Start of the decline

Partly, it is american legislative tradition to support pro-farmer bills

Almost universal

Is the public motivation driven by tradition?

True only within the sample, not in this case

Gonna be real depressed when we learn that its to stay in power

Lobbyists

All three

With what money remains a question

Seems most plausible to me

To avoid taxation themselves

How would lobbying help if its a declining sector

The political support of the labor base perhaps

La Relation École-Famille : Vers une Coéducation Concertée

Ce document de synthèse analyse les enjeux, les évolutions et les perspectives de la relation entre l'école et les parents, tels que discutés par des experts lors de l'émission « Au Périscope » de l'IH2EF.

Résumé Exécutif

La relation école-famille est aujourd'hui considérée comme un levier essentiel de la réussite de l'enfant et de la cohésion sociale.

Historiquement marquée par un cloisonnement issu de l'ère Jules Ferry, cette relation a évolué vers un modèle de partenariat institutionnalisé.

Cependant, le concept central de « coéducation », bien qu'inscrit dans la loi de 2013, demeure flou et manque de stabilisation sémantique et opérationnelle.

L'analyse met en évidence que l'école ne peut plus être conçue comme un espace clos, mais comme le cœur d'un écosystème incluant les familles, les collectivités territoriales et divers partenaires sociaux.

Le défi majeur réside dans le passage d'une approche normative — où l'on attend du parent qu'il se conforme aux attentes de l'institution — à une relation de réciprocité et de reconnaissance mutuelle.

Les experts soulignent la nécessité de dépasser le mythe du « parent démissionnaire », les recherches montrant un investissement réel, bien que parfois invisible ou maladroit, des familles les plus modestes.

La réussite de cette transition repose sur une formation accrue des professionnels, une meilleure lisibilité des compétences de chaque acteur et une adaptation aux réalités territoriales.

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1. La Coéducation : Un Concept en Quête de Définition

Bien que le terme soit entré dans le cadre réglementaire avec la loi de 2013 pour la refondation de l'école de la République, la « coéducation » reste un horizon de sens plutôt qu'un concept opérationnel précis.

• Le « halo sémantique » : Une enquête mentionnée par Pierre Perrier révèle que les enseignants et les parents associent des centaines de mots différents à ce terme, témoignant d'un flou persistant.

• Manque d'indicateurs : Il n'existe pas, au niveau national, de politique générale déclinée en objectifs opérationnels ou en indicateurs de progrès (par exemple dans l'état de l'école de la DEPP).

• Définition proposée : La coéducation peut être comprise comme une action réciproque et concertée entre les acteurs (école, famille, partenaires) dans l'intérêt exclusif de l'enfant.

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2. Évolution Historique et Institutionnelle

La relation a transitionné d'un cloisonnement strict vers une ouverture progressive.

• L'héritage de Jules Ferry : À l'origine, l'école visait à fédérer la nation et à moraliser les citoyens, créant une séparation entre la sphère privée (famille) et la sphère publique (état). Toutefois, dès 1883, Ferry recommandait déjà le respect des convictions des pères de famille.

• L'institutionnalisation des parents : Depuis la loi Haby de 1975, la place des parents est gravée dans les textes, leur conférant des droits (participation aux instances, information) et des devoirs en tant que membres de la communauté éducative.

• Persistance des représentations : Malgré les évolutions législatives, un champ sémantique de la réserve et de la prudence persiste, notamment lors des moments de décision (orientation, redoublement).

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3. L'École au Cœur d'un Écosystème Global

La journée d'un élève ne se limite pas au temps scolaire. La réussite dépend de la synergie entre plusieurs acteurs.

Les trois piliers de l'intervention territoriale

Selon Thierry Vasse, les collectivités territoriales assurent la cohérence de l'accueil de l'enfant à travers :

1. La continuité éducative : Créer des liens fluides entre les temps périscolaires (accueil du matin, soir) et le temps de la classe.

2. La complémentarité éducative : Les interventions des animateurs et des ATSEM (langage, règles de vie) complètent l'action pédagogique des enseignants.

3. La cohérence éducative : Partager des concepts de bienveillance et de respect au sein d'un projet éducatif de territoire (PEDT).

La diversité des acteurs

Le document identifie de nombreux professionnels gravitant autour de l'enfant :

• Animateurs périscolaires et personnels de restauration.

• ATSEM (Agents territoriaux spécialisés des écoles maternelles).

• Concierges d'école (rôle de médiateurs au portail).

• Médiateurs sociaux, chargés de mission handicap et acteurs de la politique de la ville (dans les quartiers prioritaires).

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4. Obstacles et Malentendus Sociologiques

L'analyse pointe des décalages importants entre les attentes de l'institution et la réalité des familles.

• Le mythe du parent démissionnaire : Pierre Perrier et Frédéric Wexler réfutent fermement cette idée. Les études (notamment pendant le confinement) montrent que les parents des milieux populaires consacrent souvent plus de temps au suivi scolaire que les autres, en raison de la moindre autonomie de leurs enfants.

• Le « métier » de parent d'élève : L'institution attend souvent un « parent idéal » qui maîtrise les codes scolaires. Or, ces attentes normatives peuvent exclure les parents dont la culture est éloignée de celle de l'école.

• Rapport de pouvoir : La relation est souvent perçue comme descendante (l'école explique au parent ce qu'il doit faire). Un véritable changement de paradigme impliquerait de concevoir les projets avec les parents dès le départ.

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5. Cadre Juridique : Droits et Obligations

Le droit de la parentalité dans le cadre scolaire repose sur l'autorité parentale exercée en commun, indépendamment de la situation matrimoniale.

| Type d'acte | Définition | Exemples | | --- | --- | --- | | Actes usuels | Présomption d'accord entre les parents. L'accord d'un seul suffit. | Justification d'absences brèves, réinscription, demande de dérogation. | | Actes non usuels | Actes rompant avec le passé et engageant l'avenir. Accord conjoint nécessaire. | Changement d'orientation, inscription dans le privé. |

Obligations des parents :

• Veiller à l'instruction obligatoire (de 3 à 16 ans) et justifier les absences.

• Respecter l'institution et ses personnels (loi sur l'école de la confiance).

• Prendre connaissance et signer le règlement intérieur.

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6. Leviers pour une Relation Renforcée

Pour transformer la relation école-famille, plusieurs pistes d'action sont identifiées par les intervenants :

• La formation professionnelle : Les enseignants sont souvent formés à la didactique, mais peu à la relation avec les familles.

Il est nécessaire d'apprendre à « lâcher une part de pouvoir » pour favoriser la réciprocité.

• La reconnaissance et l'autorisation :

◦ Reconnaissance mutuelle : Identifier les parents comme des interlocuteurs de valeur dès le début de l'année.   

◦ Autorisation : Donner une voix aux parents, les considérer comme des « auteurs » de la relation et non de simples exécutants.

• L'accessibilité et la convivialité :

◦ Ouvrir physiquement l'école (semaines de la maternelle, cafés des parents).  

◦ Créer des espaces dédiés aux parents au sein des établissements pour favoriser la parole entre pairs.

• La lisibilité institutionnelle : Les familles peinent parfois à distinguer les compétences de l'État (pédagogie) de celles des communes (matériel, périscolaire).

Une parole unifiée est nécessaire, particulièrement en période de crise.

• Adaptation territoriale : La coéducation doit se décliner localement (cités éducatives, quartiers prioritaires) pour tenir compte de la mixité sociale ou de la ségrégation.

Its amazing how this way of thinking is applied to our everyday lives!

I agree with this, however I think that sometimes it would be helpful when trying to see previous knowledge

I agree with this, and thats why its important to teach in a way that fits everyones learning style.

Briefing : L'Éducation à la Vie Affective, Relationnelle et à la Sexualité (EVARS) en Milieu Scolaire

Résumé Exécutif

Ce document synthétise les enjeux, les contenus et les modalités de mise en œuvre du nouveau programme d'éducation à la vie affective et relationnelle (1er degré) et à la sexualité (2d degré) au sein de l'Éducation nationale.

Face au constat d'une application inégale de la loi de 2001 (trois séances annuelles obligatoires) et aux défis sociétaux contemporains — accès facilité à la pornographie, cyberviolences, prise de conscience des violences sexuelles intrafamiliales —, le ministère a élaboré un cadre pédagogique clarifié.

Le programme s'articule autour de trois axes fondamentaux : la connaissance de soi et de son corps, la construction de relations respectueuses, et l'insertion dans la société en tant que citoyen responsable.

Il repose sur une approche interdisciplinaire et pluricatégoriale, visant à passer d'une logique de « cours » à un espace de réflexion et de transfert de connaissances scientifiques validées.

L'objectif est de sécuriser les pratiques des personnels tout en garantissant un accès équitable des élèves à cette éducation, essentielle à la prévention des violences et à la promotion de l'égalité.

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1. Contexte Historique et Justification de la Réforme

Un long processus législatif et réglementaire

L'éducation sexuelle en milieu scolaire est une préoccupation ministérielle depuis plus de 50 ans, marquée par des étapes clés :

• 1967 & 1975 : Lois sur la contraception et la dépénalisation de l'avortement.

• 1973 : Circulaire Fontana instaurant une politique d'information sexuelle.

• 2001 : Loi sur l'IVG imposant trois séances annuelles d'éducation à la sexualité par tranche d'âge.

• Juin 2023 : Saisine du Conseil Supérieur des Programmes (CSP) pour élaborer un programme structuré.

• Janvier 2025 : Vote favorable à l'unanimité (60 voix pour, 0 contre) du Conseil Supérieur de l'Éducation sur le projet de programme.

Les nouveaux défis sociétaux

Le besoin de clarification des objectifs de formation est accentué par plusieurs facteurs :

• Révolution numérique : Accès massif et précoce des jeunes à l'information et à la désinformation, ainsi qu'à la pornographie via les réseaux sociaux.

• Sécurité et violences : Constat qu'en France, un enfant ou un jeune est victime d'agression sexuelle toutes les trois minutes. Les mouvements comme "Me Too" ont également sensibilisé la société aux violences dans les sphères professionnelles et intrafamiliales.

• Inégalités territoriales : Disparités importantes dans la mise en œuvre effective des séances selon les établissements.

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2. Architecture et Philosophie du Programme

Le programme est conçu pour être adapté à la maturité des élèves, avec une distinction sémantique entre les degrés :

• 1er degré : Éducation à la vie affective et relationnelle (VAR).

• 2d degré : Éducation à la vie affective et relationnelle et à la sexualité (EVARS).

Les trois axes structurants (de la maternelle au lycée)

| Axe | Thématique Centrale | Objectif Pédagogique | | --- | --- | --- | | Axe 1 | Se connaître, vivre et grandir avec son corps | Relation à soi-même, compréhension des évolutions physiques et émotionnelles. | | Axe 2 | Rencontrer les autres, construire des relations | Épanouissement relationnel, respect mutuel, amitié, amour et consentement. | | Axe 3 | Trouver sa place dans la société | Liberté, responsabilité, droits, citoyenneté et égalité genres. |

Principes directeurs

• Équilibre santé et citoyenneté : Le programme vise le développement de l'esprit critique pour permettre des choix favorables à sa santé et à celle d'autrui.

• Approche scientifique et objective : Les contenus s'appuient sur des données validées et non sur des jugements de valeur ou des opinions personnelles d'adultes.

• Respect de l'intime : L'école ne traite pas des pratiques sexuelles privées, mais fournit des repères définitionnels et comportementaux.

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3. Cadre Juridique et Protection des Mineurs

La minute du juriste précise les fondements légaux entourant la sexualité des mineurs en France :

• Majorité sexuelle (15 ans) : Seuil à partir duquel un mineur peut consentir à des relations avec un majeur, hors position d'autorité de ce dernier.

• Loi du 21 avril 2021 :

◦ Crée un seuil de non-consentement pour les moins de 15 ans face à un majeur (le consentement est juridiquement inopérant).     ◦ Introduit la clause "Roméo et Juliette" (pas de pénalisation si l'écart d'âge est inférieur à 5 ans, hors inceste ou contrainte).   

◦ Renforce la lutte contre la "sextorsion" et l'incitation de mineurs à des pratiques sexuelles en ligne.

• Définition du consentement : Il doit être volontaire, libre, éclairé, spécifique, réversible, exprimé et perçu.

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4. Modalités de Mise en Œuvre et Pilotage

La réussite du programme repose sur un engagement collectif et une organisation anticipée.

Rôles des acteurs

• Chefs d'établissement et directeurs d'école : Pilotes de la mise en œuvre, ils constituent des équipes inter-catégorielles, assurent la communication avec les parents et garantissent la protection des personnels.

• Équipes pédagogiques : Travail interdisciplinaire (SVT, Lettres, Philosophie, EPS, EMC, etc.).

• Personnels sociaux et de santé : Rôle central d'expertise et de co-animation.

• Partenaires extérieurs : Les interventions associatives (prioritairement au second degré) doivent être agréées et préparées conjointement avec l'école.

