drosophila brain. This AZ profiling approach was implemented in a high-throughput quantification process, allowing for the comparison of synapse profiles within single cells, cell types, MB compartments, and between different individuals. The aim is to analyse in more detail neuronal connectivity and circuits in this centre of associative learning. These are notoriously difficult to investigate due to the density of cells and structures within a cell. The authors detect and characterize cell-type-specific differences in BRP-dependent profiling of presynapses in different compartments of the MB, while intracellular AZ distribution was found to be stereotyped. Next to the descriptive part characterizing various AZ profiles in the MB, the authors apply an associative learning assay and detect consequent AZ re-organisation.

Strengths:

The strength of this study lies in the outstanding resolution of synapse profiling in the extremely dense compartments of the MB. This detailed analysis will be the entry point for many future analyses of synapse diversity in connection with functional specificity to uncover the molecular mechanisms underlying learning and memory formation and neuronal network logics. Therefore, this approach is of high importance for the scientific community and a valuable tool to investigate and correlate AZ architecture and synapse function in the CNS.

Weaknesses:

The results and conclusions presented in this study are, in many aspects, well-supported by the data presented. To further support the key findings of the manuscript, additional controls, comments, and possibly broader functional analysis would be helpful. In particular:

(1) All experiments in the study are based on spilt-GFP lines (BRP:GFP11 and UAS-GFP1-10).The Materials and Methods section does not contain any cloning strategy (gRNA, primer, PCR/sequencing validation, exact position of tag insertion, etc.) and only refers to a bioRxiv publication. It might be helpful to add a Materials and Methods section (at least for the BRP:GFP11 line). Additionally, as this is an on locus insertion the in BRP-ORF, it needs a general validation of this line, including controls (Western Blot and correlative antibody staining against BRP) showing that overall BRP expression is not compromised due to the GFP insertion and localizes as BRP in wild type flies, that flies are viable, have no defects in locomotion and learning and memory formation and MB morphology is not affected compared to wild type animals.

We thank the reviewer for suggesting these important validations. We included details of the design of the construct and insertion site to the Methods section, performed several new experiments to validate the split-GFP tagging of Brp, and present the data in the revision.

First, to examine whether the transcription of the brp gene is unaffected by the insertion of GFP₁₁, we conducted qRT-PCR to compare the brp mRNA levels between brp::GFP₁₁, UAS-GFP1-10 and UAS-GFP1-10 and found no difference (Figure 1 - figure supplement 1A).

To further verify the effect of GFP₁₁ tagging at the protein level, we performed anti-Brp (nc82) immunohistochemistry of brains where GFP is reconstituted pan-neuronally. We found unaltered neuropile localization of nc82 signals (Figure 1 - figure supplement 1C). In presynaptic terminals of the mushroom body calyx, we found integration of Brp::rGFP to nc82 accumulation (Figure 1D). We performed super-resolution microscopy to verify the configuration of Brp::rGFP and confirmed the donut-shape arrangement of Brp::rGFP in the terminals of motor neurons (see Wu, Eno et al., 2025 PLOS Biology), corroborating the nanoscopic assembly of Brp::rGFP at active zones (Kittel et al., 2006 Science).

Furthermore, co-expression of RFP-tagged voltage-gated calcium channel alpha subunit Cacophony (Cac) and Brp::rGFP in PAM-γ5 dopaminergic neurons revealed strong presynaptic colocalization of their punctate clusters (Figure 1E), suggesting that rGFP tagging of Brp did not damage key protein assembly at active zones (Kawasaki et al., 2004 J Neuroscience; Kittel et al., Science).

These lines of evidence suggest that the localization of endogenous Brp is barely affected by the C-terminal GFP₁₁ insertion or GFP reconstitution therewith. This is in line with a large body of studies confirming that the N-terminal region and coiled-coil domains, but not the C-terminal, region of Brp are necessary and sufficient for active zone localization (Fouquet et al., 2009 J Cell Biol; Oswald et al., 2010 J Cell Biol; Mosca and Luo, 2014 eLife; Kiragasi et al., 2017 Cell Rep; Akbergenova et al., 2018 eLife; Nieratschker et al., 2009 PLoS Genet; Johnson et al., 2009 PLoS Biol; Hallermann et al., 2010 J Neurosci). We nevertheless report homozygous lethality and found the decreased immunoreactive signals in flies carrying the GFP₁₁ insertion (Figure 1 - figure supplement 1B).

For these reasons, we always use heterozygotes for all the experiments therefore there is no conspicuous defect in locomotion as reported in the original study (Wagh et al., 2005 Neuron). To functionally validate the heterozygotes, we measured the aversive olfactory memory performance of flies where GFP reconstitution was induced in Kenyon cells using R13F02-GAL4. We found that all these transgenes did not alter mushroom body morphology (Figure 7 - figure supplement 1) or memory performance as compared to wild-type flies (Figure 7 - figure supplement 2), suggesting the synapse function required for short-term memory formation is not affected by split-GFP tagging of Brp.

(2) Several aspects of image acquisition and high-throughput quantification data analysis would benefit from a more detailed clarification.

(a) For BRP cluster segmentation it is stated in the Materials and Methods state, that intensity threshold and noise tolerance were "set" - this setting has a large effect on the quantification, and it should be specified and setting criteria named and justified (if set manually (how and why) or automatically (to what)). Additionally, if Pyhton was used for "Nearest Neigbor" analysis, the code should be made available within this manuscript; otherwise, it is difficult to judge the quality of this quantification step.

(b) To better evaluate the quality of both the imaging analysis and image presentation, it would be important to state, if presented and analysed images are deconvolved and if so, at least one proof of principle example of a comparison of original and deconvoluted file should be shown and quantified to show the impact of deconvolution on the output quality as this is central to this study.

We thank the reviewer for suggesting these clarifications. We have included more description to the revised manuscript to clarify the setting of segmentation, which was manually adjusted to optimize the F-score (previous Figure 1D, now moved to Figure 1 -figure supplement 5). We have included the code used for analyzing nearest neighbor distance, AZ density and local Brp density in the revised manuscript (Supplementary file 1), together with a pre-processed sample data sheet (Supplementary file 2).

Regarding image deconvolution, we have clarified the differential use of deconvolved and not-deconvolved images in the revised manuscript. We have also included a quantitative evaluation of Richardson-Lucy iterative deconvolution (Figure 1 - figure supplement 4). We used 20 iterations due to only marginal FWHM improvement beyond this point (Figure 1 - figure supplement 4).

(3) The major part of this study focuses on the description and comparison of the divergent synapse parameters across cell-types in MB compartments, which is highly relevant and interesting. Yet it would be very interesting to connect this new method with functional aspects of the heterogeneous synapses. This is done in Figure 7 with an associative learning approach, which is, in part, not trivial to follow for the reader and would profit from a more comprehensive analysis.

(a) It would be important for the understanding and validation of the learning induced changes, if not (only) a ratio (of AZ density/local intensity) would be presented, but both values on their own, especially to allow a comparison to the quoted, previous AZ remodelling analysis quantifying BRP intensities (ref. 17, 18). It should be elucidated in more detail why only the ratio was presented here.

We thank the reviewer for the suggestion on the presentation of learning-induced Brp remodeling. The reported values in Figure 7C are the correlation coefficient of AZ density and local intensity in each compartment, but not the ratio. These results suggest that subcompartment-sized clusters of AZs with high Brp accumulation (Figure 6) undergo local structural remodeling upon associative learning (Figure 7). For clarity, we have included a schematic of this correlation and an example scatter plot to Figure 6. Unlike the previous studies (refs 17 and 18), we did not observe robust learning-dependent changes in the Brp intensity, possibly due to some confounding factors such as overall expression levels and conditioning protocols as described in the previous and following points, respectively.

(b) The reason why a single instead of a dual odour conditioning was performed could be clarified and discussed (would that have the same effects?).

(c) Additionally, "controls" for the unpaired values - that is, in flies receiving neither shock nor odour - it would help to evaluate the unpaired control values in the different MB compartments.

We use single odor conditioning because it is the simplest way to examine the effect of odor-shock association by comparing the paired and unpaired group. Standard differential conditioning with two odors contains unpaired odor presentation (CS-) even in the ‘paired’ group. We now show that single-odor conditioning induces memory that lasts one day as in differential conditioning (Figure 7B; Tully and Quinn, J Comp Phys A 1985).

(d) The temporal resolution of the effect is very interesting (Figure 7D), and at more time points, especially between 90 and 270 min, this might raise interesting results.

The sampling time points after training was chosen based on approximately logarithmic intervals, as the memory decay is roughly exponential (Figure 7B). This transient remodeling is consistent with the previous studies reporting that the Brp plasticity was short-lived (Zhang et al., 2018 Neuron; Turrel et al., 2022 Current Biol).

(e) Additionally, it would be very interesting and rewarding to have at least one additional assay, relating structure and function, e.g. on a molecular level by a correlative analysis of BRP and synaptic vesicles (by staining or co-expression of SV-protein markers) or calcium activity imaging or on a functional level by additional learning assays.

We thank the reviewer for raising this important point. We have performed calcium imaging of KC presynaptic terminals to correlate the structure and function in another study (see Figure 2 in Wu, Eno et al., 2025 PLOS Biology for more detail). The basal presynaptic calcium pattern along the γ compartments is strikingly similar to the compartmental heterogeneity of Brp accumulation (see also Figure 2 in this study). Considering colocalization of other active-zone components, such as Cac (Figure 1E), we propose that the learning-induced remodeling of local Brp clusters should transiently modulate synaptic properties.

As a response to other reviewers’ interest, we used Brp::rGFP to measure different forms of Brp-based structural plasticity upon constant light exposure in the photoreceptors and upon silencing rab3 in KCs. Since these experiments nicely reproduced the results of previous studies (Sugie et al., Neuron 2013; Graf et al., Neuron 2009), we believe the learning-induced plasticity of Brp clustering in KCs has a transient nature.

Reviewer #3 (Public review):

Summary:

The authors develop a tool for marking presynaptic active zones in Drosophila brains, dependent on the GAL4 construct used to express a fragment of GFP, which will incorporate with a genome-engineered partial GFP attached to the active zone protein bruchpilot - signal will be specific to the GAL4-expressing neuronal compartment. They then use various GAL4s to examine innervation onto the mushroom bodies to dissect compartment-specific differences in the size and intensity of active zones. After a description of these differences, they induce learning in flies with classic odour/electric shock pairing and observe changes after conditioning that are specific to the paired conditioning/learning paradigm.

Strengths:

The imaging and analysis appear strong. The tool is novel and exciting.

Weaknesses:

I feel that the tool could do with a little more characterisation. It is assumed that the puncta observed are AZs with no further definition or characterisation.

We performed additional validation on the tool, including (1) nanoscopic localization of Brp::rGFP using STED imaging; (2) colocalization between Brp::rGFP and anti-Brp signals/VGCCs (Figure 1D-E); 3) activity-dependent active zone remodeling in R8 photoreceptors (Figure 1F). These will be detailed in our point-by-point response below.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) The authors keep stating, they profile or assess synaptic structure by analyzing BRP localization, cluster volume, and intensity. However, I do not think that BRP cluster volume and intensity warrant an educated statement about presynaptic structure as a whole. I do not challenge the usefulness of BRP cluster analysis for synapse evaluation, but as there are so many more players involved in synaptic function, BRP analysis certainly cannot explain it all. This should at least be discussed.

It is correct that Brp is not the only player in the active zone. We have included more discussion on the specific role of Brp (line 84 to 89) and other synaptic markers (line 250) and edited potentially misunderstanding text.

(2) I do see that changes in BRP expression were observed following associative learning, but is it certain, that synaptic plasticity is generally unaffected by the large GFP fluorophore? BRP is grabbing onto other proteins, both with its C- and N-termini. As the GFP is right before the stop codon, it should be at the N-terminus. How far could BRP function be hampered by this? Is there still enough space for other proteins to interact?

We thank the reviewer for sharing the concerns. We here provided three lines of evidence to demonstrate that the Brp assembly at active zones required for synaptic plasticity is unaffected by split-GFP tagging.

First, we assessed olfactory memory of flies that have Brp::rGFP labeled in Kenyon cells and found the performance comparable to wild-type (Figure 7 - figure supplement 2), suggesting the Brp function required for olfactory memory (Knapek et al., J Neurosci 2011) is unaffected by split-GFP tagging.

Second, we measured Brp remodeling in photoreceptors induced by constant light exposure (LL; Sugie et al., 2015 Neuron). Consistent with the previous study, we found that LL decreased the numbers of Brp::rGFP clusters in R8 terminals in the medulla, as compared to constant dark condition (DD). This result validates the synaptic plasticity involving dynamic Brp rearrangement in the photoreceptors. We have included this result into the revised manuscript (Figure 1F).

To further validate protein interaction of Brp::rGFP, we focused on Rab3, as it was previously shown to control Brp allocation at active zones (Graf et al., 2009 Neuron). To this end, we silenced rab3 expression in Kenyon cells using RNAi and measured the intensity of Brp::rGFP clusters in γ Kenyon cells. As previously reported in the neuromuscular junction, we found that rab3 knock-down increased Brp::rGFP accumulation to the active zones, suggesting that Brp::rGFP represents the interaction with Rab3. We have included all the new data to the revised manuscript (Figure 1 - figure supplement 3).

(3) It may well be that not only active-zone-associated BRP is labeled but possibly also BRP molecules elsewhere in the neuron. I would like to see more validation, e.g., the percentage of tagged endogenous BRP associated with other presynaptic proteins.

To answer to what extent Brp::rGFP clusters represent active zones, we double-labelled Brp::rGFP and Cac::tdTomato (Cacophony, the alpha subunit of the voltage-gated calcium channels). We found that 97% of Brp::rGFP clusters showed co-localization with Cac::tdTomato in PAM-γ5 dopamine neurons terminals (Figure 1E), suggesting most Brp::rGFP clusters represent functional AZs.