Leviers opérationnels

• Temps dédiés : Utilisation des heures de vie de classe, de l'enseignement moral et civique (EMC) ou intégration transversale dans les disciplines.

• Instances de coordination : Conseil d'école, conseil pédagogique, CESCE (Comité d'éducation à la santé, à la citoyenneté et à l'environnement) et instances de liaison école-collège.

• Label ÉduSanté : Promotion du développement des compétences psychosociales.

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5. Accompagnement, Formation et Communication

Le ministère déploie un dispositif de soutien complet pour lever les freins (peurs des familles, manque de légitimité ressenti par les enseignants).

Dispositif de formation

• Plan National de Formation (PNF) : Formations pour les pilotes et formateurs académiques dès mars 2025.

• Parcours Magistère : Cinq modules d'auto-formation pour tous les personnels.

• Ressources pédagogiques : Publication de livrets par niveau proposant trois séances types et des pistes d'activités disciplinaires (disponibles sur Éduscol).

Stratégies de communication

• Transparence avec les familles : Utilisation de plaquettes d'information, de foires aux questions (FAQ) et de capsules vidéo pour expliciter les contenus et rassurer sur l'adaptation aux âges.

• Gestion des contestations : Dialogue en première intention, avec possibilité de s'appuyer sur les cellules "Valeurs de la République" des rectorats. Le document souligne que cet enseignement est obligatoire et soumis à l'obligation d'assiduité.

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6. Synthèse des Perspectives

L'introduction de ce programme est perçue comme une « opportunité institutionnelle » pour l'école républicaine. Au-delà de la prévention, les enjeux sont multiples :

1. Culture commune : Offrir un espace de réflexion sur des notions complexes (intimité, consentement, respect).

2. Équité territoriale : Garantir que chaque élève reçoive la même éducation, quel que soit son lieu de scolarisation.

3. Intelligence collective : Encourager l'inventivité pédagogique des équipes pour accueillir la parole des élèves tout en respectant le cadre de la transmission des connaissances.

« Ce programme ne porte pas atteinte à la vie privée des élèves... il n'est pas là pour imposer un modèle de bonheur... il est là pour faire réfléchir les élèves et réfléchir avec eux. » — Franck Durbage, IGESR honoraire.

Santé et bien-être des élèves : Vers une École Promotrice de Santé

Ce document de synthèse analyse les interventions et les conclusions issues de l'émission « Opériscope » de l'IH2EF consacrée à la santé et au bien-être des élèves.

Il détaille les cadres institutionnels, les fondements scientifiques et les modalités de mise en œuvre sur le terrain.

Synthèse de la problématique

La santé et le bien-être ne sont plus considérés comme des préoccupations périphériques à l'école, mais comme des conditions essentielles de la réussite scolaire.

L'institution s'éloigne d'une vision purement médicale pour adopter une approche globale et systémique. La stratégie nationale s'appuie sur deux piliers : le Parcours Éducatif de Santé (PES) et la démarche École Promotrice de Santé (EPSA).

L'enjeu majeur est de passer d'actions ponctuelles à une culture d'établissement durable, intégrant le développement des compétences psychosociales (CPS), l'amélioration du climat scolaire et une coopération étroite avec les partenaires territoriaux.

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1. Cadres conceptuels et institutionnels

Une vision globale de la santé

Conformément à la définition de l'OMS, la santé à l'école est perçue comme un état de complet bien-être physique, mental et social.

• Lien avec la réussite : Les données (Talis, OCDE, Cnesco) confirment que le stress et le mal-être pèsent sur les apprentissages. Inversement, un environnement favorable réduit l'absentéisme et améliore la concentration.

• Engagement des personnels : Le bien-être des enseignants, lié à leur sentiment de reconnaissance, est indissociable de la qualité du climat scolaire.

Le Parcours Éducatif de Santé (PES)

Le PES structure l'accompagnement de l'élève de la maternelle au lycée autour de trois axes :

1. Éducation à la santé : Développer des connaissances et des capacités pour faire des choix éclairés.

2. Prévention : Agir sur les facteurs de risque (conduites à risque, écrans, alimentation).

3. Protection : Garantir un environnement sécurisant et orienter vers les soins si nécessaire.

La démarche École Promotrice de Santé (EPSA)

Lancée en 2020 en France (mais existant depuis 1995 à l'international), l'EPSA est une démarche systémique visant à :

• Coordonner les actions de promotion de la santé préexistantes.

• Améliorer l'environnement physique et social de la scolarité.

• Favoriser les comportements favorables à la santé dès le plus jeune âge.

• La labellisation : Elle agit comme un catalyseur et un levier de reconnaissance des projets, plutôt que comme une fin en soi.

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2. Les leviers de l'efficacité selon la recherche

Karine Simar souligne que 30 ans de recul scientifique permettent d'identifier les critères d'une démarche « de qualité ».

Les trois dimensions de l'efficacité (Référentiel Santé Publique France)

• Pratiques éducatives : Elles doivent être intégrées, positives, expérientielles et actives, combinant rituels et approches informelles.

• Environnement soutenant : Qualité des relations sociales et sécurité affective dans les espaces physiques.

• Démarche collective : Les actions doivent devenir un « objet commun » au sein de l'établissement, soutenu par une formation de qualité.

Le projet "Alliance" : Un modèle de recherche-action

Ce projet, couvrant 101 écoles et 10 000 élèves, a démontré l'importance de :

• Le diagnostic partagé : Identifier les problèmes spécifiques à chaque école, car « chaque école est unique ».

• Le protocole de signalement : Articuler le pédagogique et le médical (services de santé scolaire) selon la dégradation des indicateurs.

• La durabilité : Une étude sur 10 ans montre que la pérennité des projets dépend de l'implication collective dès le départ et de la planification des actions dans le temps.

« 50 % des déterminants de la mise en œuvre d'une démarche de qualité sont en lien avec la qualité du travail collectif. » — Karine Simar

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3. Mise en œuvre opérationnelle en établissement

L'expérience de terrain montre que le passage à l'action nécessite une méthodologie rigoureuse pilotée par le chef d'établissement.

Construction du diagnostic

Il s'appuie sur des données fiables et croisées :

• Bilans infirmiers et sociaux.

• Indicateurs de vie scolaire (absentéisme, violences verbales, accidents).

• Enquêtes locales de climat scolaire.

• Auto-évaluation de l'établissement impliquant le conseil pédagogique et le CESCE.

Transformation des espaces et des pratiques : Exemples concrets

| Domaine | Action exemplaire | Impact attendu | | --- | --- | --- | | Espaces de vie | Aménagement d'un hall interdit en galerie d'art et lieu de mentorat. | Responsabilisation, sentiment d'appartenance, autonomie. | | Pédagogie | "Classe dehors", médiation artistique, innovation pédagogique. | Engagement, réduction du stress, plaisir d'apprendre. | | Climat scolaire | Installation d'un piano en libre-service, rénovation des toilettes. | Sécurité affective, entraide entre pairs, bien-être quotidien. | | Citoyenneté | Formations GQS (Gestes qui sauvent), PSC1, dispositif Sentinelles (PHARE). | Solidarité, pouvoir d'agir, engagement républicain. |

Le rôle du chef d'établissement

Il est le garant de la cohérence globale. Son action se décline en trois axes :

1. Donner du sens : Inscrire la santé dans le projet d'établissement.

2. Fédérer : Mobiliser les instances (CVC, CVL, CESCE) et coordonner les acteurs.

3. Piloter et évaluer : Ajuster les actions en fonction des indicateurs de réussite et de participation.

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4. Stratégie et pilotage à l'échelle départementale

Christian Mindivé (DASEN) souligne l'urgence de traiter la dégradation de la santé psychique et physique des élèves (sédentarité, troubles du comportement dès la maternelle).

Le Pôle Santé Départemental

La création d'un pôle unique permet de dépasser le travail en « silos » :

• Réunir médecins, psychologues, conseillers techniques et inspecteurs.

• Apporter une réponse globale aux chefs d'établissement.

• Accompagner le diagnostic et valider les ressources de formation.

Observatoire de la santé mentale

Cet outil novateur vise à objectiver les besoins du terrain à travers :

• L'élaboration de questionnaires types.

• L'expérimentation dans des réseaux de collèges/lycées cibles.

• L'accompagnement opérationnel des protocoles nationaux.

Synergie avec l'activité physique

L'apprentissage est indissociable du mouvement.

• Objectif : Généraliser les 30 minutes d'activité physique quotidienne (APQ) et encourager les pédagogies actives.

• Partenariats : Coopération nécessaire entre l'UNSS, l'USEP et les collectivités territoriales pour l'accès aux équipements sportifs.

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5. Partenariats et formation : Les clés de la réussite

Une responsabilité partagée

L'école ne peut agir seule. La frontière de sa responsabilité s'arrête là où commence le soin, mais elle doit collaborer avec :

• L'ARS et la CPAM : Pour les enjeux de prévention et de santé publique.

• La CAF : Pour le soutien à la parentalité et la coéducation.

• Les collectivités : Pour l'aménagement des locaux et les temps périscolaires.

Enjeux de la formation

• Inter-catégorialité : Former ensemble enseignants, personnels de santé, agents et acteurs du périscolaire (ex: former les ATSEM avec les professeurs).

• Formation initiale et continue : La légitimité des acteurs doit se construire dès le début de la carrière à travers un curriculum dédié aux compétences psychosociales.

• Acculturation : Clarifier le rôle de chacun pour éviter que les enseignants ne se sentent investis d'une mission médicale qui n'est pas la leur.

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Conclusion : Les prérequis d'une démarche durable

Pour éviter l'écueil du « saupoudrage » ou des actions sans lendemain, quatre conseils majeurs ressortent :

1. Le diagnostic préalable : Ne pas agir sans avoir identifié les besoins spécifiques du terrain.

2. L'intégration au projet d'établissement : La santé doit être le socle, pas une option.

3. La valorisation et la communication : Communiquer en interne et en externe pour stabiliser la nouvelle identité de l'établissement.

4. L'écoute des acteurs : Placer le pouvoir d'agir des élèves et des équipes au centre du processus.

« Prendre soin du bien-être, c'est agir sur la réussite et l'égalité des chances. » — Sabine Carotti

we hate to see IQ mentioned.

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".

Le Climat Scolaire : Enjeux Pédagogiques, Sociaux et Institutionnels

Résumé Exécutif

Ce document synthétise l'intervention de M. Canvel (septembre 2017) concernant le climat scolaire au sein de l'institution éducative française. Les points fondamentaux sont les suivants :

• Mutation de la profession : L'enseignement doit être perçu non plus comme un simple métier, mais comme une mission complexe visant à faire de l'élève l'adulte de demain.

• Priorité à l'apprentissage : Le climat scolaire n'est pas une fin en soi, mais une condition et un résultat de l'apprentissage. L'objectif premier de l'enseignant doit être de « faire apprendre » plutôt que d'« enseigner ».

• Lutte contre le décrochage : Le sentiment d'injustice, le désintérêt pour les matières et la qualité de la relation enseignant-élève sont les principaux leviers du décrochage scolaire, qualifié de « cancer de l'école ».

• Déficits institutionnels : Selon les données de l'OCDE, les enseignants français souffrent d'un manque de formation pédagogique et d'une insuffisance de coopération interprofessionnelle.

• Approche systémique : Le climat scolaire repose sur cinq piliers : relationnel, éducatif, sécurité, justice et appartenance.

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1. La Mission de l'Enseignant et l'École comme Nation

L'école est définie comme le « creuset de la République ». Avec 12 millions d'élèves, 24 millions de parents et plus d'un million de personnels, elle représente l'incarnation même de la nation.

De la profession à la mission

L'enseignement est un métier d'une complexité extrême, comparable aux parcours d'ingénieurs ou de médecins, car il traite de l'humain de manière collective.

Un enseignant rencontrera entre 7 000 et 8 000 élèves au cours de sa carrière.

L'investissement total dans cette mission est une nécessité absolue, car sans enseignants, il n'y a pas de jeunesse structurée pour l'avenir.

« Enseigner » versus « Faire apprendre »

Une distinction cruciale est opérée entre l'enseignement d'une discipline et l'acte de faire apprendre cette discipline aux élèves.

• L'expert : Se concentre sur l'observation de l'activité de l'élève et adapte son geste professionnel.

• L'enjeu : Un cours jugé « bon » par l'enseignant peut se solder par une absence totale d'apprentissage chez les élèves. L'interlocuteur prioritaire doit toujours rester l'élève et son cheminement mental.

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2. Analyse du Décrochage et du Bien-être Scolaire

Malgré une statistique positive (9 élèves sur 10 se disent satisfaits de l'école), le système scolaire fait face à des zones de rupture critiques.

Les chiffres clés de la souffrance scolaire

| Phénomène | Impact statistique | | --- | --- | | Élèves se déclarant harcelés | 1 sur 10 | | Sorties sans diplôme ni qualification | 1 sur 5 (soit environ 150 000 jeunes par an) | | Taille d'une génération d'élèves | 750 000 enfants |

Les causes du décrochage (Étude Catherine Blaya)

L'analyse des raisons invoquées par les décrocheurs révèle une responsabilité directe de l'institution et de ses acteurs :

1. Désintérêt pour la matière (17 %) : Souvent lié à un défaut de lien entre la discipline et l'élève.

2. Relation au professeur (15,5 %) : Une rencontre négative peut être le déclencheur d'un processus irréversible.

3. Désamour de l'école (13,7 %) : Souvent lié à la peur (harcèlement) et au manque de sécurité.

4. Sentiment d'être mal aimé (7 %) : Blessures liées aux appréciations sur les bulletins scolaires.

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3. Le Climat Scolaire : Une Approche Théorique et Systémique

Le climat scolaire n'est pas une simple perception individuelle, mais un jugement collectif et subjectif porté par les élèves, les parents et les éducateurs sur leur expérience de vie à l'école.

La théorie de la complexité appliquée à la classe

S'appuyant sur les travaux d'Edgar Morin, le climat scolaire est analysé selon trois axes :

• L'imprévisibilité : L'humain est imprévisible ; l'erreur de l'enseignant fait partie du système.

• La récursivité : Les apprentissages améliorent le climat, et un bon climat favorise les apprentissages. C'est une boucle rétroactive permanente.

• La totalité : Un établissement n'est pas la simple somme des classes qui le composent. Un incident dans un couloir peut déstabiliser l'ensemble du système (effet papillon).

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4. Les Cinq Facteurs Constitutifs du Climat Scolaire

Le climat scolaire est un matériau composite façonné par l'homme.

| Facteur | Description et enjeux | | --- | --- | | Relationnel | Qualité de l'accueil, propreté des sanitaires (besoin primaire), et qualité de la restauration. | | Éducatif | Cohérence des valeurs partagées par l'ensemble des adultes (enseignants, direction, agents). | | Sécurité | Attention portée à l'autre par l'adulte de référence, présence dans les couloirs et la cour. | | Justice | Perception d'équité. 70 % des élèves jugent l'école injuste, souvent à cause de sanctions inexpliquées. | | Appartenance | Sentiment d'être contributeur d'un projet collectif et d'une communauté éducative. |

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5. Critiques et Leviers d'Amélioration Institutionnels

L'analyse souligne des lacunes majeures dans le système français, notamment via les rapports de l'OCDE (Eric Charbonnier).

Les points de faiblesse

• Formation pédagogique : Les enseignants français seraient parmi les moins bien formés à la pédagogie (comment mettre les élèves au travail) par rapport à la didactique.

• Coopération interprofessionnelle : Travailler ensemble est jugé « très insuffisant ». La collaboration entre pairs est pourtant un facteur clé de la réussite des élèves.

• Isolement : Le modèle français repose trop sur le diplôme initial, au détriment de la formation continue tout au long de la carrière.

Recommandations pour les personnels

• Pratiquer l'éthologie scolaire : Observer l'élève au travail plutôt que de se focaliser sur sa propre prestation.

• Investir le « Devoirs Faits » : Se mettre « côte à côte » avec l'élève pour comprendre son cheminement mental, une pratique trop souvent réservée aux classes préparatoires.

• Sortir de l'entre-soi : Éviter l'enfermement en salle des professeurs ; aller à la rencontre des CPE, des agents et vivre une journée dans la peau d'un élève pour comprendre la « totalité » de l'établissement.

• Recherche et formation : Adopter une posture d'enseignant-chercheur, en utilisant les outils comme les enquêtes locales de climat scolaire et les ressources ministérielles (Eduscol).

Conclusion

La violence scolaire la plus insidieuse est l'incapacité irréversible d'un enfant à apprendre.

Le rôle de l'enseignant est de garantir les conditions de cet apprentissage par la construction d'un climat de confiance.

Comme le souligne l'intervention, l'école ne doit pas faire de mal ; elle doit accompagner, sécuriser et inclure chaque élève dans une dynamique collective de réussite.

sudden confusion as a result of excess alcohol consumption

Father of medical Neurology; pioneered the 'rest cure'

eLife Assessment

This important study provides a comprehensive comparison of the mechanisms through which different inhibitors affect the SARS-CoV-2 main protease, a pivotal antiviral drug target, and suggests a potentially broad-spectrum strategy to inhibit this critical viral enzyme by disrupting its dimerization states. However, whereas the biophysical analyses of the dimer stability are convincing, evidence supporting this new mode of mechanism to inhibit the main protease is incomplete and would benefit from a correlation of the biophysical observations with functional activity. With the functional validation part strengthened, this work would be of interest to biochemists and virologists working on anti-coronavirus drug discovery.

Reviewer #1 (Public review):

Summary:

Since dimerization is essential for SARS-CoV-2 Mpro enzymatic activity, the authors investigated how different classes of inhibitors, including peptidomimetic inhibitors (PF-07321332, PF-00835231, GC376, boceprevir), non-peptidomimetic inhibitors (carmofur, ebselen, and its analog MR6-31-2), and allosteric inhibitors (AT7519 and pelitinib), influence the Mpro monomer-dimer equilibrium using native mass spectrometry. Further analyses with isotope labeling, HDX-MS, and MD simulations examined subunit exchange and conformational dynamics. Distinct inhibitory mechanisms were identified: peptidomimetic inhibitors stabilized dimerization and suppressed subunit exchange and structural flexibility, whereas ebselen covalently bound to a newly identified site at C300, disrupting dimerization and increasing conformational dynamics. This study provides detailed mechanistic evidence of how Mpro inhibitors modulate dimerization and structural dynamics. The newly identified covalently binding site C300 represents novelty as a druggable allosteric hotspot.

Strengths:

This manuscript investigates how different classes of inhibitors modulate SARS-CoV-2 main protease dimerization and structural dynamics, and identifies a newly observed covalent binding site for ebselen.

Weaknesses:

The major concern is the absence of mutagenesis data to support the proposed inhibitory mechanisms, particularly regarding the role of the inhibitor binding site.

Reviewer #2 (Public review):

Summary:

This is a mechanistic study that provides new insights into the inhibition of SARS-CoV-2 Mpro.

Strengths:

The identification of dimer interface stabilization/destabilization as distinct inhibitory mechanisms and the discovery of C300 as a potential allosteric site for ebselen are important contributions to the field. The experimental approach is modern, multi-faceted, and generally well-executed.

Weaknesses:

The primary weaknesses relate to linking the biophysical observations more directly to functional enzymatic outcomes and providing more quantitative rigor in some analyses. While the study is overall strong, addressing its weaknesses and limitations would elevate the impact and translational relevance of the current manuscript.

(1) Correlation with Functional Activity:

The most significant gap is the lack of direct enzymatic activity assays under the exact conditions used for MS and HDX. While EC50 values are listed from literature, demonstrating how the observed dimer stabilization (by peptidomimetics) or dimer disruption (by ebselen) directly correlates with inhibition of proteolytic activity in the same experimental setup would solidify the functional relevance of the biophysical observations. For instance, does the fraction of monomer measured by native MS quantitatively predict the loss of activity? Also, the single inhibitor concentration used in each MS experiment needs to be specified in the main text and legends. A discussion on whether the inhibitor concentrations required to observe these dimerization effects (in native MS) or structural dynamics (in HDX-MS) align with EC50 values would be helpful for contextualizing the findings.

(2) For the two Cys residues found to be targeted by ebselen, what are their respective modification stoichiometry related to the ebselen concentration? Especially for the covalent binding site C300, which is proposed in this study to represent a novel allosteric inhibition mechanism of ebselen, more direct experimental evidence is needed to support this major hypothesis. Does mutation or modification of C300 affect the Mpro dimerization/monomer equilibrium and alter the enzymatic activity? If ebselen acts as a covalent inhibitor linked to multiple Cys, why is its activity only in the uM range?

(3) For the allosteric inhibitor pelitinib with low-uM activity, no significant differences in deuterium uptake of Mpro were observed. In terms of the binding affinity, what is the difference between pelitinib and ebselen? Some explanations could be provided about the different HDX-MS results between the two non-peptidomimetic inhibitors with similar activities.

(4) Native MS Quantification:

The analysis of monomer-dimer ratios from native MS spectra appears qualitative or semi-quantitative. A more rigorous and quantified analysis of the percentage of dimer/monomer species under each condition, with statistical replicates, would strengthen the equilibrium shift claims. For native MS analysis of each inhibitor, the representative spectrum can be shown in the main figure together with quantified dimer/monomer fractions from replicates to show significance by statistical tests.

(5) Changes of HDX rates in certain regions seem very subtle. For example, as it states 'residues 296-304 in the C-terminal region of M pro were more flexible upon ebselen binding (Figure 4c)', the difference is barely observable. The percentage of HDX rate changes between two conditions (with p values) can be specified in the text for each fragment discussed, and any change below 5% or 10% is negligible.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

Since dimerization is essential for SARS-CoV-2 Mpro enzymatic activity, the authors investigated how different classes of inhibitors, including peptidomimetic inhibitors (PF-07321332, PF-00835231, GC376, boceprevir), non-peptidomimetic inhibitors (carmofur, ebselen, and its analog MR6-31-2), and allosteric inhibitors (AT7519 and pelitinib), influence the Mpro monomer-dimer equilibrium using native mass spectrometry. Further analyses with isotope labeling, HDX-MS, and MD simulations examined subunit exchange and conformational dynamics. Distinct inhibitory mechanisms were identified: peptidomimetic inhibitors stabilized dimerization and suppressed subunit exchange and structural flexibility, whereas ebselen covalently bound to a newly identified site at C300, disrupting dimerization and increasing conformational dynamics. This study provides detailed mechanistic evidence of how Mpro inhibitors modulate dimerization and structural dynamics. The newly identified covalently binding site C300 represents novelty as a druggable allosteric hotspot.

Strengths:

This manuscript investigates how different classes of inhibitors modulate SARS-CoV-2 main protease dimerization and structural dynamics, and identifies a newly observed covalent binding site for ebselen.

Weaknesses:

The major concern is the absence of mutagenesis data to support the proposed inhibitory mechanisms, particularly regarding the role of the inhibitor binding site.

We thank the reviewer for the comments and recognition of our study. We agree that mutagenesis experiments are very helpful to validate the proposed mechanisms. We will perform site-directed mutagenesis of the key residue C300 and assess the effects of those C300 mutants on dimerization and enzymatic activity of Mpro, and integrate the results and discussion into the revised manuscript.

Reviewer #2 (Public review):

Summary:

This is a mechanistic study that provides new insights into the inhibition of SARS-CoV-2 Mpro.

Strengths:

The identification of dimer interface stabilization/destabilization as distinct inhibitory mechanisms and the discovery of C300 as a potential allosteric site for ebselen are important contributions to the field. The experimental approach is modern, multi-faceted, and generally well-executed.

We thank the reviewer for the positive comments and recognition of our study.

Weaknesses:

The primary weaknesses relate to linking the biophysical observations more directly to functional enzymatic outcomes and providing more quantitative rigor in some analyses. While the study is overall strong, addressing its weaknesses and limitations would elevate the impact and translational relevance of the current manuscript.

We thank the reviewer for the comments that are very helpful for improving the quality and impact of our manuscript.

(1) Correlation with Functional Activity:

The most significant gap is the lack of direct enzymatic activity assays under the exact conditions used for MS and HDX. While EC50 values are listed from literature, demonstrating how the observed dimer stabilization (by peptidomimetics) or dimer disruption (by ebselen) directly correlates with inhibition of proteolytic activity in the same experimental setup would solidify the functional relevance of the biophysical observations. For instance, does the fraction of monomer measured by native MS quantitatively predict the loss of activity? Also, the single inhibitor concentration used in each MS experiment needs to be specified in the main text and legends. A discussion on whether the inhibitor concentrations required to observe these dimerization effects (in native MS) or structural dynamics (in HDX-MS) align with EC50 values would be helpful for contextualizing the findings.

We thank the reviewer for the points and agree that directly linking our biophysical observations to functional outcomes under identical conditions would be more meaningful. We will perform enzymatic activity assays to investigate whether the fraction of monomer measured by native MS can predict the loss of activity. The inhibitor concentrations used in each MS experiment will be explicitly stated in the main text and figure legends, and we will also discuss how these concentrations relate to the EC50/IC50 values, providing content for the biophysical observations.

(2) For the two Cys residues found to be targeted by ebselen, what are their respective modification stoichiometry related to the ebselen concentration? Especially for the covalent binding site C300, which is proposed in this study to represent a novel allosteric inhibition mechanism of ebselen, more direct experimental evidence is needed to support this major hypothesis. Does mutation or modification of C300 affect the Mpro dimerization/monomer equilibrium and alter the enzymatic activity? If ebselen acts as a covalent inhibitor linked to multiple Cys, why is its activity only in the uM range?