(4) Z-size is ~200 nm, while x/y pixel size is ~75 nm during acquisition. How far down does the resolution go after deconvolution?

The Z-step was 370 nm and XY pixel size was 79 nm for image acquisition. We performed 20 iterations of Richarson-Lucy deconvolution using an empirical point spread function (PSF). We found that the effect of deconvolution on the full-width at half maximum (FWHM) of Brp::rGFP clusters improves only marginally beyond 20 iterations, when the XY FWHM is around 200 nm and the XZ FWHM is around 450 nm (Figure 1 - figure supplement 4).

(5) Figure Legend 7: What is a "cytoplasm membrane marker"? Does this mean membrane-bound tdTom is sticking into the cytoplasm?

We apologize for the typo and have corrected it to “plasma membrane marker”.

(6) At the end of the introduction: "characterizing multiple structural parameters..." - which were these parameters? I was under the assumption that BRP localization, cluster volume, and intensity were assessed. I do not see how these are structural parameters. Please define what exactly is meant by "structural parameters".

We apologize for the confusion. By "structural parameters”, we indeed referred to the volume, intensity and molecular density of Brp::rGFP clusters. We have revised the sentence to “Characterizing the distinct parameters and localization of Brp::rGFP cluster.”

(7) Next to last sentence of the introduction: "Characterizing multiple structural parameters revealed a significant synaptic heterogeneity within single neurons and AZ distribution stereotypy across individuals." What do the authors mean by "significant synaptic heterogeneity"?

By “synaptic heterogeneity”, we refer to the intracellular variability of active zone cytomatrices reported by Brp clusters. For instance, the intensities of Brp::rGFP clusters within Kenyon cell subtypes were variable among compartments (Figure 2). Intracellular variability of the Brp concentration of individual active zones was higher in DPM and APL neurons than Kenyon cells (Figure 3). These variabilities demonstrate intracellular synaptic heterogeneity. We have revised the sentence to be more specific to the different characters of Brp clusters.

(8) I do not understand the last sentence of the introduction. "These cell-type-specific synapse profiles suggest that AZs are organized at multiple scales, ranging from neighboring synapses to across individuals." What do the authors mean by "ranging from neighboring synapses to across individuals"? Does this mean that even neighboring synapses in the same cell can be different?

We have revised the sentence to “These cell-type-specific synapse profiles suggest that AZs are spatially organized at multiple scales, ranging from interindividual stereotypy to neighboring synapses in the same cells.”

By “neighboring synapses", we refer to the nearest neighbor similarity in Brp levels in some cell-types (Figure 6A-C), and also the sub-compartmental dense AZ clusters with high Brp level in Kenyon cells (Figure 6D-H). By “across individuals”, we refer to the individually conserved active zone distribution patterns in some neurons (Figure 5).

(9) The title talks about cell-type-specific spatial configurations. I do not understand what is meant by "spatial configurations"? Do you mean BRP cluster volume? I think the title is a little misleading.

By “spatial configuration”, we refer to the arrangement of Brp clusters within individual mushroom body neurons. This statement is based on our findings on the intracellular synaptic heterogeneity (see also response to comment #7). We have streamlined the text description in the revised manuscript for clarity.

Reviewer #2 (Recommendations for the authors):

(1) For Figure 3A: exemplary two AZs are compared here, a histogram comparing more AZs would aid in making the point that in general, AZ of similar size have different BRP level (intensities) and how much variation exists.

We have included histograms for Brp::rGFP intensity and cluster volumes to Figure 3 in the revised manuscript.

(2) Line 52: "endogenous synapses" is a confusing term; it's probably meant that the protein levels within the synapse are endogenous and not overexpressed. 

We apologize for the confusion and have revised the term to “endogenous synaptic proteins.”

(3) It is not clear from the Materials and Methods section, whether and where deconvolved or not-deconvolved images were used for the quantification pipeline. Please comment on this. 

We have now revised the Method section to clarify how deconvolved or not-deconvolved images were differently used in the pipeline.

(4) Line 664 (C) not bold.

We have corrected the error.

(5) 725 "Files" should be Flies.

We have corrected the error.

(6) 727 two times "first".

We have corrected the error.

(7) Figure 7. All (A) etc., not bold - there should be consistent annotation. 

We want to thank the reviewer for the detailed proof and have corrected all the errors spotted.

Reviewer #3 (Recommendations for the authors):

(1) Has there been an expression of the construct in a non-neuronal cell? Astrocyte-like cell? Any glia? As some sort of control for background and activity?

As the reviewer suggested, we verified the neuronal expression specificity of Brp::rGFP. Using R86E01-GAL4 and Amon-GAL4, we compared Brp::rGFP in astrocyte-like glia and neuropeptide-releasing neurons. We found no Brp::rGFP puncta in the neuropils in astrocyte-like glia compared to neurons, suggesting Brp::rGFP is specific to neurons. We have included this new dataset to the revised manuscript (Figure 1 - figure supplement 2).

(2) Similarly, expression of the construct co-expressed with a channelrhodopsin, and induction of a 'learning'-like regime of activity, similarly in a control type of experiment, expression of an inwardly rectifying channel (e.g. Kir2.1) to show that increases in size of the BRP puncta are truly activity dependent? The NMJ may be an optimal neuron to use to see the 'donut' structures of the AZs and their increase with activity. Also, are these truly AZs we are seeing here? Perhaps try co-expressing cacophony-dsRed? If the GFP Puncta are active zones, then they should be surrounded by cacophony.

We would like to clarify that we did not find Brp::rGFP size increase upon learning. Instead, we demonstrated that associative training transiently remodelled sub-compartment-sized AZ “hot spots” in Kenyon cells, indicated by the correlation of local intensity and AZ density (Figure 6-7).

To demonstrate split-GFP tagging does not affect activity-dependent plasticity associated with Brp, we measured Brp remodeling in photoreceptors induced by constant light exposure (LL; Sugie et al., 2015 Neuron). Consistent with the previous study, we found that LL decreased the numbers of Brp::rGFP clusters in R8 terminals in the medulla, as compared to constant dark condition (DD). This result validates the synaptic plasticity involving dynamic Brp rearrangement in the photoreceptors (Figure 1F).

As the reviewer suggested, we performed the STED microscopy for the larval motor neuron and confirmed the donut-shape arrangement of Brp::rGFP (Wu, Eno et al., PLOS Biol 2025).

Also following the reviewer’s suggestion, we double-labelled Brp::rGFP and Cac::tdTomato (Cacophony, the alpha subunit of the voltage-gated calcium channels). We found that 97% Brp::rGFP clusters showed co-localization with Cac::tdTomato in PAM-γ5 dopamine neurons terminals (Figure 1E), suggesting most Brp::rGFP clusters represent functional AZs.

(3) In the introduction: Intro, a sentence about BRP - central organiser of the active zone, so a key regulator of activity.

We have included a few more sentences about the role Brp in the active zones to the revised manuscript.

(4) Figure 1 E, line 650 'cite the resource here'. 

We thank the reviewer for pointing out the error and we have corrected it.

(5) Many readers may not be MB aficionados, and to make the data more accessible, perhaps use a cartoon of an MB with the cell bodies of the neurons around the MB expressing the constructs highlighted so that the reader can have a wider idea of the anatomy in relation to the MB.

We appreciate these comments and have appended cartoons of the MB to figures to help readers understand the anatomy.

Author response:

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

eLife Assessment

This useful study uses creative scalp EEG decoding methods to attempt to demonstrate that two forms of learned associations in a Stroop task are dissociable, despite sharing similar temporal dynamics. However, the evidence supporting the conclusions is incomplete due to concerns with the experimental design and methodology. This paper would be of interest to researchers studying cognitive control and adaptive behavior, if the concerns raised in the reviews can be addressed satisfactorily.

We thank the editors and the reviewers for their positive assessment of our work and for providing us with an opportunity to strengthen this manuscript. Please see below our responses to each comment raised in the reviews.

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study focuses on characterizing the EEG correlates of item-specific proportion congruency effects. In particular, two types of learned associations are characterized. One being associations between stimulus features and control states (SC), and the other being stimulus features and responses (SR). Decoding methods are used to identify SC and SR correlates and to determine whether they have similar topographies and dynamics.

The results suggest SC and SR associations are simultaneously coactivated and have shared topographies, with the inference being that these associations may share a common generator.

Strengths:

Fearless, creative use of EEG decoding to test tricky hypotheses regarding latent associations. Nice idea to orthogonalize the ISPC condition (MC/MI) from stimulus features.

Thank you for acknowledging the strength in EEG decoding and design. We have addressed all your concerns raised below point by point.

Weaknesses:

(1a) I'm relatively concerned that these results may be spurious. I hope to be proven wrong, but I would suggest taking another look at a few things.

While a nice idea in principle, the ISPC manipulation seems to be quite confounded with the trial number. E.g., color-red is MI only during phase 2, and is MC primarily only during Phase 3 (since phase 1 is so sparsely represented). In my experience, EEG noise is highly structured across a session and easily exploited by decoders. Plus, behavior seems quite different between Phase 2 and Phase 3. So, it seems likely that the classes you are asking the decoder to separate are highly confounded with temporally structured noise.

I suggest thinking of how to handle this concern in a rigorous way. A compelling way to address this would be to perform "cross-phase" decoding, however I am not sure if that is possible given the design.

Thank you for raising this important issue. To test whether decoding might be confounded by temporally structured noise, we performed a control decoding analysis. As the reviewer correctly pointed out, cross-phase decoding is not possible due to the experimental design. Alternatively, to maximize temporal separation between the training and test data, we divided the EEG data in phase 2 and phase 1&3 into the first and second half chronologically. Phase 1 and 3 were combined because they share the same MC and MI assignments. We then trained the decoders on one half and tested them on the other half. Finally, we averaged the decoding results across all possible assignments of training and test data. The similar patterns (Supplementary Fig.1) observed confirmed that the decoding results are unlikely to be driven by temporally structured noise in the EEG data. The clarification has been added to page 13 of the revised manuscript.

(1b) The time courses also seem concerning. What are we to make of the SR and SC timecourses, which have aggregate decoding dynamics that look to be <1Hz?

As detailed in the response to your next comment, some new results using data without baseline correction show a narrower time window of above-chance decoding. We speculate that the remaining results of long-lasting above-chance decoding could be attributed to trials with slow responses (some responses were made near the response deadline of 1500 ms). Additionally, as shown in Figure 6a, the long-lasting above-chance decoding seems to be driven by color and congruency representations. Thus, it is also possible that the binding of color and congruency contributes to decoding. This interpretation has been added to page 17 of the revised manuscript.

(1c) Some sanity checks would be one place to start. Time courses were baselined, but this is often not necessary with decoding; it can cause bias (10.1016/j.jneumeth.2021.109080), and can mask deeper issues. What do things look like when not baselined? Can variables be decoded when they should not be decoded? What does cross-temporal decoding look like - everything stable across all times, etc.?

As the reviewer mentioned, baseline-corrected data may introduce bias to the decoding results. Thus, we cited the van Driel et al (2021) paper in the revised manuscript to justify the use of EEG data without baseline-correction in decoding analysis (Page 27 of the revised manuscript), and re-ran all decoding analysis accordingly. The new results revealed largely similar results (Fig. 2, 4, 6 and 8 in the revised manuscript) with the following exceptions: narrower time window for separatable SC subspace and SR subspace (Fig. 4b), narrower time window for concurrent representations of SC and SR (Fig. 6a-b), and wider time window for the correlations of SC/SR representations with RTs (Fig. 8).

(2) The nature of the shared features between SR and SC subspaces is unclear.

The simulation is framed in terms of the amount of overlap, revealing the number of shared dimensions between subspaces. In reality, it seems like it's closer to 'proportion of volume shared', i.e., a small number of dominant dimensions could drive a large degree of alignment between subspaces.

What features drive the similarity? What features drive the distinctions between SR and SC? Aside from the temporal confounds I mentioned above, is it possible that some low-dimensional feature, like EEG congruency effect (e.g., low-D ERPs associated with conflict), or RT dynamics, drives discriminability among these classes? It seems plausible to me - all one would need is non-homogeneity in the size of the congruency effect across different items (subject-level idiosyncracies could contribute: 10.1016/j.neuroimage.2013.03.039).

Thank you for this question. To test what dimensions are shared between SC and SR subspaces, we first identify which factors can be shared across SC and SR subspaces. For SC, the eight conditions are the four colors × ISPC. Thus, the possible shared dimensions are color and ISPC. Additionally, because the four colors and words are divided into two groups (e.g., red-blue and green-yellow, counterbalanced across subjects, see Methods), the group is a third potential shared dimension. Similarly, for SR decoders, potential shared dimensions are word, ISPC and group. Note that each class in SC and SR decoders has both congruent and incongruent trials. Thus, congruency is not decodable from SC/SR decoders and hence unlikely to be a shared dimension in our analysis. To test the effect of sharing for each of the potential dimensions, we performed RSA on decoding results of the SC decoder trained on SR subspace (SR | SC) (Supplementary Fig. 4a) and the SR decoder trained on SC subspace (SC | SR) (Supplementary Fig. 4b), where the decoders indicated the decoding accuracy of shared SC and SR representations. In the SC classes of SR | SC, word red and blue were mixed within the same class, same were word yellow and green. The similarity matrix for “Group” of SR | SC (Supplementary Fig. 4a) shows the comparison between two word groups (red & blue vs. yellow & green). The similarity matrix for “Group” of SC | SR (Supplementary Fig. 4b) shows the comparison between two color groups (red & blue vs. yellow & green).