We thank the reviewer for the insightful comments. To address the stoichiometry of ebselen modification, we will further analyze the data and discuss accordingly. To display more direct evidence of C300 as a novel allosteric inhibition site of ebselen, we will perform site-directed mutagenesis and investigate whether these C300 mutants affect the Mpro dimerization and enzymatic activity. Regarding the modification of C300, several independent studies have been cited in this manuscript and showed that oxidation (by glutathione, Davis et., 2021) or chemical modification of C300 (by glutathione bismuth drugs, Tao et al., 2021, and Tixocortol, Davis et., 2024) leads to Mpro inactivation and promotes monomer formation. We will cite and further discuss these studies in the Discussion. The µM-range activity of ebselen can be explained by its multi-target covalent binding to multiple cysteines. The variable efficacy of cysteine modification may account for ebselen's moderate potency, as not all modifications equally inhibit their targets.

(3) For the allosteric inhibitor pelitinib with low-uM activity, no significant differences in deuterium uptake of Mpro were observed. In terms of the binding affinity, what is the difference between pelitinib and ebselen? Some explanations could be provided about the different HDX-MS results between the two non-peptidomimetic inhibitors with similar activities.

Pelitinib has non-covalent binding with Mpro, while the binding between ebselen and Mpro is covalent. We will add some explanations and discussion about their different HDX-MS results in the revised version.

(4) Native MS Quantification:

The analysis of monomer-dimer ratios from native MS spectra appears qualitative or semi-quantitative. A more rigorous and quantified analysis of the percentage of dimer/monomer species under each condition, with statistical replicates, would strengthen the equilibrium shift claims. For native MS analysis of each inhibitor, the representative spectrum can be shown in the main figure together with quantified dimer/monomer fractions from replicates to show significance by statistical tests.

We thank the reviewer for the suggestion, and we will perform a more rigorous and quantitative analysis of the monomer-dimer equilibrium. For each condition (unbound Mpro and Mpro bound to each inhibitor), native MS experiments will be shown in triplicate. As suggested, we will include a representative native MS spectrum for each condition. The quantified monomer/dimer ratios from replicates will be added. The results with statistical analysis will be provided to show significance.

(5) Changes of HDX rates in certain regions seem very subtle. For example, as it states 'residues 296-304 in the C-terminal region of M pro were more flexible upon ebselen binding (Figure 4c)', the difference is barely observable. The percentage of HDX rate changes between two conditions (with p values) can be specified in the text for each fragment discussed, and any change below 5% or 10% is negligible.

We agree with the reviewer about the need for quantitative rigor in reporting HDX changes. We will calculate the fractional deuterium uptake difference for each peptide fragment discussed in the text between the inhibitor-bound and unbound states. These values, along with their statistical significance (p-values from a two-tailed t-test), will be provided in the revised figures.

eLife Assessment

This work presents a brain-wide atlas of vasopressin (Avp) and vasopressin receptor 1A (Avpr1a) mRNA expression in mouse brains using high-resolution RNAscope in situ hybridization. The single-transcript approach provides precise localization and identifies additional brain regions expressing Avpr1a, creating a valuable resource for the field. The revised manuscript is clearer and more impactful, with improved figures, stronger data organization, and enhanced scholarship through added context and citations. Overall, the evidence is compelling, and the atlas should be broadly of use to researchers studying vasopressin signaling and related neural circuits.

Reviewer #1 (Public review):

Summary:

Despite accumulating prior studies on the expressions of AVP and AVPR1a in the brain, a detailed, gender-specific mapping of AVP/AVPR1a neuronal nodes has been lacking. Using RNAscope, a cutting-edge technology that detects single RNA transcripts, the authors created a comprehensive neuroanatomical atlas of Avp and Avpr1a in male and female brains.

Strengths:

This well-executed study provides valuable new insights into gender differences in the distribution of Avp and Avpr1a. The atlas is an important resource for the neuroscience community.

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

Reviewer #2 (Public review):

Summary:

The authors conducted a brain-wide survey of Avp (arginine vasopressin) and its Avpr1a gene expression in the mouse brain using RNAscope, a high-resolution in situ hybridization method. Overall, the findings are useful and important because they identify brain regions that express the Avpr1a transcript. A comprehensive overview of Avpr1a expression in the mouse brain could be highly informative and impactful. The authors used RNAscope (a proprietary in situ hybridization method) to assess transcript abundance of Avp and one of its receptors, Avpr1a. The finding of Avp-expressing cells outside the hypothalamus and the extended amygdala is novel and is nicely demonstrated by new photomicrographs in the revised manuscript. The Avpr1a data suggest expression in numerous brain regions. In the revised manuscript, reworked figures make the data easier to interpret.

Strengths:

A survey of Avpr1a expression in the mouse brain is an important tool for exploring vasopressin function in the mammalian brain and for developing hypotheses about cell- and circuit-level function.

[Editors' note: The authors have substantially addressed all the reviewers' concerns and comments.]

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.

Epistemology seems important here. It means understanding how knowledge is created in a certain field.

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eLife Assessment

Recent studies have shown that mRNA can be acetylated (ac4c), altering mRNA stability and translation efficiency; however, the role of mRNA acetylation in the brain remains unexplored. In this important study, the authors demonstrate that ac4c occurs in synaptically localised mRNAs, mediated by NAT10. Conditional reduction of NAT10 protein levels led to decreases in ac4c of mRNAs and deficits in synaptic plasticity and memory. These solid results suggest that mRNA acetylation may play a role in memory consolidation.

Reviewer #1 (Public review):

Summary:

RNA modification has emerged as an important modulator of protein synthesis. Recent studies found that mRNA can be acetylated (ac4c), which can alter mRNA stability and translation efficiency. The role of ac4c mRNA in the brain has not been studied. In this paper, the authors convincingly show that ac4c occurs selectively on mRNAs localized at synapses, but not cell wide. The ac4c "writer" NAT10 is highly expressed in hippocampal excitatory neurons. Using NAT10 conditional KO mice, decreasing levels of NAT10 resulted in decreases in ac4c of mRNAs and also showed deficits in LTP and spatial memory. These results reveal a potential role for ac4c mRNA in memory consolidation.

This is a new type of mRNA regulation that seems to act specifically at synapses, which may help elucidate the mechanisms of local protein synthesis in memory consolidation. Overall, the studies are well carried out and presented. The precise mRNAs that require ac4c to carry out memory consolidation is not clear, but is an important focus of future work. The specificity of changes occurring only at the end of training, rather than after each day of training is interesting and also warrants further investigation. This timeframe is puzzling because the authors show that ac4c can dynamically increase within 1hr after cLTP.

Strengths:

(1) The studies show that mRNA acetylation (ac4c) occurs selectively at mRNAs localized to synaptic compartments (using synaptoneurosome preps).

(2) The authors identify a few key mRNAs acetylated involved in plasticity and memory - eg Arc.

(3) The authors show that Ac4c is induced by learning and neuronal activity (cLTP).

(4) The studies show that the ac4c "writer" NAT10 is expressed in hippocampal excitatory neurons and may relocated to synapses after cLTP/learning induction.

(5) The authors used floxed NAT10 mice injected with AAV-Cre in the hippocampus (NAT10 cKO) to show that NAT10 may play a role in LTP maintenance and memory consolidation (using the Morris Water Maze).

Weaknesses:

(1) The NAT10 cKO mice are useful to test the causal role of NAT10 in ac4a and plasticity/memory but all the experiments used AAV-CRE injections in the dorsal hippocampus that showed somewhat modest decreases in total NAT10 protein levels. For these experiments, it would be better to cross the NAT10 floxed animals to CRE lines where better knock down of NAT10 can be achieved postnatally in specific neurons, with less variability.

(2) Because knock down is only modest (~50%), it is not clear if the remaining ac4c on mRNAs is due to remaining NAT10 protein or due to alternative writer (as the authors pose).

Reviewer #2 (Public review):

This is an interesting study that shows that mRNA acetylation at synapses is dynamically regulated at synapses by spatial memory in the mouse hippocampus. The dynamic changes of ac4C-mRNAs regulated by memory were validated by methods including ac4C dot-blot and liquid 13 chromatography-tandem mass spectrometry (LC-MS/MS).

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

(1) The authors use a confusing timeline for their behavioral experiments, i.e., day 1 is the first day of training in the MWM, and day 6 is the probe trial, but in reality, day 6 is the first day after the last training day. So this is really day 1 post-training, and day 20 is 14 days post-training.

We have revised the timeline accordingly. Briefly, mice were trained in the Morris water maze (MWM) with a hidden platform for five consecutive days (training days 1–5). Probe tests were then conducted on day 6 and day 20, which correspond to post-training day 1 and post-training day 15, respectively. We clearly stated as such in the revised manuscript (see results, line 108 – 113) and figure S1 (see figure legend, line 1747 – 1749).

(2) The authors inaccurately use memory as a term. During the training period in the MWM, the animals are learning, while memory is only probed on day 6 (after learning). Thus, day 6 reflects memory consolidation processes after learning has taken place.

We have revised the manuscript to distinguish between "learning" and "memory". We refer to the performance during the 5-day training period as "spatial learning" and restrict the term "memory" to the probe tests on day 6, which reflect memory consolidation after learning has taken place.

(3) The NAT10 cKO mice are useful... but all the experiments used AAV-CRE injections in the dorsal hippocampus that showed somewhat modest decreases... For these experiments, it would be better to cross the NAT10 floxed animals to CRE lines where a better knockdown of NAT10 can be achieved, with less variability.

We want to clarify the reason for using AAV-Cre injection rather than Cre lines. Indeed, we attempted to generate Nat10 conditional knockouts by crossing Nat10^flox/flox mice with several CNS-specific Cre lines. Crossing with Nestin-Cre and Emx1-Cre resulted in embryonic and premature lethality, respectively, consistent with the essential housekeeping function of NAT10 during neurodevelopment. We will use the Camk2α-Cre line which starts to express Cre after postnatal 3 weeks specifically in hippocampal pyramidal neurons (Tsien et al., 1996).

(4) Because knockdown is only modest (~50%), it is not clear if the remaining ac4c on mRNAs is due to remaining NAT10 protein or due to an alternative writer (as the authors pose).

Our results suggest the existence of alternative writers. As shown in Figure 6D, we identified a population of "NAT10-independent" MISA mRNAs (present in MISA but not downregulated in NASA). Remarkably, these mRNAs possess a consensus motif (RGGGCACTAACY) that is fundamentally different from the canonical NAT10 motif (AGCAGCTG). This distinct motif usage suggests that the residual ac4C signals are not merely due to incomplete knockdown of NAT10, but reflect the activity of other, as-yet-unidentified ac4C writers. We will perform ac4C immunostaining in Nat10-reporter mice which express red fluorescent proteins in Nat10-positive cells. The results that ac4C is expressed in both Nat10-positive and negative cells will support the presence of as-yet-unidentified ac4C writers.

Reviewer #2 (Public review):

(1) It is known that synaptosomes are contaminated with glial tissue... So the candidate mRNAs identified by acRIP-seq might also be mixed with glial mRNAs. Are the GO BP terms shown in Figure 3A specifically chosen, or unbiasedly listed for all top ones?

This reviewer is correct that some ac4C-mRNAs identified by acRIP-seq from the synaptosomes are highly expressed in astrocytes, such as Aldh1l1, ApoE, Sox9 and Aqp4 (see list of ac4C-mRNAs in the synaptosomes, Table S3). In agreement, we found that NAT10 was also expressed in astrocyte in addition to neurons. We have provided a representative image showing NAT10-Cre expression in astrocytes in the revised manuscript (Figure 4F and H). In the figure 3A of original submission, we showed 10 out of 16 top BP items for MISA mRNAs. In the figure 3A of revised manuscript, we showed all the top 16 BP items for MISA mRNAs, which are unbiasedly chosen (also see Table S4).

(2) Where does NAT10-mediated mRNA acetylation take place within cells generally? Is there evidence that NAT10 can catalyze mRNA acetylation in the cytoplasm?

The previous studies from non-neuronal cells showed that NAT10 can catalyze mRNA acetylation in the cytoplasm and enhance translational efficiency (Arango et al., 2018; Arango et al., 2022). In this study, we showed that mRNA acetylation occurred both in the homogenates and synapses (see ac4C-mRNA lists in Table S2 and S3). However, spatial memory upregulated mRNA acetylation mainly in the synapses rather than in the homogenates (Fig. 2 and Fig. S2).

(3) "The NAT10 proteins were significantly reduced in the cytoplasm (S2 fraction) but increased in the PSD fraction..." The small increase in synaptic NAT10 might not be enough to cause a decrease in soma NAT10 protein level.

We showed that the NAT10 protein levels were increased by one-fold in the PSD fraction, but were reduced by about 50% in the cytoplasm after memory formation (Fig. 5J and K). The protein levels of NAT10 in the homogenates and nucleus were not altered after memory formation (Fig. 5F and I). Due to these facts, we hypothesized that NAT10 proteins may have a relocation from cytoplasm to synapses after memory formation, which was also supported by the immunofluorescent results from cultured neurons (Fig. S4). However, we agree with this reviewer that drawing such a conclusion may require the time-lapse imaging of NAT10 protein trafficking in living animals, which is technically challenging at this moment.

(4) It is difficult to separate the effect on mRNA acetylation and protein mRNA acetylation when doing the loss of function of NAT10.

This is a good point. We agree with this reviewer that NAT10 may acetylate both mRNA and proteins. We examined the acetylation levels of a-tubulin and histone H3, two substrate proteins of NAT10 in the hippocampus of Nat10 cKO mice. As shown in Fig S5C, E, and F, the acetylation levels of a-tubulin and histone H3 remained unchanged in the Nat10 cKO mice, likely due to the compensation by other protein acetyltransferases. In contrast, mRNA ac4C levels were significantly decreased in the Nat10 cKO mice (Figure S5G–H). These results suggest that the memory deficits seen in Nat10 cKO mice may be largely due to the impaired mRNA acetylation. Nonetheless, we believe that developing a new technology which enables selective erasure of mRNA acetylation would be helpful to address the function of mRNA acetylation. We discussed these points in the MS (see discussion, line 582-589).

Reference

Arango, D., Sturgill, D., Alhusaini, N., Dillman, A. A., Sweet, T. J., Hanson, G., Hosogane, M., Sinclair, W. R., Nanan, K. K., & Mandler, M. D. (2018). Acetylation of cytidine in mRNA promotes translation efficiency. Cell, 175(7), 1872-1886. e1824.

Arango, D., Sturgill, D., Yang, R., Kanai, T., Bauer, P., Roy, J., Wang, Z., Hosogane, M., Schiffers, S., & Oberdoerffer, S. (2022). Direct epitranscriptomic regulation of mammalian translation initiation through N4-acetylcytidine. Molecular cell, 82(15), 2797-2814. e2711.

Tsien, J. Z., Chen, D. F., Gerber, D., Tom, C., Mercer, E. H., Anderson, D. J., Mayford, M., Kandel, E. R., & Tonegawa, S. (1996). Subregion-and cell type–restricted gene knockout in mouse brain. Cell, 87(7), 1317-1326.

A messenger who brings news specifically about the end of the current world order or a major divine intervention that will change everything

eLife Assessment

The work convincingly demonstrates the role of the mycobacterial secreted effector protein MmpE, which translocates to the host nucleus and exhibits phosphatase activity. The study is particularly valuable in showing that both the nuclear localization signal sequences and residues critical for phosphatase function are essential for host gene regulation, lysosomal biogenesis, and intracellular survival. Future studies will be important to explore additional host pathways modulated by MmpE, particularly in the context of infection with a fully virulent Mycobacterium tuberculosis strain.

Reviewer #1 (Public review):

Summary:

The study provides 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.

(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.

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

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

Comments on revisions:

I appreciate the work the authors have done to address reviewers comments. The revised manuscript looks significantly improved. My major concern in the revised version is the microscopy data where the BCG staining using the DiD fluorescent stain does not bring out the rod-shaped bacilli structure. I suggest the authors either use a GFP reporter or some other fluorescent stain to address this issue.

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 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 mice 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 strain. The relative levels of ~ 175 transcripts were altered in mmpE mutant infected macrophages and 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 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.

Comments on revisions:

Thanks to the authors for addressing the concerns raised during the review of the original manuscript. The data is now presented with clarity, and discrepancies in mouse experiments have also been addressed with additional experiments.

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 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 regulation of the VDR gene expression.

Weaknesses:

There are significant differences in lysosomal retention between M. tuberculosis and M. bovis BCG. This study uses BCG and MMPE overexpression to draw conclusions about the impact of the MMPE gene on host gene expression and the bacteria's lysosomal localisation. While the authors have convincingly supported their claims with this model system, the relevance of this mechanism in M. tuberculosis infection remains unaddressed.

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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He is starting to think about converting them and using them

He's asking the queen if he should take them to learn their language, as well that they weren't well educated on weapons

shows his desire for looking and getting gold.

Columbus is describing them specifically saying that they're beautiful.

shows that they never seen the blades they have and they are ignorant

he is looking for how to use them

Columbus thought they were easy to control because of their friendliness

ceci est un test

typo: minima

Falta valor esperado en B

This correlates with my pervious annotation on pushing someone out of your comfort zone. that may be different for everyone and what might be different for someone else. Its always a good idea to try.

this statement is a good push for someone out of their comfort zone and to aim to challenge themselves.

This one is nice -- is it the same in the PQ table?

Make it clear that 'speculative' is OK here

"Provide evidence suggesting" -- not "show"

Effective Altruism in caps

,jv,jhv,jhv

perjured – Introduces betrayal and broken oaths, we may expect deception or treachery in the plot. boiling lead – Violent, graphic imagery. Suggests the play will not shy away from brutality.

Implies unimaginable horror. Raises suspense, what horrors are coming? Suggests emotional and psychological intensity.

Introduces romance. A tragic love story is expected. Love appears central to the character’s identity.

Suggests we are entering the underworld, not the earthly world. A dark, serious, possibly fatalistic tone is signalled.

Graphic but poetic description of dying. Emphasises violence and physical suffering. Suggests that death was dramatic and possibly unjust.

“Secret” signals a hidden love. Hidden relationships often create a sense of something eventually being exposed or jealousy. These aspects could signal conflict or a scandal.

Raises spiritual or religious expectations, something to do with the afterlife.

the audience now remembers that "hey, there's this other person here, they speak.".

kuyfuyf,jy.

danser

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.

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 solid evidence suggests that Uev1a works with the E3 ligase APC/C to degrade Cyclin A. This work would be of interest to researchers in germline biology and cancer.

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 over-expression 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 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.

Comments on revisions:

The authors have greatly improved the manuscript and satisfactorily addressed all of my concerns.

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 homolog, UBE2V1 and UBE2V2, suppresses tumor growth in human colorectal cancer xenografts and cell lines. Notably, these genes' expression correlates with the survival of colorectal cancer patients carrying 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.

Comments on revisions:

The authors have addressed several of the major concerns, including the addition of new data and improved figure presentation. However, some issues remain insufficiently resolved, particularly regarding control reuse (Major Comment 3) and experimental interpretation (Major Comments 5 and 8).

Regarding Major Comment 5, the authors state that UAS copy number affects the frequency of egg chamber degradation in Fig. 2D, and thus explains the reduced phenotype in RasG12V + GFP-RNAi compared to RasG12V alone. However, this explanation is not consistent with other data in the manuscript. UAS-RasG12V combined with UAS-lacZ in Fig. 2G shows a phenotype comparable to UAS-RasV12 alone, despite also increasing the UAS copy number. This suggests that the effect is not simply due to copy number.

I understand that the authors used UAS-RasG12V + GFP-RNAi as a control for the RNAi experiments and UAS-RasG12V + lacZ for the overexpression experiments. I suggest examining the phenotype frequency of UAS-RasG12V + UAS-GFP, to figure the reason out. Overall, these results indicate that there is a spectrum of phenotype frequencies, and therefore appropriate controls should be included for each experiment rather than reusing the same dataset across different experiments, as also noted in Major Comment 3.

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).

Briefing : L’Accrochage Scolaire – Perspectives Pédopsychiatriques et Enjeux de Persévérance

Ce document de synthèse analyse les interventions et les réflexions issues de la conférence donnée dans le cadre de la "Semaine de la persévérance".

Il explore les dynamiques de l'accrochage scolaire à travers le prisme de la pédopsychiatrie, en mettant l'accent sur les mécanismes psychologiques de l'adolescence, l'importance des interactions humaines et les leviers pratiques de l'apprentissage.

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Synthèse de la Direction (Executive Summary)

Le paradigme de la lutte contre l'échec scolaire évolue : l'accent est désormais mis sur l'accrochage (l'adhésion active à la scolarité) plutôt que sur le simple traitement du décrochage.

Cette transition s'inscrit dans un contexte post-crise sanitaire où le nombre de décrocheurs, particulièrement en lycée, a fortement augmenté.

Les points clés à retenir sont les suivants :

• La dynamique naturelle de l'autonomie : L'éducation doit naviguer entre l'accrochage nécessaire (protection) et le décrochage salutaire (individualisation), à l'image des modèles observés dans la nature.

• L'ambivalence adolescente : Les comportements et les discours des jeunes sont souvent des messages codés. Un « je m'en fiche » traduit fréquemment un investissement émotionnel trop lourd à porter.

• Le blocage par la « peur de penser » : Les bouleversements de la puberté peuvent entraîner une inhibition intellectuelle volontaire pour se protéger de pensées (agressives ou sexuelles) jugées effrayantes.

• L'impact déterminant des figures adultes : La réussite ou l'échec d'un parcours tient souvent à des rencontres humaines spécifiques qui valident l'existence et la valeur du jeune.

• L'optimisation des rythmes biologiques : Une meilleure prise en compte des types de mémoire (visuelle/auditive) et des rythmes de sommeil individuels est essentielle pour favoriser l'accrochage.

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I. La Dialectique de l'Accrochage : Entre Protection et Autonomie

L'expert propose une réflexion sémantique et biologique sur le terme "accrochage", en utilisant l'analogie des lémuriens malgaches pour illustrer le développement vers l'autonomie.

1. Le modèle biologique de l'indépendance

• L'accrochage initial : À la naissance, le petit est physiquement accroché à sa mère pour sa survie. C'est le temps de la dépendance absolue.

• Le décrochage progressif : À la puberté (très brève chez le lémurien), la mère laisse le jeune explorer les branches basses. Elle n'intervient que si le danger est réel.

• La fonction de l'adulte : L'adulte doit se situer à "mi-hauteur" dans l'arbre : observer sans étouffer, et n'intervenir (attraper par la peau du cou) que pour empêcher une chute grave.

2. La difficulté du curseur humain

L'équilibre entre vigilance et lâcher-prise est complexe pour les humains, qui oscillent souvent entre deux extrêmes :

• L'hyper-vigilance : Une intervention permanente qui entrave l'apprentissage de la prudence.

• Le désintérêt : Une absence de regard qui laisse le jeune seul face à des dangers qu'il ne sait pas encore évaluer.

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II. Comprendre le Langage et les Blocages de l'Adolescence

L'adolescence est une période de décalage entre l'évolution rapide du corps et la stabilisation de l'image de soi. Ce processus impacte directement l'investissement scolaire.

1. L'ambivalence et la "Traduction Interne"

L'adulte doit disposer d'un "traducteur interne" pour interpréter les signaux adolescents :

• Négation de l'intérêt : Dire « j'en ai rien à faire » signifie souvent « j'en ai trop à faire et cela me submerge ».

• Indécision d'orientation : L'absence d'idée pour l'avenir cache fréquemment un trop-plein de centres d'intérêt ou une peur de s'engager alors que le présent est incertain.

• Provocation et retard : Saboter un rendez-vous ou arriver en retard peut être une mise à l'épreuve pour vérifier si l'adulte se soucie réellement du jeune.

2. Le mécanisme de la "Peur de Penser"

L'inhibition intellectuelle et la chute des résultats scolaires peuvent découler d'un mécanisme de défense :

• Le corps pubère génère des pensées nouvelles (agressivité, sexualité) qui peuvent effrayer le jeune.

• Par peur que ces pensées ne se transforment en actes, l'adolescent "pose un couvercle" sur l'ensemble de son activité mentale.

• Conséquence : Une inhibition des apprentissages. Le jeune ne pense plus du tout pour ne pas risquer de penser à ce qui l'effraie. L'autorisation de penser (distinguée de l'autorisation d'agir) est un levier de délivrance majeur.

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III. Le Rôle des Interactions Humaines et Institutionnelles

L'accrochage scolaire ne dépend pas uniquement de facteurs pédagogiques, mais d'une reconnaissance de la valeur de l'individu par ses pairs et ses mentors.

1. Les rencontres déterminantes

Le parcours scolaire est marqué par quelques figures adultes clés. Ces "transmetteurs" déclenchent le plaisir d'apprendre par identification.

À l'inverse, la violence ou l'indifférence (le professeur qui "débite son cours" sans voir l'élève) peuvent briser l'investissement.

2. Le besoin de considération exprimée

• La sphère familiale : La sécurité affective doit être verbalisée. L'adolescent a besoin d'entendre qu'il a de la valeur, même s'il feint l'indifférence.

• L'existence aux yeux de l'institution : Un simple appel d'un CPE après une absence peut suffire à faire exister de nouveau un élève qui s'était "effacé".

• Valorisation multi-facettes : L'exemple du "livret de compétences municipales" (Issy-les-Moulineaux) montre l'intérêt de ne pas réduire un jeune à son statut d'élève, mais d'intégrer ses réussites sportives, associatives ou artistiques.

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IV. Facteurs Pratiques et Physiologiques de la Réussite

Le document souligne l'importance de diagnostiquer les besoins individuels pour éviter une dégradation de l'image de soi liée à des efforts inefficaces.

| Facteur | Enjeux pour l'Accrochage | | --- | --- | | Sommeil | Les besoins varient (6h à 10h) et les pics de performance diffèrent (matinal vs tardif). Copier le rythme d'un autre peut mener au surmenage et à l'échec. | | Mémoire Auditive | Privilégie l'écoute active en classe ; peu de travail personnel nécessaire si l'attention est maintenue. | | Mémoire Visuelle | Nécessite des prises de notes attractives, des couleurs et des fiches pour une mémorisation efficace. |

Conclusion de l'analyse : L'accrochage scolaire est un investissement énergétique et émotionnel.