The RSA results revealed that the contributions of group to the SC decoder (Supplementary Fig. 5a) and the SR decoder (Supplementary Fig. 5b) were significant. Meanwhile, a wider time window showed significant effect of color on the SC decoder (approximately 100 - 1100 ms post-stimulus onset, Supplementary Fig. 5a) and a narrower time window showed significant effect of word on SR decoder (approximately 100 - 500 ms post-stimulus onset, Supplementary Fig. 5b). However, we found no significant effect of ISPC on either SC or SR decoders. We also performed the same analyses on response-locked data from the time window -800 to 200 ms. The results showed shared representation of color in the SC decoder (Supplementary Fig. 5c) and group in both decoders (Supplementary Fig. 5c-d). Overall, the above results demonstrated that color, word and group information are shared between SC and SR subspaces.

Lastly, we would like to stress that our main hypothesis for the cross-subspace decoding analysis is that SR and SC subspaces are not identical. This hypothesis was supported by lower decoding accuracy for cross-subspace than within-subspace decoders and enables following analyses that treated SC and SR as separate representations.

We have added the interpretation to page 13-14 of the revised manuscript.

(3) The time-resolved within-trial correlation of RSA betas is a cool idea, but I am concerned it is biased. Estimating correlations among different coefficients from the same GLM design matrix is, in general, biased, i.e., when the regressors are non-orthogonal. This bias comes from the expected covariance of the betas and is discussed in detail here (10.1371/journal.pcbi.1006299). In short, correlations could be inflated due to a combination of the design matrix and the structure of the noise. The most established solution, to cross-validate across different GLM estimations, is unfortunately not available here. I would suggest that the authors think of ways to handle this issue.

Thank you for raising this important issue. Because the bias comes from the covariance between the regressors and the same GLM was applied to all time points in our analysis, we assume that the inflation would be similar at different time points. Therefore, we calculated the correlation of SC and SR betas ranging from -200 to 0 ms relative to stimulus onset as a baseline (i.e., no SC or SR representation is expected before the stimulus onset) and compared the post-stimulus onset correlation coefficients against this baseline. We hypothesized that if the positively within-trial correlation of SC and SR betas resulted from the simultaneous representation instead of inflation, we should observe significantly higher correlation when compared with the baseline. To examine this hypothesis, we first performed the linear discriminant analysis (Supplementary Fig. 7a) and RSA regression (Supplementary Fig. 7b) on the -200 - 0 ms window relative to stimulus onset. We then calculated the average r_baseline of SC and SR betas on that time window for each participant (group results at each time point are shown in Supplementary Fig. 7c) and computed the relative correlation at each post-stimulus onset time point using (fisher-z (r) - fisher-z (r_baseline)). Finally, we performed a simple t test at the group level on baseline-corrected correlation coefficients with Bonferroni correction. The results (Fig. 6c) showed significantly more positive correlation from 100 - 500 ms post-stimulus onset compared with baseline, supporting our hypothesis that the positive within-trial correlation of SC and SR betas arise from simultaneous representation rather than inflation. The related interpretation was added to page 17 of the revised manuscript.

(4) Are results robust to running response-locked analyses? Especially the EEG-behavior correlation. Could this be driven by different RTs across trials & trial-types? I.e., at 400 ms poststim onset, some trials would be near or at RT/action execution, while others may not be nearly as close, and so EEG features would differ & "predict" RT.

Thanks for this question. We now pair each of the stimulus-locked EEG analysis in the manuscript with response-locked analysis. To control for RT variations among trial types, when using the linear mixed model (LMM) to predict RTs from trial-wise RSA results, we included a separate intercept for each of the eight trial types in SC or SR. Furthermore, at each time point, we only included trials that have not generated a response (for stimulus-locked analysis) or already started (for response-locked analysis). All the results (Fig. 3, 5, 7, 9 in the revised manuscript) are in support of our hypothesis. We added these detailed to page 31 of the revised manuscript.

(5) I suggest providing more explanation about the logic of the subspace decoding method - what trialtypes exactly constitute the different classes, why we would expect this method to capture something useful regarding ISPC, & what this something might be. I felt that the first paragraph of the results breezes by a lot of important logic.

In general, this paper does not seem to be written for readers who are unfamiliar with this particular topic area. If authors think this is undesirable, I would suggest altering the text.

To improve clarity, we revised the first paragraph of the SC and SR association subspace analysis to list the conditions for each of the SC and SR decoders and explain more about how the concept of being separatable can be tested by cross-decoding between SC and SR subspaces. The revised paragraph now reads:

“Prior to testing whether controlled and non-controlled associations were represented simultaneously, we first tested whether the two representations were separable in the EEG data.

In other words, we reorganized the 16 experimental conditions into 8 conditions for SC (4 colors × MC/MI, while collapsing across SR levels) and SR (4 words × 2 possible responses per word, while collapsing across SC levels) associations separately. If SC and SR associations are not separable, it follows that they encode the same information, such that both SC and SR associations can be represented in the same subspace (i.e., by the same information encoded in both associations). For example, because (1) the word can be determined by the color and congruency and (2) the most-likely response can be determined by color and ISPC, the SR association (i.e., association between word and most-likely response) can in theory be represented using the same information as the SC association. On the other hand, if SC and SR associations are separable, they are expected to be represented in different subspaces (i.e., the information used to encode the two associations is different). Notably, if some, but not all, information is shared between SC and SR associations, they are still separable by the unique information encoded. In this case, the SC and SR subspaces will partially overlap but still differ in some dimensions. To summarize, whether SC and SR associations are separable is operationalized as whether the associations are represented in the same subspace of EEG data. To test this, we leveraged the subspace created by the LDA (see Methods). Briefly, to capture the subspace that best distinguishes our experimental conditions, we trained SC and SR decoders using their respective aforementioned 8 experimental conditions. We then projected the EEG data onto the decoding weights of the LDA for each of the SC and SR decoders to obtain its respective subspace. We hypothesized that if SC and SR subspaces are identical (i.e., not separable), SC/SR decoding accuracy should not differ by which subspace (SC or SR) the decoder is trained on. For example, SC decoders trained in SC subspace should show similar decoding performance as SC decoders trained in SR subspace. On the other hand, if SC and SR association representations are in different subspaces, the SC/SR subspace will not encode all information for SR/SC associations. As a result, decoding accuracy should be higher using its own subspace (e.g., decoding SC using the SC subspace) than using the other subspace (e.g., decoding SC using the SR subspace). We used cross-validation to avoid artificially higher decoding accuracy for decoders using their own subspace (see Methods).” (Page 11-12).

We also explicitly tested what information is shared between SC and SR representations (see response to comment #2). Lastly, to help the readers navigate the EEG results, we added a section “Overview of EEG analysis” to summarize the EEG analysis and their relations in the following manner:

“EEG analysis overview. We started by validating that the 16 experimental conditions (8 unique stimuli × MC/MI) were represented in the EEG data. Evidence of representation was provided by above-chance decoding of the experimental conditions (Fig. 2-3). We then examined whether the SC and SR associations were separable (i.e., whether SC and SR associations were different representations of equivalent information). As our results supported separable representations of SC and SR association (Fig. 4-5), we further estimated the temporal dynamics of each representation within a trial using RSA. This analysis revealed that the temporal dynamics of SC and SR association representations overlapped (Fig. 6a-b, Fig. 7a-b). To explore the potential reason behind the temporal overlap of the two representations, we investigated whether SC and SR associations were represented simultaneously as part of the task representation, independently from each other, or competitively/exclusively (e.g., on some trials only SC association was represented, while on other trials only SR association was represented). This was done by assessing the correlation between the strength of SC and SR representations across trials (Fig. 6c, Fig. 7c). Lastly, we tested how SC and SR representations facilitated performance (Fig.8-9).” (Page 8-9).

Minor suggestions:

(6) I'd suggest using single-trial RSA beta coefficients, not t-values, as they can be more stable (it's a t-value based on 16 observations against 9 or so regressors.... the SE can be tiny).

Thank you for your suggestion. To choose between using betas and t-values, we calculate the proportion of outliers (defined as values beyond mean ± 5 SD) for each predictor of the design matrix and each subject. We found that outliers were less frequent for t-values than for beta coefficients (t-values: mean = 0.07%, SD = 0.009%; beta-values: mean = 0.19%, SD = 0.033%). Thus, we decided to stay with t-values.

(7) Instead of prewhitening the RTs before the HLM with drift terms, try putting those in the HLM itself, to avoid two-stage regression bias.

Thank you for your suggestion. Because our current LMM included each of the eight trial types in SC or SR as separate predictors with their own intercepts (as mentioned above), adding regressors of trial number and mini blocks (1-100 blocks) introduced collinearity (as ISPC flipped during the experiment). We therefore excluded these regressors from the current LMM (Page 31).

(8) The text says classical MDS was performed on decoding *accuracy* - is this accurate?

We now clarify in the manuscript that it is the decoders’ probabilistic classification results (Page 28).

(9) At a few points, it was claimed that a negative correlation between SC and SR would be expected within single trials, if the two were temporally dissociable. Wouldn't it also be possible that they are not correlated/orthogonal?

We agree with the reviewer and revised the null hypothesis in the cross-trial correlation analysis to include no correlation as SC and SR association representations may be independent from each other (Page 17, 22).

Reviewer #2 (Public review):

Summary:

In this EEG study, Huang et al. investigated the relative contribution of two accounts to the process of conflict control, namely the stimulus-control association (SC), which refers to the phenomenon that the ratio of congruent vs. incongruent trials affects the overall control demands, and the stimulus-response association (SR), stating that the frequency of stimulusresponse pairings can also impact the level of control. The authors extended the Stroop task with novel manipulation of item congruencies across blocks in order to test whether both types of information are encoded and related to behaviour. Using decoding and RSA, they showed that the SC and SR representations were concurrently present in voltage signals, and they also positively co-varied. In addition, the variability in both of their strengths was predictive of reaction time. In general, the experiment has a solid design, but there are some confounding factors in the analyses that should be addressed to provide strong support for the conclusions.

Strengths:

(1) The authors used an interesting task design that extended the classic Stroop paradigm and is potentially effective in teasing apart the relative contribution of the two different accounts regarding item-specific proportion congruency effect, provided that some confounds are addressed.

(2) Linking the strength of RSA scores with behavioural measures is critical to demonstrating the functional significance of the task representations in question.

Thank you for your positive feedback. We hope our responses below address your concerns.

Weakness:

(1) While the use of RSA to model the decoding strength vector is a fitting choice, looking at the RDMs in Figure 7, it seems that SC, SR, ISPC, and Identity matrices are all somewhat correlated. I wouldn't be surprised if some correlations would be quite high if they were reported. Total orthogonality is, of course, impossible depending on the hypothesis, but from experience, having highly covaried predictors in a regression can lead to unexpected results, such as artificially boosting the significance of one predictor in one direction, and the other one to the opposite direction. Perhaps some efforts to address how stable the timed-resolved RSA correlations for SC and SR are with and without the other highly correlated predictors will be valuable to raising confidence in the findings.

Thank you for this important point. The results of proportion of variability explained shown in the Author response table 1 below, indicated relatively higher correlation of SC/SR with Color and Identity. We agree that it is impossible to fully orthogonalize them. To address the issue of collinearity, we performed a control RSA by removing predictors highly correlated with others. Specifically, we calculated the variance inflation factor (VIF) for each predictor. The Identity predictor had a high VIF of 5 and was removed from the RSA. All other predictors had VIFs < 4 and were kept in the RSA. The results (Supplementary Fig. 6) showed patterns similar to the results with the Identity predictor, suggesting that the findings are not significantly influenced by collinearity. We have added the interpretation to page 17 of the revised manuscript.

Author response table 1.

Proportion of variability explained (r²) of RSA predictors.

(2) In "task overview", SR is defined as the word-response pair; however, in the Methods, lines 495-496, the definition changed to "the pairing between word and ISPC" which is in accordance with the values in the RDMs (e.g., mccbb and mcirb have similarity of 1, but they are linked to different responses, so should they not be considered different in terms of SR?). This needs clarification as they have very different implications for the task design and interpretation of results, e.g., how correlated the SC and SR manipulations were.

Thank you for pointing out this important issue with how our operationalization captures the concept in questions. In the revised manuscript, we clarified the stimulus-response (SR) association is the link between the word and the most-likely response (i.e., not necessarily the actual response on the current trial). This association is likely to be encoded based on statistical learning over several trials. On each trial, the association is updated based on the stimulus and the actual response. Over multiple trials, the accumulated association will be driven towards the most-common (i.e., most-likely) response. In our ISPC manipulation, a color is presented in mostly congruent/incongruent (MC/MI) trials, which will also pair a word with a most-likely response. For example, if the color blue is MC, the color blue, which leads to the response blue, will co-occur with the word blue with high frequency. In other words, the SR association here is between the word blue and the response blue. As the actual response is not part of the SR association, in the RDM two trial types with different responses may share the same SR association, as long as they share the same word and the same ISPC manipulation, which, by the logic above, will produce the same most-likely response. These clarifications have been added to page 4 and 29 of the revised manuscript.

In the revised manuscript (Page 17), we addressed how much the correlated SC and SR predictors in the RDM could affect the correlation analysis between SC and SR association representation strength. Specifically, we conducted the RSA using the same GLM on EEG data prior to stimulus onset (Supplementary Fig. 7a-b). As no SC and SR associations are expected to be present before stimulus onset, the correlation between SC and SR representation would serve as a baseline of inflation due to correlated predictors in the GLM (Supplementary Fig. 7c, also see comment #3 of R1). The SC-SR correlation coefficients following stimulus onset was then compared to the baseline to control for potential inflation (Fig. 6c). Significantly above-baseline correlation was still observed between ~100-500 ms post-stimulus onset, providing support for the hypothesis that SC and SR are encoded in the same task representation.

Minor suggestions:

(3) Overall, I find that calling SC-controlled and SR-uncontrolled representations unwarranted. How is the level controlledness defined? Both are essentially types of statistical expectation that provide contextual information for the block of tasks. Is one really more automatic and requires less conscious processing than the other? More background/justification could be provided if the authors would like to use these terms.