Il nécessite que le jeune se sente en sécurité pour penser, qu'il soit reconnu dans sa globalité humaine et qu'il comprenne son propre fonctionnement biologique pour transformer la contrainte scolaire en source de satisfaction.

eLife Assessment

This study presents valuable findings on the role of KLF6 in in vitro endothelial cells exposed to altered (high or low) shear stress with a customized microfluidic device to investigate mechanisms of atherosclerosis. The finding that altered shear stress results in endothelial cell ferroptosis through reduced expression of KLF6 is compelling and adds a new layer of complexity to the pathogenesis of atherosclerotic plaques. However, more detailed characterization of ferroptosis is needed.

Reviewer #1 (Public review):

Summary:

The authors used an in vitro microfluidic system where HUVECs are exposed to high, low or physiologic (normal) shear stress to demonstrate that both high and low shear stress for 24 hours resulted in decreased KLF6 expression, decreased lipid peroxidation and increased cell death which was reversible upon treatment with Fer-1, the ferroptosis inhibitor. RNA sequencing (LSS vs normal SS) revealed decreased steroid synthesis and UPR signaling in low shear stress conditions, which they confirmed by showing reduced expression of proteins that mitigate ER stress under both LSS and HSS. Decreased KLF6 expression after exposure to HSS/LSS was associated with decreased expression of regulators of ER stress (PERK, BiP, MVD) which was restored with KLF6 overexpression. Overexpression of KLF6 also restored SLC7A11 expression, Coq10 and reduced c11 bodipy oxidation state- all markers of lipid peroxidation and ferroptosis. The authors then used vascular smooth muscle cells (atherosclerotic model) with HUVECs and monocytes to show that KLF6 overexpression reduces the adhesion of monocytes and lipid accumulation in conditions of low shear stress.

Strengths:

(1) The use of a microfluidic device used to simulate shear stress while keeping the pressure constant when varying shear stress applied is improved and more physiologic compared to traditional cone and shearing devices. Similarly, the utilization of both low and high shear stress in most experiments is a strength.

(2) This study provides a link between disturbed shear stress and ferroptosis, which is novel, and fits nicely with existing knowledge that endothelial cell ferroptosis promote atherosclerosis. This concept was also recently reported Sept 2025 when a publication also demonstrated that LSS trigger ferroptosis in vascular endothelial cells (PMID: 40939914), which partly validates these findings.

Weaknesses:

(1) While HUVECs are commonly used in endothelial in vitro studies, it would be preferable to confirm the findings using an arterial cell line such as human coronary artery cells when studying mechanisms of early atherosclerosis. Furthermore, physiologic arterial shear stress is higher than venous shear stress, and different vascular beds have varying responses to altered shear stress and as such, the up and downregulated pathways in HUVECs should be confirmed in an arterial system.

(2) The authors provide convincing evidence of disturbances in shear stress inducing endothelial ferroptosis with assays for impaired lipid peroxidation and increased cell death that was reversed with a ferroptosis inhibitor. However more detailed characterization of ferroptosis with iron accumulation assays, as well as evaluating GPX4 activity as a consequence of the impaired mevalonate pathway, and testing for concomitant apoptosis in addition to ferroptosis would add to the data.

(3) The authors state that KLF2 and KLF4 are not amongst the differentially expressed genes downregulated by reduced shear stress, which is contrary to previous data, where both KLF2 and KLF4 are well studied to be upregulated by physiologic laminar shear stress. While this might be due to the added pressure in their microfluidic system, it also might be due to changes in gene expression over time. In this case, a time course experiment would be needed. It is possible that KLF2, KLF4 and KLF6 are all reduced in low (and high) shear stress and cooperatively regulate the endothelial cell phenotype. Both KLF2 and KLF4 have been shown to be protective against atherosclerosis.

Comments on revisions:

The authors have failed to respond to all the preceding critiques with supporting experimental data. Recommend a reassessment of the initial critiques.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #2 (Public review):

Points to be addressed:

(1) As a statistical test, the authors report having used unpaired t-tests; however, often three groups are compared for which t-tests are inadequate. This is faulty as, amongst other things, it does not take multiple comparison testing into account.

We have adopted the reviewers' suggestions and conducted a variance analysis (ANOVA) to reanalyze the experimental results with three or more different condition groups. At the same time, we have retained the t-test results for experiments with only two condition groups.

(2) Both B-Actin and GAPDH seem to have been used for protein-level normalization. Why? The Figure 2HL first panel reports B-actin, whereas the other three report GAPDH. The same applies to Figures 3E-F, where both are shown, and it is not mentioned which of the two has been used. Moreso, uncropped blots seem to be unavailable as supplementary data for proper review. These should be provided as supplementary data.

In Figures 2G and 3E-F, β-actin and GAPDH both have been used for protein level normalization. The main issue is the mixed use of these two housekeeping proteins, without taking consistency into account in advance. In addition, the expression levels of these two proteins show no significant differences in response to different fluid shear stresses. The uncropped blot images have been organized and provided in the supplementary data.

(3) LSS and MSS were compared based on transcriptomic analysis. Conversely, RNA sequencing was not reported for the HSS. Why is this data missing? It would be valuable to assess transcriptomics following HSS, and also to allow transcriptomic comparison of LSS and HSS.

In the current study, we have only conducted the transcriptomic comparative analysis between LSS and MSS conditions, mainly considering that most of current researches focuses on the endothelial dysfunction and atherosclerosis under LSS. Since our HSS condition is overall about 24 dyn/cm², which is also recognized within the normal physiological range in some reports. Moreover, the transcriptomic data are primarily used to identify the targets in our study. Interestingly, for these selected genes, they share the same trend involved in endothelial cell ferroptosis induced by LSS and HSS. At the same time, we strongly agree with the reviewer’s claim that the RNA sequencing results under HSS are also valuable. Therefore, in the future, we are planning to perform the transcriptomic sequencing analysis under the HSS or higher level of shear stress, aiming to discover new insights.

(4) Actual sample sizes should be reported rather than "three or more". Moreso, it would be beneficial to show individual datapoints in bar graphs rather than only mean with SD if sample sizes are below 10 (e.g., Figures 1B-H, Figure 2G, etc.).

After rechecking our original data, All analyzed results were from three biological replicates, so they are uniformly marked as 'n=3' in the article. According to the reviewer's suggestion, the position of each data point has been added in the chart of the statistical results along with the standard deviation bars.

(5) The authors claim that by modifying the thickness of the middle layer, shear stress could be modified, whilst claiming to keep on-site pressure within physiological ranges (approx. 70 mmHg) as a hallmark of their microfluidic devices. Has it been experimentally verified that pressures indeed remain around 70 mmHg.

It is a very interesting question. In this article, the cross-sectional areas of different tunnel-like channel is related to the thickness of the middle layer, resulting in different level of shear stress. Since all flow rates under three conditions keep same at 1.6 ml/min, the average pressure is calculated to be around 70 mmHg based on our previously reported formula (PMID: 37662690). To address the reviewer's question about the actual pressure values, we used a water-filled tube connected to a chip and measured the height of the water surface in the elevated end relative to the chip position, as shown in the Author response image 1. As expected, when the height of the middle layer bulging to the same value (0.7 mm) as under the LSS condition, the water level reaches to 900 mm, which is corresponding to about 70 mmHg.

Author response image 1.

Schematic diagram of on-chip pressure detection

(6) A coculture model (VSMC, EC, monocytes) is mentioned in the last part of the results section without any further information. Information on this model should be provided in the methods section (seeding, cell numbers, etc.). Moreover, comparison of LSS vs LSS+KLF6 OE and HSS vs HSS+KLF6 OE is shown. It would benefit the interpretation of the outcomes if MSS were also shown. It would also be beneficial to demonstrate differences between LSS, MSS, and HSS in this coculture model (without KLF6 OE).

The specific methods for constructing the co-culture models (vascular smooth muscle cells, endothelial cells, monocytes) mentioned in the results section have been introduced in our previous paper. For the convenience for reading this article, we have added a brief description in the section of “Methods and materials” in this paper, including cell seeding and numbers. In this study, the results of LSS vs LSS+KLF6 OE and HSS vs HSS+KLF6 OE are presented to verify the role of KLF6 in LSS- or HSS-induced promotion of early atherosclerotic events. In our previously published paper (PMID: 37662690), we have showed the effects of three different shear stresses on the atherosclerotic events (shown in Fig. 4 in that paper). Those results have demonstrated that both LSS and HSS significantly promote early atherosclerotic events compared with the MSS.

(7) The experiments were solely performed with a venous endothelial cell line (HUVECs). Was the use of an arterial endothelial cell line considered? It may translate better towards atherosclerosis, which occurs within arteries. HUVECs are not accustomed to the claimed near-physiological pressures.

The human umbilical vein endothelial cell (HUVEC) is a commonly used cell line for many in vitro studies of vascular endothelium under fluid shear stress conditions. Although human arterial endothelial cells (HAECs) may be more suitable than HUVECs for responding to physiologically relevant pressure, HUVECs are more easy to obtain and maintain. However, we are going to order HAECs and will use them to validate the conclusion for the potential translatability.

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

(1) Information on seeding of the microfluidic device is absent in the methods section (i.e., seeding, cell density, passage number, confluence, etc.). Moreso, treatment with Fer-1 is not reported in the methods section.

We have described the cell seeding information in‘Preparation of cell culture in the microfluidic chip’ and the Fer-1 treatment in ‘Cell death assay’ in the Method section.

(2) Figure 3F has "MSS", "HSS", and "LSS+KLF6" as groups on the x-axis; the latter should probably be "HSS+KLF6".

Thank you for pointing out this error in Figure 3F. We have made the correction.

(3) Data should be made available in online repositories rather than "making it available upon reasonable request". As it was not provided, the sequencing data could not be reviewed. In addition, it was stated that a preprint was available on BioRxiv, but I could not find it.

Thank you for the suggestion. We have uploaded the RNA-seq data to the NCBI GEO database, which was publicly available on December 9, 2025.

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.

eLife Assessment

Using genome databases, the authors performed solid bioinformatic analyses to trace the genomic history of the clinically relevant Staphylococcus aureus tetracycline resistance plasmid pT181 over the last seven decades. They discovered that this element has transitioned from a multicopy plasmid to a chromosomally integrated element, and the work represents a valuable demonstration of the use of publicly available data to investigate plasmid biology and inform clinical epidemiology. This work will appeal to researchers interested in staphylococcal evolution and plasmid biology.

Reviewer #1 (Public review):

The study provides a robust bioinformatic characterization of the evolution of pT181. My main criticism of the work is the lack of experimental validation for the hypotheses proposed by the authors.

Comments on the study:

(1) One potential reason for the decline in pT181 copy number over time may be a high cost associated with the multicopy state. In this sense, it would be interesting if the authors could use (or construct) isogenic strains differing only in the state of the plasmid (multicopy/integrated). With this system, the authors could measure the fitness of the strains in the presence and absence of tetracycline, and they could be able to understand the benefit associated with the plasmid transition. The authors discuss these ideas, but it would be nice to test them.

(2) It would be interesting to know the transfer frequencies of the multicopy mobilizable pT181 plasmid, compared to the transfer frequency of the plasmid integrated into the SSCmec element (which can be co-transferred, integrated in conjugative plasmids, or by transduction).

(3) One important limitation of the study that should be mentioned is that inferring pT181 PCN from whole genome data can be problematic. For example, some DNA extraction methods may underestimate the copy number of small plasmids because the small, circular plasmids are preferentially depleted during the process (see, for example, https://www.nature.com/articles/srep28063).

Reviewer #2 (Public review):

Summary:

The authors performed bioinformatic analyses to trace the genomic history of the clinically relevant pT181 plasmid. Specifically, they:

(1) tracked the presence of pT181 across different S. aureus strain backgrounds through time. It was first found in one, later multiple strains, though this may reflect changes in sampling over time.

(2) estimated the mutation rate of the chromosome and plasmid.

(3) estimated the plasmid copy number of pT181, and found that it decreased over time. The latter was supported by two sets of statistical analyses, first showing that the number of single-copy isolates increased over time, and second, that the multicopy isolates demonstrated a lower PCN over time.

(4) reported the different integration sites at which pT181 integrated into the genome.

As a caveat, they mentioned that identical plasmid sequences have variable plasmid copy numbers across different genomes in their dataset.

Strengths:

This is a very solid, well-considered bioinformatic study on publicly available data. I greatly appreciate the thoughtful approach the authors have taken to their subject matter, neither over- nor underselling their results. It is a strength that the authors focussed on a single plasmid in a single bacterial species, as it allowed them to take into account unique knowledge about the biology of this system and really dive deep into the evolution of this specific plasmid. It makes for a compelling case study. At the same time, I think the introduction and discussion can be strengthened to demonstrate what lessons might be drawn from this case study for other plasmids.