Following your advice, we have added more discussion on how controlledness is conceptualized in this work and in the literature, which reads:

“We consider SC and SR as controlled and uncontrolled respectively based on the literature investigating the mechanism of ISPC effect. The SC account posits that the ISPC effect results from conflict and involves conflict adaptation, which requires the regulation of attention or control (Bugg & Hutchison, 2013; Bugg et al., 2011; Schmidt, 2018; Schmidt & Besner, 2008). On the other hand, the SR account argues that ISPC effect does not require conflict adaptation but instead reflects contingency leaning. That is, the response can be directly retrieved from the association between the stimulus and the most-likely response without top-down regulation of attention or control. As more empirical evidence emerged, researchers advocating control view began to acknowledge the role of associative learning in cognitive control regarding the ISPC effect (Abrahamse et al., 2016). SC association has been thought to include both automatic that is fast and resource saving and controlled processes that is flexible and generalizable (Chiu, 2019). Overall, we do not intend to claim that SC is entirely controlled or SR is completely automatic. We use SC-controlled and SR-uncontrolled representations to align with the original theoretical motivation and to highlight the conceptual difference between SC and SR associations.” (Page 24-25)

(4) Figures 3c and d: the figures could benefit from more explanation of what they try to show to the readers. Also for 3d, the dimensions were aligned with color sets and congruencies, but word identities were not linearly separable, at least for the first 3 axes. Shouldn't one expect that words can be decoded in the SR subspace if word-response pairs were decodable (e.g., Figure 3b)?

Thank you for the insightful observation. We now clarified that Fig. 3c and d in the original manuscript (Fig. 4c and d in the current manuscript) aim to show how each of the 8 trial types in the SC and SR subspaces are represented. The MDS approach we used for visualization tries to preserve dissimilarity between trial types when projecting from data from a high dimensional to a low dimensional space. However, such projection may also make patterns linearly separatable in high dimensional space not linearly separatable in low dimensional space. For example, if the word blue has two points (-1, -1) and (1, 1) and the word red has two points (-1, 1) and (1, -1), they are not linearly separatable in the 2D space. Yet, if they are projected from a 3D space with coordinates of (-1, -1, -0.1), (1, 1, -0.1), (-1, 1, 0.1) and (1, -1, 0.1), the two words can be linearly separatable using the 3^rd dimension. Thus, a better way to test whether word can be linearly separated in SR subspace is to perform RSA on the original high dimensional space. We performed the RSA with word (Supplementary Fig. 2) on the SR decoder trained on the SR subspace. Note that in Fig. 3c and d of the original script (Fig. 4c and d in the current manuscript) there are two pairs of words that are not linearly separable: red-blue and yellow-green. Thus, we specifically tested the separability within the two pairs using the one predictor for each pair, as shown in Supplementary Fig. 2. The results showed that within both word pairs individual words were presented above chance level (Supplementary Fig. 3). Considering that the decoders are linear, this finding indicates linear separability of the word pairs in the original SR subspace. The clarification has been added to page 13 (the end of the second paragraph) of the revised manuscript.

References

Abrahamse, E., Braem, S., Notebaert, W., & Verguts, T. (2016). Grounding cognitive control in associative learning. Psychological Bulletin, 142(7), 693-728.doi:10.1037/bul0000047.

Bugg, J. M., & Hutchison, K. A. (2013). Converging evidence for control of color-word Stroop interference at the item level. Journal of Experimental Psychology:Human Perception and Performance, 39(2), 433-449. doi:10.1037/a0029145.

Bugg, J. M., Jacoby, L. L., & Chanani, S. (2011). Why it is too early to lose control in accounts of item-specific proportion congruency effects. Journal of Experimental Psychology: Human Perception and Performance, 37(3), 844-859. doi:10.1037/a0019957.

Chiu, Y.-C. (2019). Automating adaptive control with item-specific learning. In Psychology of Learning and Motivation (Vol. 71, pp. 1-37).

Schmidt, J. R. (2018). Evidence against conflict monitoring and adaptation: An updated review. Psychonomic Bulletin & Review, 26(3), 753-771. doi:10.3758/s13423018-1520-z.

Schmidt, J. R., & Besner, D. (2008). The Stroop effect: Why proportion congruent has nothing to do with congruency and everything to do with contingency. Journal of Experimental Psychology: Learning, Memory, and Cognition, 34(3), 514-523. doi:10.1037/0278-7393.34.3.514.

not much has changed since back then, there is still this decline for new immigrants now

Me gusta en un 50/50 esta forma de pensar, si bien nada es perfecto y todo puede mejorar y evolucionar con el tiempo, no me cerraría a pensar de que estoy lejos o que no podría llegar a la meta que me propuse en cierto momento. Todo en la vida es resiliencia y mejora continua de procesos de manera progresiva y alcanzable.

Me parece algo totalmente genial (dejando de lado de que no todo el mundo tiene un récord guinness y todos los requisitos que solicitan para tenerlo) sino por el hecho de que no es algo aleatorio.

Recuerdo haber leído sobre ese récord hace un tiempo y si no me equivoco era sobre recorrer cierta cantidad de estaciones de metro (unas 400 y algo) en un tiempo de 24 horas ("pasaditas") y que estuvo vigente hasta cierto tiempo, lo que me parece fascinante es que cómo dije anteriormente, no fue algo esporádico, fue algo planeado, algo planificado en equipo

Cómo toda gran idea novedosa o innovadora que nace de la incomodidad ...

Ser disruptivo y crear algo que cambie y mejore las reglas convencionales es algo que siempre he de admirar. Tener la convicción de diseñar algo que se sabe que reúne lo mejor de varios sistemas es algo que no todo el mundo hace, si bien quisieron hacer algo más "pequeño, propio y privado" (que se entiende muy bien, no por el tema de envidia o privatización sino porque quizá uno cómo persona no dimensiona el impacto de sus creaciones), algo que me llamó la atención es que fueron de lleno a crear algo a la altura de los lenguajes de alto nivel (básicamente que se pueden hacer más y mejores cosas sin tantas líneas de código), ósea que simplemente no fue un típico proyecto que ya existía, sino que intentaron ir más allá de una vez, simplemente adelantados a su tiempo, es increíble

La verdadera ruptura fue la transición de un proyecto entre amigos a una compañía auténtica, para Shah y su equipo, el desafío no consistía en el aspecto técnico del lenguaje, que ya era excelente; sino en cómo hacer que sobreviviera: convertir un código gratuito en una empresa sostenible. Fue una decisión, abandonar el experimento académico y transformarlo en un instrumento profesional que hoy emplean empresas de gran tamaño en cualquier parte del mundo.

Básicamente, fue una "frustración colectiva" entre amigos, que unió a mentes brillantes Karpinski expresó su inconformidad a Shah, quien a su vez convenció a Bezanson y Edelman, decidieron dejar de esperar que no existiera una la herramienta tecnológica perfecta y crearon su propia solución .

Esta anécdota de Karpinski lo dice todo, Julia fue creado por gente que odiaba perder el tiempo, en otras palabras Julia es el resultado de expertos que se cansaron de esperar y decidieron construir un diseño mas ágil en el mundo de programación.

A veces la frustración en cualquier campo es el combustible de las mejores innovaciones tecnológicas y el caso Julia y sus creadores no es exención.

La colaboración entre comunidad y desarrolladores es imprescindible ya que estos mismos usuarios al encontrarse con necesidades propias y la adaptabilidad de las herramientas en condición de software libre genera una distribución y una evolución del proyecto de una manera que el equipo original jamás hubiera logrado. Es importante contar con el desarrollo y ayuda de cualquier persona interesada ya que al fin y al cabo de esa misma manera se hace la ciencia, mediante comunicación y aportando a lo ya existente.

Hasta ahora no sabía de lenguajes de programación creados a partir de combinar varios lenguajes existentes y establecidos para transformar algo nuevo, ya ahora se me hace una idea muy interesante que se alinea con el objetivo que se planteó al principio el cual era hacer un lenguaje accesible para todos, cómodo para los principiantes y completo para los veteranos, por lo que este esfuerzo de crear algo nuevo con bases de otros se entiende como tomar los mejor de cada uno y crear algo nuevo mas allá de la suma de sus partes.

Cuando Julia es puesta en código abierto, se convierte en un espacio donde muchas personas pueden contribuir y también proponer una mejora continua. Considero que de esta manera los usuarios dejan de solo consumir información a crear nuevo conocimiento a partir de sus intervenciones.

Creo que el hecho de siempre crear algo para hacer un cambio mayor en el mundo, trae muchos problemas como el hecho de no querer usarlo por ser nuevo, pero por lo que se nos cuenta, realmente era algo diferente a lo que ya habia

Es curioso como en algunas historias tambien se escuchan temas similares, una persona que tiene un trabajo en una empresa haciendo una labor, pero cuando tienen tiempo a solas, y esa libertad de crear, es cuando avanzan en su proyecto personal

Me encanta esta frase ya que resalta que lo que realmente une a las personas no es solo la tecnología en sí, sino un propósito compartido y no individual.

¿Un lenguaje se vuelve grande solo con ser bueno? Considero firmemente que necesita de una comunidad que lo respalde.

Esto es bastante real, ya que la mayoría de lenguas de programación nacen de una necesidad.

El trabajo colaborativo del código abierto es lo que permite que evolucione contantemente, si algo a ellos los creadores no se les ocurre, puede que a otro se le ocurra algo mejor. y ese constante cambio puede llegar a mejores cosas.

Ellos lo saben, con tantos desarrollos en proceso y la tecnología corriendo a grandes pasos, su proyecto no tendrá final, siempre estará en constante evolución, e impactando en su proceso.

Es curioso como se dio el comienzo de este lenguaje, sin tanto tramite o burocracia, solo una charla entre colegas con correos y pasaron manos a la obra, es gratificante ver como un proyecto así nace sin tanto papeleo, solo personas, una idea y ganas de desarrollar algo.

Las grandes invenciones son desde una necesidad, para estas personas que en su practica de uso de estos lenguajes y querer combinar y crear algún modelo mucho mejor y que fuera fácil, es como todo desde una idea o un pensamiento pequeño puede llegar a cambiar algo en gran medida.

Las iniciativas que se amplían y permiten ver múltiples perspectivas, crecen debido a que el talento se puede esconder en las manos que no tienen la oportunidad.

La invención de grandes cosas inició con grandes frustraciones. Por ello, actualmente existe un dilema social y estructural: si la inteligencia artificial resuelve por nosotros, ¿qué pasará con nuestro desarrollo, especialmente en el ámbito cognitivo?

Esta frase me recuerda a ese dicho "se el cambio que quieres ver", y es que supongo que así es como se produce el avance, como el ejemplo que vimos en clase con Opera y como su fundador tras ver los cambios que le hicieron con su venta, lo llevaría a la creación de vivaldi. Al final supongo que para cambiar algo, es necesario crear un nuevo modelo que haga obsoleto el modelo existente.

Es bastante bueno, diferencial e innovador que se permita crear y dirigir la creación de páginas web de acuerdo a lo que desean y quieren las personas. Mientras que otros servicios se encargan de ser generadores netamente de sitios web empresariales o de market, esta permite dirigir su servicio a otro del mercado, cómo el de los videojuegos

Si bien esto puede ser un ganchazo comercial muy bueno, es una razón valida para optar por herramientas gratuitas y propias que de verdad se preocupen y sean garantes de la protección de los datos, datos personales y contenidos de las páginas web que creen las personas

Si bien comparten ciertas características cómo algunos referentes que son lideres en la generación rápida de sitios web cómo GoDaddy o Wix, me llamó la atención que sean laxos en cuanto a funciones, contenidos e infraestructura, mientras que otros sitios que de cara ya son de pago o restringen el uso de cierto tipo de contenido, esta promesa que compite contra estos lideres potencia y libera la capacidad creativa de los usuarios de forma gratuita, propia e intuitiva

En mi caso personal lo que importa es la versatilidad y la eficiencia que se logra con una base bien diseñada.

Considero que esto es lo que fortalece la pluralidad y expansión del conocimiento en todos sus ámbitos, la iteración. Poder volver atrás cuando algo no está bien o incluso volver a empezar, reconocer la frustración y los nuevos principios como nuevos comienzos.

NOTE DE SYNTHÈSE : Dans la tête d'un colérique

Introduction

Cette note de synthèse explore les facettes de la colère présentées dans les extraits de l'émission "Dans la tête d'un colérique".

Le documentaire examine la nature de cette émotion souvent perçue négativement, sa gestion individuelle et collective, ses manifestations, ses fonctions insoupçonnées, et les conséquences de son refoulement ou de son expression violente.

À travers des témoignages, des analyses de spécialistes et des expériences, l'émission offre un éclairage nuancé sur une émotion complexe et puissante.

Thèmes Principaux

La perception sociale de la colère et son contrôle: La colère est largement considérée comme une émotion négative et "mal vue".

L'éducation et les règles de bienséance nous incitent à la contrôler.

La fonction intrinsèque et l'utilité de la colère: Malgré sa mauvaise réputation, les spécialistes affirment que la colère est nécessaire.

Elle peut nous protéger et, de manière surprenante, augmenter considérablement nos performances physiques et cognitives.

Les manifestations et mécanismes de la colère individuelle:

Le témoignage d'Eduardo illustre comment la colère peut être déclenchée par la peur (face à une agression) ou la frustration (avec ses enfants), entraînant des réactions physiques intenses ("tout ton corps qui change si ce mr bouillir... les yeux qui devient pour les rouges tout ça").

Chez un colérique, la tension monte vite et très haut, rendant difficile la prise en compte d'autres perspectives ("il est plus capable de penser... il n'ya plus que son point de vue qui compte").

La colère dans les relations proches:

Les émotions, y compris la colère, débordent davantage avec ceux qu'on aime.

L'anticipation des réactions de l'autre ("je savais que tu allais faire ça") et le système d'attachement (sécurisé ou insécure) influencent la gestion de la colère dans les relations intimes.

Un système d'attachement insécure peut amplifier les émotions négatives ("ça va alimenter active est encore plus mes émotions de tristesse de colère").

La réaction face à la colère d'autrui:

Être confronté à la colère d'un inconnu provoque souvent la "pétrification" ou la "sidération".

La colère fait peur car elle "menace potentiellement l'intégrité d'autrui", mais aussi "l'intégrité des règles de civilité".

On ne sait jamais quelles sont les "limites" de la personne en colère, d'où la peur et le retrait.

Les conséquences de la colère non maîtrisée et violente:

La colère, surtout lorsqu'elle devient violente, fait souffrir non seulement les proches mais aussi les personnes colériques elles-mêmes.

Dans les cas extrêmes, elle peut empêcher de vivre et mener à des comportements autodestructeurs ou hétéro-agressifs.

Le témoignage de Mischa, une femme violente, met en lumière la honte et le caractère dévastateur de sa colère ("c'est très violent c'est c'est comme un tremblement de terre").

La gestion et la canalisation de la colère:

Refouler la colère est comparé à un "cancer" qui peut créer des "pathologies physiques".

Apprendre à réguler sa colère ne signifie pas l'éradiquer, mais trouver d'autres stratégies pour soulager la tension interne.

L'utilité fonctionnelle de la colère: Communication et positionnement:

La colère est un "outil de communication" dès l'enfance et permet, à l'âge adulte, de "mettre des limites" et de ne pas se faire "bouffer par les autres".

Performance physique: Une expérience a démontré que la colère (induite par un sentiment d'injustice) peut augmenter la force physique ("augmentent leur force physique").

La colère agit comme un "moteur", un "booster" qui "permet d'optimiser les performances" physiques car elle est associée à un niveau d'"activation" et d'"éveil" élevé.

Performance cognitive (inconsciente):

Une autre expérience suggère que l'activation inconsciente du concept de colère (par des images subliminales) peut faciliter les performances cognitives et rendre le cœur plus efficace ("économise son énergie et devient plus efficace").

Ce processus doit être inconscient pour fonctionner.

Performance sociale et politique: La colère peut être utilisée consciemment pour "faire passer un message", comme le fait l'entraîneur Bernard Challandes.

La "colère sociale" est perçue comme un "moteur" essentiel pour "changer les choses" et maintenir l'engagement dans les mouvements collectifs (comme la grève du climat).

Les colères collectives ont historiquement fait peur car elles sont "synonyme d'émeutes" et de "révolution", mobilisant les individus en une force collective qui peut ébranler l'ordre établi.

Le traitement de la colère:

L'approche pour gérer la colère dépend de sa nature.

La psychiatrie peut être pertinente si la difficulté à contrôler la colère est constante, survient dans différents contextes et est associée à des comportements délétères, suggérant un trouble psychiatrique sous-jacent (comme une dépression).

Cependant, pour des difficultés comportementales, des approches se concentrant sur l'apprentissage de la gestion des émotions et le renforcement de l'estime de soi sont également efficaces, notamment pour les femmes violentes qui recherchent souvent de l'aide comportementale plutôt que psychiatrique.

Idées ou Faits Importants et Citations Clés

La colère, une émotion nécessaire:

"pourtant les spécialistes l'affirment nous ne pourrions pas vivre sans colère elle nous protège et vous le verrez dans cette émission elle augmente considérablement nos performances physiques et cognitives".

La montée rapide de la colère chez certains:

"[chez Eduardo] la tension monte très vite un très rapidement mais aussi très haut et qu'à ce moment là sur une émotion qui semble être de la colère il est plus capable de penser".

La colère dans les relations proches:

"les psychologues le disent les émotions déborde davantage avec ceux qu'on aime".

L'influence du système d'attachement:

Un système d'attachement insécure "va très vite interpréter les signaux que me donne l'autre comme il est en train de me laisser tomber... ça va alimenter active est encore plus mes émotions de tristesse de colère ou tout autre émotion négative".

La peur face à la colère d'autrui:

"les émotions de colère elles elles font peur parce que menace potentiellement l'intégrité d'autrui... face à la colère d'autrui surtout des personnes inconnues on est plutôt dans un état de pétrification de sidération".

Les conséquences du refoulement:

"je pense que la colère c'est comme un cancer c'est que si on la garde à l'intérieur qu'on laval caen laval caen laval... réellement je pense que oui ça rend malade sa c'est sûr ça j'en suis sûre et certaine".

La fonction de communication de la colère:

"c'est un outil de communication déjà depuis la petite la prime enfance c'est un outil qui me sera utile aussi plus tard à l'âge adulte pour me positionner dans la vie mettre des limites... Si je me mets jamais en colère je me fais bouffer par les autres".

La colère comme moteur de performance physique:

"la colère c'est une sorte de moteur un booster qui va permettre d'optimiser les performances mais certaines performances performance physique".

La colère et la performance cognitive inconsciente:

"cette activation d'une idée de la colère de penser à la colère peut influencer comment je m'applique pendant une tâche... la colère peut faciliter nos actions". "tout ça doit être inconscient".

La colère collective comme moteur du changement social: "il faut une colère sociale pour changer les choses Sinon c'est quoi le moteur". "les colères collective elles ont toujours fait extrêmement peur parce que ça a été synonyme d' émeutes synonyme de l'annoncé de révolution".

Conclusion

L'émission "Dans la tête d'un colérique" démystifie la colère en la présentant non pas uniquement comme une émotion destructive, mais aussi comme une force intrinsèque et potentiellement utile.

Si sa manifestation violente ou constante peut avoir des conséquences dévastatrices pour l'individu et ses proches, la colère, lorsqu'elle est comprise et gérée de manière appropriée, peut servir d'outil de communication, de motivation et même de catalyseur pour le changement social.

Il est crucial d'apprendre à canaliser cette énergie plutôt que de la refouler ou de la laisser déborder de manière incontrôlée.

heuristics for personal responsibiity

that's why I resonate with Hermetic principles

Rejet, victimisation par les pairs et émotions négatives : Synthèse des dynamiques d'influence en milieu scolaire

Synthèse opérationnelle

Ce document présente une analyse approfondie des recherches récentes menées par l'Institut universitaire Jeunes en difficulté concernant les liens entre l'isolement social, la victimisation par les pairs et les émotions négatives chez les élèves du primaire.

Les points saillants de cette étude sont les suivants :

• Prévalence élevée : Un nombre significatif de jeunes, particulièrement les filles, éprouvent une détresse émotionnelle quotidienne et un sentiment de non-acceptation dès le début du secondaire, des tendances amorcées au primaire.

• Renversement de la perspective traditionnelle :

Contrairement à l'idée reçue voulant que les problèmes relationnels causent les émotions négatives, les résultats indiquent que les émotions négatives (tristesse, désespoir) précèdent et prédisent souvent la victimisation.

• Boucle de rétroaction pour l'isolement : Il existe une relation bidirectionnelle entre l'isolement et les émotions négatives, créant un cycle d'aggravation mutuelle.

• Stabilité des traits vs États changeants : L'étude distingue les caractéristiques chroniques des élèves des fluctuations momentanées, révélant que si les relations sociales peuvent se réinitialiser partiellement entre deux années scolaires, les émotions négatives ont tendance à persister, voire à s'intensifier lors des transitions.

• Nécessité d'interventions multidimensionnelles : La simple prévention de l'intimidation est jugée insuffisante.

Les interventions doivent impérativement intégrer la promotion du bien-être et la gestion des émotions pour rompre les cycles de victimisation.

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1. État des lieux : Un portrait préoccupant chez les jeunes

Les données statistiques issues d'enquêtes canadiennes et québécoises révèlent une réalité complexe pour les élèves :

| Indicateur | Garçons | Filles | | --- | --- | --- | | Tristesse ou désespoir quotidien (début secondaire) | 19 % | 36 % | | Sentiment de ne pas être accepté tel que l'on est | 36 % | 52 % | | Victimes d'intimidation (12 derniers mois - Québec) | ~11 % | ~11 % |

Note sur la victimisation : Bien que le chiffre de 11 % soit cité, la proportion peut grimper jusqu'à 20 %, voire 40 % pour des événements isolés, soulignant la difficulté de cerner précisément ce phénomène.

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2. Définition des concepts fondamentaux

L'étude s'articule autour de trois réalités distinctes mais interconnectées :

• Émotions négatives : Comprennent la tristesse, le sentiment de désespoir et les idées négatives.

Elles sont considérées comme des précurseurs de la dépression, bien qu'elles ne correspondent pas nécessairement à un diagnostic clinique à ce stade (primaire).

• Isolement des pairs : Fait d'avoir peu d'interactions sociales, que ce soit par choix ou par rejet subi. Le rejet est la forme d'isolement non volontaire la plus fréquente.

• Victimisation : Actes d'agressivité intentionnels et répétitifs caractérisés par un déséquilibre des forces (physiques ou de réputation).

Elle peut être directe (frapper, insulter) ou indirecte (nuire à la réputation, propager des rumeurs).

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3. Modèles théoriques de la relation pairs-émotions

Trois modèles alternatifs tentent d'expliquer l'interaction entre ces variables :

1. Modèle des risques interpersonnels : Les expériences difficiles avec les pairs agissent comme des stresseurs qui s'accumulent et génèrent des émotions négatives.

C'est le modèle le plus testé et documenté à ce jour.

2. Modèle axé sur les symptômes : Les émotions négatives (ou l'affectivité négative) entraînent un retrait social ou une vulnérabilité qui fait de l'élève une cible privilégiée pour la victimisation.

3. Modèle transactionnel : Suppose une influence réciproque et un renforcement mutuel entre les émotions et les expériences sociales.

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4. Méthodologie de la recherche

L'étude a suivi 992 élèves de la 3e à la 6e année du primaire (Québec) sur deux années scolaires, avec quatre points de mesure.

L'originalité de l'approche réside dans l'utilisation de modèles statistiques ("modèles à décalage croisé avec intercept aléatoire") permettant de distinguer :

• Le Trait (stable/chronique) : La tendance d'un élève à être d'une certaine façon sur le long terme.

• L'État (changeant) : Les fluctuations d'un élève autour de sa propre tendance stable à un moment précis.

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5. Analyse des résultats : Des dynamiques différenciées

Interrelations stables (Traits)

De manière chronique, les trois dimensions sont liées : un élève ayant une tendance stable à l'isolement aura également une tendance stable à la victimisation et aux émotions négatives.

Ces réalités co-occurrent sans ordre temporel défini.

Dynamiques temporelles (États changeants)

L'analyse des fluctuations d'un moment à l'autre révèle des mécanismes distincts :

• Émotions négatives et Isolement : Suivent un modèle transactionnel.

Un niveau élevé d'émotions négatives en début d'année prédit un isolement accru en fin d'année, et inversement. C'est une boucle d'accentuation.

• Émotions négatives et Victimisation : Suivent un modèle axé sur les symptômes.

Les émotions négatives en début d'année prédisent une victimisation accrue plus tard, mais la victimisation ne semble pas augmenter les émotions négatives de manière immédiate.

Ce lien est direct et ne passe pas par l'intermédiaire de l'isolement.

• Stabilité temporelle :

◦ La victimisation et l'isolement sont plus stables au sein d'une même année qu'entre deux années.

Le changement de classe ou d'enseignant atténue l'effet de réputation.    ◦

Les émotions négatives sont plus stables entre les années scolaires, suggérant une anticipation anxieuse de la rentrée ou une persistance des traits internes malgré les changements d'environnement.

Constat important : Ces mécanismes sont identiques pour les garçons et les filles, ainsi que pour les élèves plus jeunes ou plus vieux au sein du primaire.

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6. Conclusions et orientations pour l'action

Pour la recherche

Les résultats de cette étude québécoise, bien que novateurs, ne font pas encore consensus au niveau international, d'autres études montrant parfois des résultats inverses ou sexués.

Une réplication du modèle est prévue en Belgique (Flandre) pour valider ces observations.

Pour l'intervention en milieu scolaire

L'étude remet en question les stratégies d'intervention uniquement centrées sur le comportement social :

• Insuffisance de la lutte contre l'intimidation seule : Retirer un élève d'une situation de victimisation ne garantit pas la disparition de ses émotions négatives.

• Approche multifactorielle : Il est impératif d'agir simultanément sur l'environnement social et sur le bien-être psychologique interne.

• Priorité à la promotion du bien-être : La prévention de la dépression et la gestion des émotions négatives dès le primaire sont des leviers essentiels pour réduire, par ricochet, les risques de victimisation et d'isolement.

"Les efforts de prévenir la victimisation sont essentiels, mais nos résultats suggèrent qu'ils ne sont potentiellement pas suffisants parce qu'il y a une dynamique plus large."

Briefing : L’autorégulation chez les enfants victimes d’agression sexuelle

Résumé exécutif

Ce document synthétise les résultats de recherches doctorales portant sur l’autorégulation des enfants ayant survécu à une agression sexuelle (AS).

L’autorégulation, définie comme la capacité à moduler ses réponses cognitives et émotionnelles pour générer des comportements adaptatifs, est un processus clé souvent altéré par le trauma.

Les conclusions principales soulignent que si l’agression sexuelle est globalement associée à des difficultés de fonctionnement exécutif (inhibition et flexibilité cognitive), l'impact n'est pas uniforme.

La recherche identifie quatre profils distincts d'autorégulation chez les victimes : disrégulé, inhibé, flexible et régulation identifiée par les parents.

L'étude démontre également que des facteurs tels que le sexe de l'enfant, l'historique de maltraitance multiple et l'environnement socio-économique (défavorisation du quartier) influencent de manière significative les capacités d'autorégulation.