Weaknesses:

The finding that the pT181 copy number declined over time is the most interesting claim of the paper to me, and not something that I have seen done before. While the authors have looked at some confounders in this analysis, I think this could be strengthened further in a revision.

For the flow of the storyline, I also think the estimation of mutation rates (starting L181) and integration into the chromosome (starting L255) could be moved to the supplement or a later position in the main text.

Clearly, the use of publicly available data prevents the authors from controlling the growth and sequencing conditions of the isolates. It is striking that they observe a clear signal in spite of this, but I would have loved to see more discussion of the metadata that came with the publicly available sequences and even more use of that metadata to control for confounding.

Author response:

eLife Assessment

Using genome databases, the authors performed solid bioinformatic analyses to trace the genomic history of the clinically relevant Staphylococcus aureus tetracycline resistance plasmid pT181 over the last seven decades. They discovered that this element has transitioned from a multicopy plasmid to a chromosomally integrated element, and the work represents a valuable demonstration of the use of publicly available data to investigate plasmid biology and inform clinical epidemiology. This work will appeal to researchers interested in staphylococcal evolution and plasmid biology.

Thank you, we agree with this overview. We also think this work is interesting to people interested in antimicrobial resistance and bacterial genome structure.

Public Reviews:

Reviewer #1 (Public review):

The study provides a robust bioinformatic characterization of the evolution of pT181. My main criticism of the work is the lack of experimental validation for the hypotheses proposed by the authors.

Comments on the study:

(1) One potential reason for the decline in pT181 copy number over time may be a high cost associated with the multicopy state. In this sense, it would be interesting if the authors could use (or construct) isogenic strains differing only in the state of the plasmid (multicopy/integrated). With this system, the authors could measure the fitness of the strains in the presence and absence of tetracycline, and they could be able to understand the benefit associated with the plasmid transition. The authors discuss these ideas, but it would be nice to test them.

We agree that the relative fitness of integrated versus multicopy plasmids is interesting and a costly multicopy state could explain the transition of independent pT181 replicons to chromosomal integration. This is a project we are exploring for a future study. However, we think that this additional experimental work goes beyond the scope of the paper.

(2) It would be interesting to know the transfer frequencies of the multicopy mobilizable pT181 plasmid, compared to the transfer frequency of the plasmid integrated into the SSCmec element (which can be co-transferred, integrated in conjugative plasmids, or by transduction).

We agree with the reviewer that this is an interesting question. However, we think inferring these rates from natural sequence data is not feasible in this case given the low heterogeneity of the plasmid sequence. A laboratory-based experimental study could not address the real transfers we observe over the course of decades, as in vitro S. aureus transfer rates are often not good proxies for in vivo (McCarthy et al., 2014). In addition, we do not know what is moving the integrated plasmid. pT181 could be moved by a phage or plasmid, so we are uncertain what the correct experiment would be to explore this.

(3) One important limitation of the study that should be mentioned is that inferring pT181 PCN from whole genome data can be problematic. For example, some DNA extraction methods may underestimate the copy number of small plasmids because the small, circular plasmids are preferentially depleted during the process (see, for example, https://www.nature.com/articles/srep28063).

We will investigate this issue further in the revisions. The kits used to extract DNA for the earlier-collected samples may possibly yield more plasmid DNA relative to the chromosome compared to newer ones on average; however, we think this is not driving the decline that we observe in multicopy pT181 copy number. Multiple BioProjects find the same result, where earlier samples have higher copy number compared to later samples. We expect extraction methods to be consistent within a BioProject, suggesting that this decline is genuine and not technical. In revisions, we intend to evaluate the effect of date of sequencing and additional metadata on copy number.

Reviewer #2 (Public review):

Summary:

The authors performed bioinformatic analyses to trace the genomic history of the clinically relevant pT181 plasmid. Specifically, they:

(1) Tracked the presence of pT181 across different S. aureus strain backgrounds through time. It was first found in one, later multiple strains, though this may reflect changes in sampling over time.

(2) Estimated the mutation rate of the chromosome and plasmid.

(3) Estimated the plasmid copy number of pT181, and found that it decreased over time. The latter was supported by two sets of statistical analyses, first showing that the number of single-copy isolates increased over time, and second, that the multicopy isolates demonstrated a lower PCN over time.

(4) Reported the different integration sites at which pT181 integrated into the genome.

As a caveat, they mentioned that identical plasmid sequences have variable plasmid copy numbers across different genomes in their dataset.

Strengths:

This is a very solid, well-considered bioinformatic study on publicly available data. I greatly appreciate the thoughtful approach the authors have taken to their subject matter, neither over- nor underselling their results. It is a strength that the authors focused on a single plasmid in a single bacterial species, as it allowed them to take into account unique knowledge about the biology of this system and really dive deep into the evolution of this specific plasmid. It makes for a compelling case study. At the same time, I think the introduction and discussion can be strengthened to demonstrate what lessons might be drawn from this case study for other plasmids.

Weaknesses:

The finding that the pT181 copy number declined over time is the most interesting claim of the paper to me, and not something that I have seen done before. While the authors have looked at some confounders in this analysis, I think this could be strengthened further in a revision.

In the revisions, we will further explore the impact that technical variation could have in contributing to copy number variation and update our claims for the decline in copy number of the independent replicon over time and variation for the same plasmid sequence accordingly. Multiple BioProjects show earlier samples have higher copy number compared to later samples; we expect extraction methods to be consistent within a BioProject, supporting our initial findings that this decline over time is not due to technical variation.

For the flow of the storyline, I also think the estimation of mutation rates (starting L181) and integration into the chromosome (starting L255) could be moved to the supplement or a later position in the main text.

We will revisit the text organization for flow and clarity of storyline.

Clearly, the use of publicly available data prevents the authors from controlling the growth and sequencing conditions of the isolates. It is striking that they observe a clear signal in spite of this, but I would have loved to see more discussion of the metadata that came with the publicly available sequences and even more use of that metadata to control for confounding.

In revisions, we will further investigate possible contributors to the observed decline in copy number of multicopy pT181 over time. We have incorporated the date of sample collection and BioProject in our analysis, but not the date of sequencing or extraction technique.

References

McCarthy, A. J., Loeffler, A., Witney, A. A., Gould, K. A., Lloyd, D. H., & Lindsay, J. A. (2014). Extensive horizontal gene transfer during Staphylococcus aureus co-colonization in vivo. Genome Biology and Evolution, 6(10), 2697–2708. https://doi.org/10.1093/gbe/evu214

eLife Assessment

This valuable study describes significant differences in prey capture behavior between PSD-95 knock-out and wild-type mice, despite prior work by the same authors showing only modest visual deficits in the former. The data convincingly demonstrated prey capture performance in PSD-95 knock-out mice to improve under monocular viewing conditions. However, this finding alone was inadequate to support the interpretation of results as revealing a deficit in binocular visual integration, especially given the lack of eye and head tracking data or consideration of alternative explanations for the observed behavior.

Reviewer #1 (Public review):

Summary:

PSD95 has long been studied in detail to understand molecular mechanisms of synaptic plasticity as related to specific cell types (excitatory), circuits (visual cortex) and circuit development and function (ocular dominance plasticity ). While much was known about the molecular and cellular details of its function, it remained unclear whether and how it might contribute to the development of specific aspects of visual perception. While overall vision is preserved in PSD95 KO (Knockout) mice, studying natural, visually-guided prey capture behavior revealed robust, yet specific, perturbations to binocular processing during the behavior.

Strengths:

A major strength of the paper is being able to quantify precise measures of the visual aspects versus the motor aspects of prey pursuit. Comparing changes in behavior due to monocular occlusion was particularly revealing that mice indeed employ binocular summation to extract visual cues useful for prey pursuit. This result further suggested that in cases with poor binocular vision, monocular input can improve perceptual and behavioral processes as it does in human subjects with comparable challenges.

The study not only provided a useful finding regarding the function of PSD95, but also outlined a useful general approach toward identifying and quantifying specific deficits in binocular summation. This is likely to broadly impact studies of visual system development, behavior, and neural circuit function. The careful attention to details, observations, and openness about subject variance will also be helpful to those studying specific visual pursuit and natural prey capture behavior in the mouse.

Weaknesses:

Lack of eye movement monitoring and detailed head movement analysis preclude total certainty for the interpretation of observed behaviors.

Reviewer #2 (Public review):

Summary:

This manuscript studies the impacts of knocking out a protein known to be involved in synapse maturation in mice, measuring their ability to hunt prey items (and to discriminate simple visual patterns) under binocular and monocular viewing conditions. The main results are that the mice with this protein knocked out are impaired when performing visual tasks with binocular viewing, but are actually better when they perform monocularly. The interpretation is that the knocked-out protein has affected binocular visual integration.

Strengths:

Overall, the attempt to connect a protein to behavior/perception, via known mechanistic effects on synapse development and visual critical periods, is admirable.

The use of multiple visual conditions and behavioral paradigms (binocular/monocular, cricket hunting/orientation discrimination, light/dark) strengthens and enriches the results.

Weaknesses:

The primary interpretation - that binocular integration is affected in the PSD-95 knockouts- is not supported by the behavioral evidence. Using behavior to isolate a particular stage in visual processing (and further, to distinguish it from elements of generating the behavioral response and/or acquiring the visual information in the first place) is notoriously difficult. Such attempts are, of course, the domain of psychophysics. In fact, the most classical and loveliest success is in the domain of binocular integration- Bela Julesz's "psychoanatomy" that used random dot stereograms to isolate stereoscopic computations.

I mention this example because it is, in fact, directly relevant to my primary concern about the evidence used as support for the favored interpretation here. Julesz's stimuli were extremely clever in isolating binocular mechanisms (i.e., binocular mechanisms MUST be used to perform the task), and any perceptual/behavioral reports are very straightforward to interpret (i.e., a stereoscopically-defined shape can be identified, or not).

Now compare this to the work described in this manuscript. KO (knockout) mice are worse than wild types at chasing prey items or at moving towards a rewarded orientation, but they get better when performing this task monocularly. No argument that that is an interesting bit of scientific phenomenology to characterize. However, the behaviors do not require binocular integration, the freely-moving paradigms involve a variety of gaze and body-movement strategies, and the metrics used to quantify performance are similarly high-dimensional. Bottom line, it is not possible to glean whether the KO's intriguing binocular-vs-monocular differences are due to binocular integration per se, or something better thought of as fundamentally sensorimotor in origin. The tasks do not isolate visual from sensorimotor processing, and the behaviors and associated metrics cannot definitely adjudicate between a multitude of possible specific interpretations.

More specifically, the KO mice may have abnormal patterns of binocular coordination. Eye movements were not tracked in these studies, despite the availability of such instrumentation and their successful application in many preceding studies of mouse prey capture. If the KO mice do not coordinate their eye movements (in task-specific/task-relevant ways), they might receive binocular input that is abnormal. Under monocular conditions, that mismatched or inappropriately coordinated binocular input is absent, which would relieve them of the confusing visual information. That is rather different than having an impairment of binocular integration, as it is basically a question of whether the visual system is impaired, or whether the inputs to the visual system are abnormal due to differences in binocular coordination.

It is also possible that the binocular deficit, as measured in behavior,r occurs in a distinct part of the sensorimotor loop. Even if the binocular eye movements are normal, and binocular visual integration is normal, PSD-95 KO mice may be confused or distracted by the larger visual field that comes from binocular viewing (quite profound in species with mostly lateralized eyes). Such a "post-sensory" interpretation related to target selection (from what could be a totally normal visual representation) is difficult to rule out as well.

In summary, this reviewer appreciates the value of trying to connect this molecular mechanism to sensory processing and behavior. The use of naturalistic tasks and freely-moving paradigms is also something to commend. However, the sorts of visual stimuli and behavioral paradigms used here are not well-suited to supporting the rather specific interpretation that has been put forth in this manuscript.

Reviewer #3 (Public review):

Summary:

Bhattacharya et al. describe significant differences in prey capture behaviour in PSD-95 KO (Knockout) and wild-type (WT) mice. This work develops logically from their previous findings that KO of PSD-95 inhibits the maturation in the primary visual cortex. However, their previous work revealed that the visual deficits in the KO mice were relatively modest. Here, by employing an ethologically-relevant behavioural task, they show that several aspects of prey capture are impaired in the KO. Importantly, the deficits in predatory behavior in the KO mouse improved with monocular deprivation, consistent with deficits in binocular vision.

Strengths:

Overall, the data presented are convincing and valuable, and support the idea that PSD-95 expression is important for the maturation of visual responses.

Weaknesses:

The manuscript could be strengthened by consideration of the following points:

(1) The deficits in predatory behavior are interpreted to reveal several possible visual defects, including the absence of binocularity, binocular summation, or binocular mismatch in V1 neurons. Yet this is done with insufficient detail about each possible mechanism and without direct neuronal evidence.