Les implications cliniques suggèrent d'abandonner les approches universelles au profit d'interventions différenciées et d'évaluations multi-méthodes (tâches cognitives et questionnaires) impliquant plusieurs répondants (parents et enseignants).

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1. Cadre théorique et définitions

L'agression sexuelle est une problématique de santé publique mondiale touchant environ une fille sur cinq et un garçon sur dix avant l'âge de 18 ans.

Elle entraîne des conséquences psychologiques variées, notamment des problèmes de comportement intériorisés (dépression, retrait) et extériorisés (agression, opposition).

L'autorégulation

Le concept d'autorégulation repose sur deux composantes interdépendantes :

• La régulation émotionnelle : Stratégies et compétences modulant l'expression et l'expérience des émotions.

• Les fonctions exécutives : Processus mentaux orientés vers un but, incluant :

◦ L'inhibition : Capacité à freiner une réponse automatique face à un stimulus (ex: répondre "nuit" quand on montre un soleil).    ◦ La flexibilité cognitive : Capacité à s'adapter au changement de règles dans l'environnement.

Le mécanisme biologique du trauma

L'exposition précoce à un stress intense (maltraitance, pauvreté) provoque une dysrégulation des hormones de stress, entraînant des atteintes structurelles et fonctionnelles au cerveau, ce qui fragilise les capacités d'autorégulation.

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2. Impact de l'agression sexuelle sur les fonctions exécutives

Les recherches présentées indiquent que l'agression sexuelle est un prédicteur significatif de difficultés exécutives, même après avoir contrôlé d'autres facteurs comme le TDAH ou la défavorisation sociale.

Constats par type de fonction

• Flexibilité cognitive : L'agression sexuelle est directement associée à une moins bonne performance dans les tâches mesurant cette capacité.

• Inhibition : Les enfants victimes montrent une performance significativement inférieure aux enfants non victimes.

Effet modérateur du sexe

L'étude révèle des différences marquées selon le sexe de l'enfant :

• Garçons : Les enseignants rapportent beaucoup plus de difficultés de fonctionnement exécutif chez les garçons victimes que chez les non-victimes. Ils affichent également des performances plus faibles aux tâches d'inhibition.

• Filles : Il y a peu de différence significative entre les filles victimes et non victimes sur le plan de l'évaluation des fonctions exécutives par les enseignants ou dans les tâches d'inhibition.

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3. Typologie des profils d'autorégulation

L'analyse a permis de dégager quatre profils types chez les enfants victimes d'agression sexuelle (échantillon de 225 enfants) :

| Profil | Proportion | Caractéristiques principales | Problèmes de comportement associés | | --- | --- | --- | --- | | Disrégulé | 39 % | Faible performance cognitive, forte labilité émotionnelle, difficultés rapportées par les parents. | Problèmes intériorisés et extériorisés élevés (comorbidité). | | Inhibé | 19 % | Excellente performance aux tâches d'inhibition, mais faibles compétences émotionnelles perçues par les parents. | Niveaux les plus élevés de problèmes intériorisés. | | Flexible | ~28 % | Autorégulation supérieure à la moyenne, profil concordant (maison/école), résilience. | Faible symptomatologie. | | Régulation (Parents) | 14 % | Performance cognitive faible, mais parents rapportant de très bonnes capacités (profil discordant). | Symptômes visibles par les enseignants mais sous-estimés par les parents. |

Analyse des profils spécifiques

• Le profil "Inhibé" : Ces enfants semblent utiliser une sur-régulation cognitive pour contrôler leurs impulsions, mais au prix d'une grande détresse interne.

Chez les filles, ce profil est un facteur de risque pour les problèmes intériorisés, tandis que chez les garçons, il semble agir comme un facteur de protection apparent contre les problèmes extériorisés.

• Le profil "Discordant" : Souvent associé à des agressions sexuelles intrafamiliales (80-90 % des cas dans ce groupe). Les parents peuvent surévaluer les compétences de l'enfant par désir de normalité ou sous l'effet d'un cadre familial trop rigide.

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4. Facteurs de risque et de protection contextuels

L'autorégulation ne dépend pas uniquement de l'acte traumatique, mais d'un écosystème de facteurs :

• Historique de maltraitance : Les profils "disrégulé" et "inhibé" sont corrélés à une exposition à un plus grand nombre de formes de maltraitance.

• Défavorisation du quartier : Les enfants vivant dans des quartiers favorisés présentent une meilleure autorégulation. Cela s'expliquerait par l'accès aux ressources (bibliothèques, musées, espaces verts) et une moindre exposition à la violence communautaire.

• Éducation parentale : Un niveau d'études plus élevé chez les parents favorise le développement des compétences langagières, lesquelles soutiennent directement l'autorégulation de l'enfant.

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5. Recommandations pour l'intervention clinique

Évaluation multidimensionnelle

Il est impératif de multiplier les sources d'information :

1. Multi-modalité : Combiner les questionnaires (perceptions) et les tâches cognitives (mesures objectives), car les résultats sont souvent divergents.

2. Multi-répondants : Inclure systématiquement le point de vue des enseignants pour identifier les difficultés qui pourraient être masquées dans le cadre familial.

Approche différenciée

L'intervention ne doit pas être identique pour tous les profils :

• Pour les enfants disregulés : Approche standard axée sur le renforcement des fonctions exécutives et de la régulation émotionnelle.

• Pour les enfants inhibés : Éviter de renforcer l'inhibition (potentiellement néfaste). Prioriser la reconnaissance, la compréhension et l'expression des émotions, ainsi que la flexibilité cognitive.

• Pour les enfants "flexibles" : L'intervention sur l'autorégulation peut être inutile. Se concentrer sur le soutien psychosocial et la prévention de la revictimisation.

• Pour le profil discordant : Évaluer la flexibilité des parents et utiliser des sources d'évaluation externes pour pallier la sous-estimation parentale des difficultés.

Pistes d'activités pratiques

• Pour l'inhibition : Jeux de type "1, 2, 3 Soleil", coloriage attentionnel (arrêter au signal), ou jeux de rôle où l'enfant doit attendre son tour face à une frustration.

• Pour la flexibilité : Jeux avec changement de règles fréquent (ex: varier qui gagne à "Roche-Papier-Ciseau"), résolution de problèmes avec des solutions multiples ou inversions de rôles.

• Implication des parents : Travailler sur l'autorégulation propre des parents et favoriser un attachement sécurisant, facteur de protection majeur pour l'enfant.

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Conclusion

La recherche souligne la complexité des trajectoires de développement après une agression sexuelle.

Le constat majeur est que le trauma n'entraîne pas systématiquement une dysrégulation.

Près de 42 % des enfants présentent des profils adaptés.

L'enjeu clinique réside dans l'identification des profils "surrégulés" ou "discordants", qui peuvent passer inaperçus tout en présentant des risques élevés de pathologie à long terme.

Author response:

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

Public Reviews:

Reviewer #1 (Public review): 

Summary:

In this study, Lamberti et al. investigate how translation initiation and elongation are coordinated at the single-mRNA level in mammalian cells. The authors aim to uncover whether and how cells dynamically adjust initiation rates in response to elongation dynamics, with the overarching goal of understanding how translational homeostasis is maintained. To this end, the study combines single-molecule live-cell imaging using the SunTag system with a kinetic modeling framework grounded in the Totally Asymmetric Simple Exclusion Process (TASEP). By applying this approach to custom reporter constructs with different coding sequences, and under perturbations of the initiation/elongation factor eIF5A, the authors infer initiation and elongation rates from individual mRNAs and examine how these rates covary.

The central finding is that initiation and elongation rates are strongly correlated across a range of coding sequences, resulting in consistently low ribosome density ({less than or equal to}12% of the coding sequence occupied). This coupling is preserved under partial pharmacological inhibition of eIF5A, which slows elongation but is matched by a proportional decrease in initiation, thereby maintaining ribosome density. However, a complete genetic knockout of eIF5A disrupts this coordination, leading to reduced ribosome density, potentially due to changes in ribosome stalling resolution or degradation.

Strengths:

A key strength of this work is its methodological innovation. The authors develop and validate a TASEP-based Hidden Markov Model (HMM) to infer translation kinetics at single-mRNA resolution. This approach provides a substantial advance over previous population-level or averaged models and enables dynamic reconstruction of ribosome behavior from experimental traces. The model is carefully benchmarked against simulated data and appropriately applied. The experimental design is also strong. The authors construct matched SunTag reporters differing only in codon composition in a defined region of the coding sequence, allowing them to isolate the effects of elongation-related features while controlling for other regulatory elements. The use of both pharmacological and genetic perturbations of eIF5A adds robustness and depth to the biological conclusions. The results are compelling: across all constructs and conditions, ribosome density remains low, and initiation and elongation appear tightly coordinated, suggesting an intrinsic feedback mechanism in translational regulation. These findings challenge the classical view of translation initiation as the sole rate-limiting step and provide new insights into how cells may dynamically maintain translation efficiency and avoid ribosome collisions.

We thank the reviewer for their constructive assessment of our work, and for recognizing the methodological innovation and experimental rigor of our study.

Weaknesses:

A limitation of the study is its reliance on exogenous reporter mRNAs in HeLa cells, which may not fully capture the complexity of endogenous translation regulation. While the authors acknowledge this, it remains unclear how generalizable the observed coupling is to native mRNAs or in different cellular contexts.

We agree that the use of exogenous reporters is a limitation inherent to the SunTag system, for which there is currently no simple alternative for single-mRNA translation imaging. However, we believe our findings are likely generalizable for several reasons.

As discussed in our introduction and discussion, there is growing mechanistic evidence in the literature for coupling between elongation (ribosome collisions) and initiation via pathways such as the GIGYF2-4EHP axis (Amaya et al. 2018, Hickey et al. 2020, Juszkiewicz et al. 2020), which might operate on both exogenous and endogenous mRNAs.

As already acknowledged in our limitations section, our exogenous reporters may not fully recapitulate certain aspects of endogenous translation (e.g., ER-coupled collagen processing), yet the observed initiation-elongation coupling was robust across all tested constructs and conditions.

We have now expanded the Discussion (L393-395) to cite complementary evidence from Dufourt et al. (2021), who used a CRISPR-based approach in Drosophila embryos to measure translation of endogenous genes. We also added a reference to Choi et al. 2025, who uses a ER-specific SunTag reporter to visualize translation at the ER (L395-397).

Additionally, the model assumes homogeneous elongation rates and does not explicitly account for ribosome pausing or collisions, which could affect inference accuracy, particularly in constructs designed to induce stalling. While the model is validated under low-density assumptions, more work may be needed to understand how deviations from these assumptions affect parameter estimates in real data.

We agree with the reviewer that the assumption of homogeneous elongation rates is a simplification, and that our work represents a first step towards rigorous single-trace analysis of translation dynamics. We have explicitly tested the robustness of our model to violations of the low-density assumption through simulations (Figure 2 - figure supplement 2). These show that while parameter inference remains accurate at low ribosome densities, accuracy slightly deteriorates at higher densities, as expected. In fact, our experimental data do provide evidence for heterogeneous elongation: the waiting times between termination events deviate significantly from an exponential distribution (Figure 3 - figure supplement 2C), indicating the presence of ribosome stalling and/or bursting, consistent with the reviewer's concern. We acknowledge in the Limitations section (L402-406) that extending the model to explicitly capture transcript-dependent elongation rates and ribosome interactions remains challenging. The TASEP is difficult to solve analytically under these conditions, but we note that simulation-based inference approaches, such as particle filters to replace HMMs, could provide a path forward for future work to capture this complexity at the single-trace level.

Furthermore, although the study observes translation "bursting" behavior, this is not explicitly modeled. Given the growing recognition of translational bursting as a regulatory feature, incorporating or quantifying this behavior more rigorously could strengthen the work's impact.

While we do not explicitly model the bursting dynamics in the HMM framework, we have quantified bursting behavior directly from the data. Specifically, we measure the duration of translated (ON) and untranslated (OFF) periods across all reporters and conditions (Figure 1G for control conditions and Figure 4G-H for perturbed conditions), finding that active translation typically lasts 10-15 minutes interspersed with shorter silent periods of 5-10 minutes. This empirical characterization demonstrates that bursting is a consistent feature of translation across our experimental conditions. The average duration of silent periods is similar to what was inferred by Livingston et al. 2023 for a similar SunTag reporter; while the average duration of active periods is substantially shorter (~15 min instead of ~40 min), which is consistent with the shorter trace duration in our system compared to theirs (~15 min compared to ~80 min, on average). Incorporating an explicit two-state or multi-state bursting model into the TASEP-HMM framework would indeed be computationally intensive and represents an important direction for future work, as it would enable inference of switching rates alongside initiation and elongation parameters. We have added this point to the Discussion (L415-417).

Assessment of Goals and Conclusions:

The authors successfully achieve their stated aims: they quantify translation initiation and elongation at the single-mRNA level and show that these processes are dynamically coupled to maintain low ribosome density. The modeling framework is well suited to this task, and the conclusions are supported by multiple lines of evidence, including inferred kinetic parameters, independent ribosome counts, and consistent behavior under perturbation.

Impact and Utility:

This work makes a significant conceptual and technical contribution to the field of translation biology. The modeling framework developed here opens the door to more detailed and quantitative studies of ribosome dynamics on single mRNAs and could be adapted to other imaging systems or perturbations. The discovery of initiation-elongation coupling as a general feature of translation in mammalian cells will likely influence how researchers think about translational regulation under homeostatic and stress conditions.

The data, models, and tools developed in this study will be of broad utility to the community, particularly for researchers studying translation dynamics, ribosome behavior, or the effects of codon usage and mRNA structure on protein synthesis.

Context and Interpretation:

This study contributes to a growing body of evidence that translation is not merely controlled at initiation but involves feedback between elongation and initiation. It supports the emerging view that ribosome collisions, stalling, and quality control pathways play active roles in regulating initiation rates in cis. The findings are consistent with recent studies in yeast and metazoans showing translation initiation repression following stalling events. However, the mechanistic details of this feedback remain incompletely understood and merit further investigation, particularly in physiological or stress contexts. 

In summary, this is a thoughtfully executed and timely study that provides valuable insights into the dynamic regulation of translation and introduces a modeling framework with broad applicability. It will be of interest to a wide audience in molecular biology, systems biology, and quantitative imaging.

We appreciate the reviewer's thorough and positive assessment of our work, and that they recognize both the technical innovation of our modeling framework and its potential broad utility to the translation biology community. We agree that further mechanistic investigation of initiation-elongation feedback under various physiological contexts represents an important direction for future research.

Reviewer #2 (Public review):

Summary:

This manuscript uses single-molecule run-off experiments and TASEP/HMM models to estimate biophysical parameters, i.e., ribosomal initiation and elongation rates. Combining inferred initiation and elongation rates, the authors quantify ribosomal density. TASEP modeling was used to simulate the mechanistic dynamics of ribosomal translation, and the HMM is used to link ribosomal dynamics to microscope intensity measurements. The authors' main conclusions and findings are:

(1) Ribosomal elongation rates and initiation rates are strongly coordinated.

(2) Elongation rates were estimated between 1-4.5 aa/sec. Initiation rates were estimated between 0.5-2.5 events/min. These values agree with previously reported values.

(3) Ribosomal density was determined below 12% for all constructs and conditions.

(4) eIF5A-perturbations (KO and GC7 inhibition) resulted in non-significant changes in translational bursting and ribosome density.

(5) eIF5A perturbations resulted in increases in elongation and decreases in initiation rates.

Strengths:

This manuscript presents an interesting scientific hypothesis to study ribosome initiation and elongation concurrently. This topic is highly relevant for the field. The manuscript presents a novel quantitative methodology to estimate ribosomal initiation rates from Harringtonine run-off assays. This is relevant because run-off assays have been used to estimate, exclusively, elongation rates.

We thank the reviewer for their careful evaluation of our work and for recognizing the novelty of our quantitative methodology to extract both initiation and elongation rates from harringtonine run-off assays, extending beyond the traditional use of these experiments.

Weaknesses:

The conclusion of the strong coordination between initiation and elongation rates is interesting, but some results are unexpected, and further experimental validation is needed to ensure this coordination is valid. 

We agree that some of our findings need further experimental investigation in future studies. However, we believe that the coordination between initiation and elongation is supported by multiple results in our current work: (1) the strong correlation observed across all reporters and conditions (Figure 3E), and (2) the consistent maintenance of low ribosome density despite varying elongation rates. While additional experimental validation would be valuable, we note that directly manipulating initiation or elongation independently in mammalian cells remains technically challenging. Nevertheless, our findings are consistent with emerging mechanistic understanding of collision-sensing pathways (GIGYF2-4EHP) that could mediate such coupling, as discussed in our manuscript.

(1) eIF5a perturbations resulted in a non-significant effect on the fraction of translating mRNA, translation duration, and bursting periods. Given the central role of eIF5a, I would have expected a different outcome. I would recommend that the authors expand the discussion and review more literature to justify these findings.

We appreciate this comment. This finding is indeed discussed in detail in our manuscript (Discussion, paragraphs 6-7). As we note there, while eIF5A plays a critical role in elongation, the maintenance of bursting dynamics and ribosome density upon perturbation can be explained by compensatory feedback mechanisms. Specifically, the coordinated decrease in initiation rates that counterbalances slower elongation to maintain homeostatic ribosome density. We also discuss several factors that complicate interpretation: (1) potential RQC-mediated degradation masking stronger effects in proline-rich constructs, (2) differences between GC7 treatment and genetic knockout suggesting altered stalling resolution kinetics, and (3) the limitations of using exogenous reporters that lack ER-coupled processing, which may be critical for eIF5A function in endogenous collagen translation (as suggested by Rossi et al., 2014; Mandal et al., 2016; Barba-Aliaga et al., 2021). The mechanistic complexity and tissue-specific nature of eIF5A function in mammals, which differs substantially from the better-characterized yeast system, likely contributes to the nuanced phenotype we observe. We believe our Discussion adequately addresses these points.

(2) The AAG construct leading to slow elongation is very surprising. It is the opposite of the field consensus, where codon-optimized gene sequences are expected to elongate faster. More information about each construct should be provided. I would recommend more bioinformatic analysis on this, for example, calculating CAI for all constructs, or predicting the structures of the proteins.

We agree that the slow elongation of the AAG construct is counterintuitive and indeed surprising. Following the reviewer's suggestion, we have now calculated the Codon Adaptation Index (CAI) for all constructs (Renilla 0.89, Col1a1 0.78, Col1a1 mutated 0.74). It is therefore unlikely that codon bias explains the slow translation, particularly since we designed the mutated Col1a1 construct with alanine codons selected to respect human codon usage bias, thereby minimizing changes in codon optimality. As we discuss in the manuscript, we hypothesize that the proline-to-alanine substitutions disrupted co-translational folding of the collagen-derived sequence. Prolines are critical for collagen triple-helix formation (Shoulders and Raines, 2009), and their replacement with alanines likely generates misfolded intermediates that cause ribosome stalling (Barba-Aliaga et al., 2021; Komar et al., 2024). This interpretation is supported by the high frequency (>30%) of incomplete run-off traces for AAG, suggesting persistent stalling events. Our findings thus illustrate an important potential caveat: "optimizing" a sequence based solely on codon usage can be detrimental when it disrupts functionally important structural features or co-translational folding pathways.

This highlights that elongation rates depend not only on codon optimality but also on the interplay between nascent chain properties and ribosome progression.

(3) The authors should consider using their methodology to study the effects of modifying the 5'UTR, resulting in changes in initiation rate and bursting, such as previously shown in reference Livingston et al., 2023. This may be outside of the scope of this project, but the authors could add this as a future direction and discuss if this may corroborate their conclusions. 

We thank the reviewer for this excellent suggestion. We agree that applying our methodology to 5'-UTR variants would provide a complementary test of initiation-elongation coupling, and we have now added this as a future direction in the Discussion (L417-420).

(4) The mathematical model and parameter inference routines are central to the conclusions of this manuscript. In order to support reproducibility, the computational code should be made available and well-documented, with a requirements file indicating the dependencies and their versions. 

We have added the Github link in the manuscript (https://github.com/naef-lab/suntag-analysis) and have also deposited the data (.ome.tif) on Zenodo (https://zenodo.org/records/17669332).

Reviewer #3 (Public review):

Disclaimer:

My expertise is in live single-molecule imaging of RNA and transcription, as well as associated data analysis and modeling. While this aligns well with the technical aspects of the manuscript, my background in translation is more limited, and I am not best positioned to assess the novelty of the biological conclusions.

Summary:

This study combines live-cell imaging of nascent proteins on single mRNAs with time-series analysis to investigate the kinetics of mRNA translation.

The authors (i) used a calibration method for estimating absolute ribosome counts, and (ii) developed a new Bayesian approach to infer ribosome counts over time from run-off experiments, enabling estimation of elongation rates and ribosome density across conditions.

They report (i) translational bursting at the single-mRNA level, (ii) low ribosome density (~10% occupancy

{plus minus} a few percents), (iii) that ribosome density is minimally affected by perturbations of elongation (using a drug and/or different coding sequences in the reporter), suggesting a homeostatic mechanism potentially involving a feedback of elongation onto initiation, although (iv) this coupling breaks down upon knockout of elongation factor eIF5A.

Strengths:

(1) The manuscript is well written, and the conclusions are, in general, appropriately cautious (besides the few improvements I suggest below).

(2) The time-series inference method is interesting and promising for broader applications. 

(3) Simulations provide convincing support for the modeling (though some improvements are possible). 

(4) The reported homeostatic effect on ribosome density is surprising and carefully validated with multiple perturbations.

(5) Imaging quality and corrections (e.g., flat-fielding, laser power measurements) are robust.

(6) Mathematical modeling is clearly described and precise; a few clarifications could improve it further.

We thank the reviewer for recognizing the novelty of the approach and its rigour, and for providing suggestions to improve it further.

Weaknesses:

(1) The absolute quantification of ribosome numbers (via the measurement of $i_{MP}$ ) should be improved.This only affects the finding that ribosome density is low, not that it appears to be under homeostatic control. However, if $i_{MP}$ turns out to be substantially overestimated (hence ribosome density underestimated), then "ribosomes queuing up to the initiation site and physically blocking initiation" could become a relevant hypothesis. In my detailed recommendations to the authors, I list points that need clarification in their quantifications and suggest an independent validation experiment (measuring the intensity of an object with a known number of GFP molecules, e.g., MS2-GFP MS2-GFP-labeled RNAs, or individual GEMs).

We agree with the reviewer that the estimation of the number of ribosomes is central to our finding that translation happens at low density on our reporters. This result derives from our measurement of the intensity of one mature protein (i_MP), that we have achieved by using a SunTag reporter with a RH1 domain in the C terminus of the mature protein, allowing us to stabilise mature proteins via actin-tethering. In addition, as suggested by the reviewer, we already validated this result with an independent estimate of the mature protein intensity (Figure 5 - figure supplement 2B), which was obtained by adding the mature protein intensity directly as a free parameter of the HMM. The inferred value of mature protein intensity for each construct (10-15 a.u) was remarkably close to the experimental calibration result (14 ± 2 a.u.). Therefore, we have confidence that our absolute quantification of ribosome numbers is accurate.

(2) The proposed initiation-elongation coupling is plausible, but alternative explanations, such as changes in abortive elongation frequency, should be considered more carefully. The authors mention this possibility, but should test or rule it out quantitatively. 

We thank the reviewer for the comment, but we consider that ruling out alternative explanations through new perturbation experiments is beyond the scope of the present work.

(3) The observation of translational bursting is presented as novel, but similar findings were reported by Livingston et al. (2023) using a similar SunTag-MS2 system. This prior work should be acknowledged, and the added value of the current approach clarified.

We did cite Livingston et al. (2023) in several places, but we recognized that we could add a few citations in key places, to make clear that the observation of bursting is not novel but is in agreement with previous results. We now did so in the Results and Discussion sections.

(4) It is unclear what the single-mRNA nature of the inference method is bringing since it is only used here to report _average_ ribosome elongation rate and density (averaged across mRNAs and across time during the run-off experiments - although the method, in principle, has the power to resolve these two aspects).

While decoding individual traces, our model infers shared (population-level) rates. Inferring transcript-specific parameters would be more informative, but it is highly challenging due to the uncertainty on the initial ribosome distribution on single transcripts. Pooling multiple transcripts together allows us to use some assumptions on the initial distribution and infer average elongation and initiation-rate parameters, while revealing substantial mRNA-to-mRNA variability in the posterior decoding (e.g. Figure 3 - figure Supplement 2C). Indeed, the inference still informs on the single-trace run-off time distribution (Figure 3 A) and the waiting time between termination events (Figure 3 - figure supplement 2C), suggesting the presence of stalling and bursting. In addition, the transcript-to-transcript heterogeneity is likely accounted for by our model better than previous methods (linear fit of the average run-off intensity), as suggested by their comparison (Figure 3 - figure supplement 2 A). In the future the model could be refined by introducing transcript-specific parameters, possibly in a hierarchical way, alongside shared parameters.

(5) I did not find any statement about data availability. The data should be made available. Their absence limits the ability to fully assess and reproduce the findings.

We have added the Github link in the manuscript (https://github.com/naef-lab/suntag-analysis) and have also deposited the data (.ome.tif) on Zenodo (https://zenodo.org/records/17669332).

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors): 

Major Comments:

(1) Lack of Explicit Bursting Model

Although translation "bursts" are observed, the current framework does not explicitly model initiation as a stochastic ON/OFF process. This limits insight into regulatory mechanisms controlling burst frequency or duration. The authors should either incorporate a two-state/more-state (bursting) model of initiation or perform statistical analysis (e.g., dwell-time distributions) to quantify bursting dynamics. They should clarify how bursting influences the interpretation of initiation rate estimates.

We agree with the reviewer that an explicit bursting model (e.g., a two-state telegraph model) would be the ideal theoretical framework. However, integrating such a model into the TASEP-HMM inference framework is computationally intensive and complex. As a robust first step, we have opted to quantify bursting empirically based on the decoded single-mRNA traces. As shown in Figure 1G (control) and Figure 4G (perturbed conditions), we explicitly measured the duration of "ON" (translated) and "OFF" (untranslated) periods. This statistical analysis provides a quantitative description of the bursting dynamics without relying on the specific assumptions of a telegraph model. We have clarified this in the text (L123-125) and, as suggested, added a discussion (L415-417) on the potential extensions of the model to include explicit switching kinetics in the Outlook section.

(2) Assumption of Uniform Elongation Rates

The model assumes homogeneous elongation across coding sequences, which may not hold for stalling-prone inserts (e.g., PPG). This simplification could bias inference, particularly in cases of sequence-specific pausing. Adding simulations or sensitivity analysis to assess how non-uniform elongation affects the accuracy of inferred parameters. The authors should explicitly discuss how ribosome stalling, collisions, or heterogeneity might skew model outputs (see point 4).

A strong stalling sequence that affects all ribosomes equally should not deteriorate the inference of the initiation rate, provided that the low-density assumption holds. The scenario where stalling events lead to higher density, and thus increased ribosome-ribosome interactions, is comparable to the conditions explored in Figure 2E. In those simulations, we tested the inference on data generated with varying initiation and elongation rates, resulting in ribosome densities ranging from low to high. We demonstrated that the inference remains robust at low ribosome densities (<10%). At higher densities, the accuracy of the initiation rate estimate decreases, whereas the elongation rate estimate remains comparatively robust. Additionally, the model tends to overestimate ribosome density under high-density conditions, likely because it neglects ribosome interference at the initiation site (Figure 2 figure supplement 2C). We agree that a deeper investigation into the consequences of stochastic stalling and bursting would be beneficial, and we have explicitly acknowledged this in the Limitations section.

(3) Interpretation of eIF5A Knockout Phenotype

The observation that eIF5A KO reduces initiation more than elongation, leading to decreased ribosome density, is biologically intriguing. However, the explanation invoking altered RQC kinetics is speculative and not directly tested. The authors should consider validating the RQC hypothesis by monitoring reporter mRNA stability, ribosome collision markers, or translation termination intermediates.

We thank the reviewer for the comment, but we consider that ruling out alternative explanations through new experiments is beyond the scope of the present work.

(4) To strengthen the manuscript, the authors should incorporate insights from three studies.

- Livingston et al. (PMC10330622) found that translation occurs in bursts, influenced by mRNA features and initiation factors, supporting the coupling of initiation and elongation.

- Madern et al. (PMID: 39892379) demonstrated that ribosome cooperativity enhances translational efficiency, highlighting coordinated ribosome behavior.

- Dufourt et al. (PMID: 33927056) observed that high initiation rates correlate with high elongation rates, suggesting a conserved mechanism across cell cultures and organisms.

Integrating these studies could enrich the manuscript's interpretation and stimulate new avenues of thought.

We thank the reviewer for the valuable comment. We added citations of Livingston et al. in the context of translational bursting. We already cited Madern et al. in multiple places and, although its observations of ribosome cooperativity are very compelling, they cannot be linked with our observations of a feedback between initiation and elongation, and it would be very challenging to see a similar effect on our reporters. This is why we did not expressly discuss cooperativity. We also integrated Dufourt et al. in the Discussion about the possibility of designing genetically-encoded reporter. We also added a sentence about the possibility of using an ER-specific SunTag reporter, as done recently in Choi et al., Nature (2025) (https://doi.org/10.1038/s41586-025-09718-0).

Minor Comments:

(1) Use consistent naming for SunTag reporters (e.g., "PPG" vs "proline-rich") throughout.

Thank you for the comment. However, the term proline-rich always appears together with PPG, so we believe that the naming is clear and consistent.

(2) Consider a schematic overview of the experimental design and modeling pipeline for accessibility.

Thank you for the suggestion. We consider that experimental design and modeling is now sufficiently clearly described and does not justify an additional scheme. 

(3) Clarify how incomplete run-off traces are handled in the HMM inference.

Incomplete run-off traces are treated identically to complete traces in our HMM inference. This is possible because our model relies on the probability of transitions occurring per time step to infer rates. It does not require observing the final "empty" state to estimate the kinetic parameters ɑ and λ. The loss of signal (e.g., mRNA moving out of the focal volume or photobleaching) does not invalidate the kinetic information contained in the portion of the trace that was observed. We have clarified this in the Methods section.

Reviewer #2 (Recommendations for the authors):

(1) Reproducibility:

(1.1) The authors should use a GitHub repository with a timestamp for the release version.

The code is available on GitHub (https://github.com/naef-lab/suntag-analysis).

(1.2) Make raw images and data available in a figure repository like Figshare.

The raw images (.ome.tif) are now available on Zenodo (https://zenodo.org/records/17669332).

(2) Paper reorganization and expansion of the intensity and ribosome quantification:

(2.1) Given the relevance of the initiation and elongation rates for the conclusions of this study, and the fact that the authors inferred these rates from the spot intensities. I recommend that the authors move Figure 1 Supplement 2 to the main text and expand the description of the process to relate spot intensity and number of ribosomes. Please also expand the figure caption for this image.

We agree with the importance of this validation. We have expanded the description of the calibration experiment in the main text and in the figure caption.

(2.2) I suggest the authors explicitly mention the use of HMM in the abstract.

We have now explicitly mentioned the TASEP-based HMM in the abstract.

(2.3) In line 492, please add the frame rate used to acquire the images for the run-off assays.

We have added the specific frame rate (one frame every 20 seconds) to the relevant section.

(3) Figures and captions:

(3.1) Figure 1, Supplement 2. Please add a description of the colors used in plots B, C. 

We have expanded the caption and added the color description.

(3.2) In the Figure 2 caption. It is not clear what the authors mean by "traceseLife". Please ensure it is not a typo.

Thank you for spotting this. We have corrected the typo.

(3.3) Figure 1 A, in the cartoon N(alpha)->N-1, shouldn't the transition also depend on lambda?

The transition probability was explicitly derived in the “Bayesian modeling of run-off traces” section (Eqs. 17-18), and does not depend on λ, but only on the initiation rate under the low-density assumption.

(3.4) Figure 3, Supplement 2. "presence of bursting and stalling.." has a typo.

Corrected.

(3.5) Figure 5, panel C, the y-axis label should be "run-off time (min)."

Corrected.

(3.6) For most figures, add significance bars.

(3.7) In the figure captions, please add the total number of cells used for each condition.

We have systematically indicated the number of traces (n_t) and the number of independent experiments (n_e) in the captions in this format (n_t, n_e).

(4) Mathematical Methods:

We greatly thank the reviewer for their detailed attention to the mathematical notation. We have addressed all points below.

(4.1) In lines 555, Materials and Methods, subsection, Quantification of Intensity Traces, multiple equations are not numbered. For example, after Equation (4), no numbers are provided for the rest of the equations. Please keep consistency throughout the whole document.

We have ensured that all equations are now consistently numbered throughout the document.

(4.2) In line 588, the authors mention "$X$ is a standard normal random variable with mean $\mu$ and standard deviation $s_0$". Please ensure this is correct. A standard normal random variable has a 0 mean and std 1. 

Thank you for the suggestion, we have corrected the text (L678).

(4.3) Line 546, Equation 2. The authors use mu(x,y) to describe a 2d Gaussian function. But later in line 587, the authors reuse the same variable name in equation 5 to redefine the intensity as mu = b_0 + I.

We have renamed the 2D Gaussian function to \mu_{2D}(x,y) in the spot tracking section

(4.4) For the complete document, it could be beneficial to the reader if the authors expand the definition of the relationship between the signal "y" and the spot intensity "I". Please note how the paragraph in lines 582-587 does not properly introduce "y".

We have added an explicit definition of y and its relationship to the underlying spot intensity I in the text to improve readability and clarity.

(4.5) Please ensure consistency in variable names. For example, "I" is used in line 587 for the experimental spot intensity, then line 763 redefines I(t) as the total intensity obtained from the TASEP model; please use "I_sim(t)" for simulated intensities. Please note that reusing the variable "I" for different contexts makes it hard for the reader to follow the text. 

We agree that this was confusing. We have implemented the suggestion and now distinguish simulated intensities using the notation I_S .

(4.6) Line 555 "The prior on the total intensity I is an "uninformative" prior" I ~ half_normal(1000). Please ensure it is not "I_0 ~ half_normal(1000)."? 

We confirm that “I” is the correct variable representing the total intensity in this context; we do not use an “I₀” variable here.

(4.7) In lines 595, equation 6. Ensure that the equation is correct. Shouldn't it be: s_0^2 = ln ( 1 + (sigma_meas^2 / ⟨y⟩^2) )? Please ensure that this is correct and it is not affecting the calculated values given in lines 598.

Thank you for catching this typo. We have corrected the equation in the manuscript. We confirm that the calculations performed in the code used the correct formula, so the reported values remain unchanged.

(4.8) In line 597, "the mean intensity square ^2". Please ensure it is not "the square of the temporal mean intensity."

We have corrected the text to "the square of the temporal mean intensity."

(4.9) In lines 602-619, Bayesian modeling of run-off traces, please ensure to introduce the constant "\ell". Used to define the ribosomal footprint?

We have added the explicit definition of 𝓁 as the ribosome footprint size (length of transcript occupied by one ribosome) in the "Bayesian modeling of run-off traces" section.

(4.10) Line 687 has a minor typo "[...] ribosome distribution.. Then, [...]"

We have corrected the punctuation.

(4.11) In line 678, Equation 19 introduces the constant "L_S", Please ensure that it is defined in the text.

We have added the explicit definition of L_S (the length of the SunTag) to the text surrounding Equation 19.

(4.12) In line 695, Equation 22, please consider using a subscript to differentiate the variance due to ribosome configuration. For example, instead of "sigma (...)^2" use something like "sigma_c ^2 (...)". Ensure that this change is correctly applied to Equation 24 and all other affected equations.

Thank you, we have implemented the suggestions.

(4.13) In line 696, please double-check equations 26 and 27. Specifically, the denominator ^2. Given the previous text, it is hard to follow the meaning of this variable. 

We have revised the notation in Equations 26 and 27 to ensure the denominator is consistent with the definitions provided in the text.

(4.14) In lines 726, the authors mention "[...], but for the purposes of this dissertation [...]", it should be "[...], but for the purposes of this study [...]"

Thank you for spotting this. We have replaced "dissertation" with "study."

(4.15) Equations 5, 28, 37, and the unnumbered equation between Equations 16 and 17 are similar, but in some, "y" does not explicitly depend on time. Please ensure this is correct. 

We have verified these equations and believe they are correct.

(4.16) Please review the complete document and ensure that variables and constants used in the equations are defined in the text. Please ensure that the same variable names are not reused for different concepts. To improve readability and flow in the text, please review the complete Materials and Methods sections and evaluate if the modeling section can be written more clearly and concisely. For example, Equation 28 is repeated in the text.

We have performed a comprehensive review of the Materials and Methods section. To improve conciseness and flow, we have merged the subsection “Observation model and estimation of observation parameters” with the “Bayesian modeling of run-off traces” section. This allowed us to remove redundant definitions and repeated equations (such as the previous Equation 28). We have also checked that all variables and constants are defined upon first use and that variable names remain consistent throughout the manuscript.

Reviewer #3 (Recommendations for the authors):

(1) Data Presentation

(1.1) In main Figures 1D and 4E, the traces appear to show frequent on-off-on transitions ("bursting"), but in supplementary figures (1-S1A and 4-S1A), this behavior is seen in only ~8 of 54 traces. Are the main figure examples truly representative?

We acknowledge the reviewer's point. In Figure 1D, we selected some of the longest and most illustrative traces to highlight the bursting dynamics. We agree that the term "representative" might be misleading if interpreted as "average." We have updated the text to state "we show bursting traces" to more accurately reflect the selection.

(1.2) There are 8 videos, but I could not identify which is which.

Thank you for pointing this out. We have renamed the video files to clearly correspond to the figures and conditions they represent.

(2) Data Availability:

As noted above, the data should be shared. This is in accordance with eLife's policy: "Authors must make all original data used to support the claims of the paper, or that are required to reproduce them, available in the manuscript text, tables, figures or supplementary materials, or at a trusted digital repository (the latter is recommended). [...] eLife considers works to be published when they are posted as preprints, and expects preprints we review to meet the standards outlined here." Access to the time traces would have been helpful for reviewers.

We have now added the Github link for the code (https://github.com/naef-lab/suntag-analysis) and deposited the raw data (.ome.tif files) on Zenodo (10.5281/zenodo.17669332).

(3) Model Assumptions:

(3.1) The broad range of run-off times (Figure 3A) suggests stalling, which may be incompatible with the 'low-density' assumption used on the TASEP model, which essentially assumes that ribosomes do not bump into each other. This could impact the validity of the assumptions that ribosomes behave independently, elongate at constant speed (necessary for the continuum-limit approximation), and that the rate-limiting step is the initiation. How robust are the inferences to this assumption?

We agree that the deviation of waiting times from an exponential distribution (Figure 3 - figure supplement 2C) suggests the presence of stalling, which challenges the strict low-density assumption and constant elongation speed. We explicitly explored the robustness of our model to higher ribosome densities in simulations. As shown in Figure 2 - figure supplement 2, while the model accuracy for single parameters deteriorates at very high densities (overestimating density due to neglected interference), it remains robust for estimating global rates in the regime relevant to our data. We have expanded the discussion on the limitations of the low density and homogeneous elongation rate assumptions in the text (L404-408).

(3.2) Since all constructs share the same SunTag region, elongation rates should be identical there and diverge only in the variable region. This would affect $\gamma (t)$ and hence possibly affect the results. A brief discussion would be helpful.

This is a valid point. Currently, our model infers a single average elongation rate that effectively averages the behavior over the SunTag and the variable CDS regions. Modeling distinct rates for these regions would be a valuable extension but adds significant complexity. While our current "effective rate" approach might underestimate the magnitude of differences between reporters, it captures the global kinetic trend. We have added a brief discussion acknowledging this simplification (L408-412).

(3.3) A similar point applies to the Gillespie simulations: modeling the SunTag region with a shared elongation rate would be more accurate.

We agree. Simulating distinct rates for the SunTag and CDS would increase realism, though our current homogeneous simulations serve primarily to benchmark the inference framework itself. We have noted this as a potential future improvement (L413-414).

(3.4) Equation (13) assumes that switching between bursting and non-bursting states is much slower than the elongation time. First, this should be made explicit. Second, this is not quite true (~5 min elongation time on Figure 3-s2A vs ~5-15min switching times on Figure 1). It would be useful to show the intensity distribution at t=0 and compare it to the expected mixture distribution (i.e., a Poisson distribution + some extra 'N=0' cells). 

We thank the reviewer for this insightful comment. We have added a sentence to the text explicitly stating the assumption that switching dynamics are slower than the translation time. While the timescales are indeed closer than ideal (5 min vs. 5-15 min), this assumption allows for a tractable approximation of the initial conditions for the run-off inference. Comparing the intensity distribution at t=0 to a zero-inflated Poisson distribution is an excellent suggestion for validation, which we will consider for future iterations of the model.

(4) Microscopy Quantifications:

(4.1) Figure 1-S2A shows variable scFv-GFP expression across cells. Were cells selected for uniform expression in the analysis? Or is the SunTag assumed saturated? which would then need to be demonstrated.