(2) The observation that binocular visual field bias is intact in the PSD-95 KO mice is interesting but appears to contradict other data suggesting the absence of binocularity in the KO visual system, and this is not discussed in sufficient detail.

(3) No consideration of previous work using constitutive PSD-95 KOs that documented a learning deficit.

(4) Throughout the manuscript, including the first paragraph of the discussion, the authors state that "This study explored whether the maturation of CP closure, inhibited by PSD-95 influences binocular visual behaviour". However, if this were the case, the current experiments would have compared cricket capture behavior at two ages across the two genotypes: pre- and post-CP closure in WTs and at matching chronological ages in KOs.

(5) Freeman and others have shown that the influence of binocular summation on orientation discrimination is highest at low stimulus contrast and short duration stimuli. How does this impact the interpretation of predatory behavior and discrimination in the VWT?

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.

eLife Assessment

In this valuable study, the authors present traces of bone modification on ~1.8 million-year-old proboscidean remains from Tanzania, which they infer to be the earliest evidence for stone-tool-assisted megafaunal consumption by hominins. Challenging published claims, the authors argue that persistent megafaunal exploitation roughly coincided with the earliest Acheulean tools. Notwithstanding the rich descriptive and spatial data, the behavioral inferences about hominin agency rely on traces (such as bone fracture patterns and spatial overlap) that are not unequivocal; the evidence presented to support the inferences thus remains incomplete. Given the implications of the timing and extent of hominin consumption of nutritious and energy-dense food resources, as well as of bone toolmaking, the findings of this study will be of interest to paleoanthropologists and other evolutionary biologists.

Reviewer #2 (Public review):

The revised manuscript does a good job of using less definitive language, particularly by adding "possible" qualifiers to several interpretations. This addresses the concern about overstatement.

The main issue raised in the original review, however, remains unresolved. Only two elephant bone specimens at EAK show green-bone breakage interpreted as anthropogenic, and the diagnostic basis for that interpretation is not demonstrated clearly on the EAK material itself. The manuscript discusses a suite of fracture attributes described as diagnostic of dynamic percussive breakage, but these attributes are not explicitly documented on the EAK specimens. Instead, the diagnostic traits are illustrated using material from other Olduvai contexts, and that behavior is then extrapolated to make similar claims at EAK. For a paper making a potentially important behavioral argument, the key diagnostic evidence is not clearly demonstrated at the focal assemblage.

This problem is evident in the presentation of the EAK specimens. In their response, the authors state that one EAK specimen shows "overlapping scars" and constitutes a "long bone flake"; however, these features are not clearly identifiable in the figures or captions as currently presented. The authors state that Figures S21-S23 clearly indicate human agency, including a long bone flake with overlapping scars and a view of the medullary surface, but it is unclear which specimens or surfaces these descriptions refer to. Figure S21 does appear to show green fracture and is described only as an "elephant-sized flat bone fragment with green-bone curvilinear break." Figure S22 shows the same bone and cortical surface in a different orientation, providing no additional information. In Figure S23, I cannot clearly identify a medullary surface or evidence of green-bone fracture from this image. None of these images clearly demonstrates overlapping scars, and the figures would be substantially improved by explicitly identifying the features described in the text. Even if both EAK specimens are accepted as green-broken, they do not demonstrate the co-occurrence of multiple diagnostic fracture traits such as multiple green breaks, large step fractures, hackle marks, and overlapping scars that the authors state is required to attribute dynamic percussive activity to hominins and address equifinality.

I appreciate that the authors are careful to state that spatial association between stone tools and fossils alone does not demonstrate hominin behavior, and that they treat the spatial analyses as supportive rather than decisive. While the association is intriguing, the problem is downstream: spatial association is used to strengthen an interpretation of butchery at EAK that still depends on fracture evidence that is not clearly documented at the assemblage level.

The critique concerning Nyayanga is not addressed in the revision. The manuscript proposes alternative explanations for the Nyayanga material but does not demonstrate why these are more plausible than the interpretation advanced by Plummer et al. (2023). I am not arguing that the Nyayanga material should be accepted as butchery; rather, showing that trampling is possible does not establish it as more probable than cut marks. In contrast, the EAK material is treated as evidence of butchery on the basis of evidence that, in my opinion, is more limited and less clearly demonstrated. Even if this is not the authors' intention, the uneven treatment removes an earlier megafaunal case from the comparison and strengthens the case for interpreting EAK as marking a behavioral shift toward megafaunal butchery by excluding other early cases.

While I remain concerned about how the EAK evidence is documented and interpreted, I think the manuscript is appropriate for publication and will generate useful discussion. Readers can then assess for themselves whether the available evidence supports the strength of the behavioral claims.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public review):

I am happy with the revisions the authors made, and believe that the manuscript is now stronger, representing an important contribution.

We are truly thankful to this reviewer for the very constructive comments

Reviewer #2 (Public review):

In their response, the authors state that they do not treat the EAK evidence as decisive, yet the manuscript repeatedly characterizes the assemblage in very definitive terms. For example, EAK is described as "the oldest unambiguous proboscidean butchery site at Olduvai" and as "the oldest secure proboscidean butchery evidence." These phrases communicate a high level of confidence that does not align with the more qualified position articulated in the rebuttal and extends beyond what the documented evidence securely supports.

We decided to sound less dogmatic and remove the emphasis by adding “potentially” the oldest…. We emphasize that even if we had documented cut marks, we would be on the same epistemological ground, since there is no 100% certainty that the marks identified as cut marks could be cut marks.

I appreciate the authors' clarification regarding the fracture features, and I agree that these are well-established outcomes of dynamic hammerstone percussion. At the same time, several of these traits have been documented in non-anthropogenic contexts, including helicoidal spiral fractures resulting from trampling and carnivore activity (Haynes 1983), adjacent or flake-like scars created by carnivore gnawing (Villa and Bartram 1996), hackled break surfaces produced by heavy passive breakage such as trampling or sediment pressure (Haynes 1983), and impact-related bone flakes observed in carnivore-modified assemblages (Coil et al. 2020).

We added this explanation to the final version of the article:

“This interpretation is epistemologically problematic because it does not satisfy the fundamental criteria for valid analogy as outlined by Bunge (1981), namely substantial, structural, and environmental affinity. Specifically, the cited examples involve agents, materials, and contexts that differ markedly in composition, mechanical properties, and loading regimes from those considered here. Experimental and actualistic studies demonstrate that carnivores—rather than trampling—are also capable of producing spiral fractures and overlapping bone scarring, but these observations are restricted to faunal remains of substantially smaller body size than elephants, which they can gnaw (Haynes 1983; see also Figures S30–S36). To date, no carnivore has been documented as producing comparable fracture morphologies or surface damage on elephant bones. Consequently, the proposed analogy is not supported. Moreover, Haynes (1983) provides no empirical evidence that sediment pressure or trampling can generate hackled fracture surfaces. Such features are instead associated with dynamic loading conditions, whereas passive breakage processes have not been shown to produce these types of modifications. This reasoning also applies to impact flakes on elephant bones, which can only be produced by the sole modern agent documented to dynamically fracture green proboscidean long bones: humans.”

One of the biggest issues is that there is no quantitative data or images of the bone fracture features that the authors refer to as the main diagnostic criteria at EAK. The only figures that show EAK specimens (S21, S22, S23) illustrate general green-bone fracture morphology but none of the specific traits listed in the text. In contrast, clear examples of similar features come from other Olduvai assemblages, which may be misleading to readers if they mistakenly interpret those as images from EAK. The manuscript also states that these traits "co-occur," but it is not defined whether this refers to multiple features on the same fragment or within the broader assemblage. Without images or counts that document these traits on EAK fossils, readers cannot evaluate the strength of the interpretation. Including that information would substantially strengthen the manuscript.

The arguments were addressed in the general criteria criticized by the reviewer in his/her previous review encompassing all green broken elephant bones documented. If we restrict the arguments now to EAK, then suffice to rescue the arguments from the previous reply. Images (Figs S21-23) show the EAK broken specimens and clearly indicate their human agency by two factors: a) at least one of them is a long bone flake with overlapping scars (FS 23 is showing its medullary side), and b) elephant bones impacted by carnivores (namely, hyenas) have always shown intensive gnawing and tooth-marking; lack thereof in both EAK specimens refutes a non-human carnivore agency. The former argument is interpreted as human agency because carnivores have not documented to produce such features on elephant bones.

Regarding the statement that "natural elephant long limb breaks have been documented only in pre or peri-mortem stages when an elephant breaks a leg, and only in femora (Haynes et al., 2021)," it is not entirely clear what this example is intended to illustrate in relation to the EAK assemblage. My understanding is that the authors are suggesting that naturally produced green bone fractures in elephants are very limited, perhaps occurring only in pre or peri-mortem broken leg cases, and that fractures on other elements should therefore be attributed to hominin activity. If that is not the intended argument, I would encourage clarifying this point. This appears to conflate pre-mortem injury with the broader issue of equifinality. My original comment was not referring to pre-mortem breaks but to the range of natural (i.e., non-hominin) and post-mortem processes that can generate spiral or green bone fractures similar to those described by the authors.

We elaborated such argument addressing exclusively the reviewer´s previous argument that natural limb breaking produced spiral breaks on elephant long bones, which is correctly, as Haynes describes it, the only way not involving human agency that can generate a helicoidal spiral fracture on an elephant long bone. Non-human post-mortem processes on fresh bone do not generate these features. Neither have extant carnivores documented to produce these features on elephant bones.

Finally, in considering the authors' response on the Nyayanga material, I still find the basis for their dismissal of that evidence difficult to follow and the contrasting treatment of the Nyayanga and EAK evidence raises concerns about interpretive consistency. Plummer et al. (2023) specify that bone surface modifications were examined using low-power magnification (10×-40×) and strong light sources to identify modifications and that they attributed agency (e.g., hominin, carnivore) to modifications only after excluding possible alternatives. The rebuttal does not engage with the procedures reported. The existence of newer analytical techniques does not diminish the validity of long-standing methods that have been applied across many studies. It is also unclear why abrasion is presented as a more likely explanation than stone tool cutmarks. The authors dismiss the Nyayanga images as "blurry," but this is irrelevant to the interpretation, since the analysis was based on the fossils, not the photographs. The Nyayanga dataset is dismissed without a thorough engagement, while the EAK material, despite similar uncertainties and potential for alternative explanations, is treated as definitive.

We believe the rebuttal engages with these arguments. The protocol “bone surface modifications were examined using low-power magnification (10×-40×) and strong light sources to identify modifications and that they attributed agency (e.g., hominin, carnivore) to modifications only after excluding possible alternatives” does not guarantee that any derived interpretation is correct. These methods have consistently been used for decades now in contexts in which different researchers draw different conclusions on the same marks. The underlying variables used are subjectively interpreted and tallied, and equifinal when not considering overlapping factors, such as sediment abrasion and trampling. As an example, the same marks on the Nyayanga hippo bones interpreted by the original authors as cut marks, we see them undifferentiable from trampling marks from the image evidence published.

It is clear in the final version of our article that the EAK evidence is not treated as definitive, since that would be dogmatic, and thus, non-scientific. We thank this reviewer for having given us the chance to reconsider our original phrasing.

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Monitoring could be beneficial if it allows platforms to intervene and provide resources to users showing signs of crisis or self-harm. However, it becomes harmful if that sensitive psychological data is sold to advertisers or used to manipulate vulnerable users when they are at their lowest points.

Social media can be detrimental when I fall into the trap of doomscrolling or comparing my everyday life to someone else’s curated highlight reel, which often fuels anxiety. On the other hand, it has been a great way for finding community and staying connected with long-distance friends who provide genuine emotional support.

R0:

Reviewer #1:

Minor comments: 1. In lines 172 and 179, PCV13 is incorrectly written as “VCP-13” and “VPC-13” and should be corrected.

1. The abbreviations PCV-13 and PCV13 are both used in the manuscript. Authors should be consistent and use a single form.

2. The title of Figure 2 contains an error, it reads “stautt” instead of “status”.

3. The WHO reference used for the pneumonia case definition links to a French-language document. Authors should change this to a link to an English version of the document.

4. The endpoint of the Cox analysis is time to hospital discharge; however, the authors use different terms to describe this outcome (e.g. “recovery”, “released cured”, and “discharge”), consistent terminology for time to hospital discharge would improve clarity.

5. The study does not capture information on siblings and their vaccination status. As vaccinated siblings may provide indirect protection (on a household-level), this unmeasured factor could influence disease severity and time to hospital discharge and should be mentioned as a limitation.

Major comments: 7. Although a dose–response analysis is performed in a secondary linear regression analysis, the primary Cox model groups children with one or two PCV13 doses together with unvaccinated children. As children with one or two doses are likely to have some level of protection, this grouping may make the effect of full vaccination appear smaller. The authors may consider a sensitivity analysis using separate vaccination dose categories in the Cox model.

1. Please clarify whether repeat hospitalizations of the same child were possible during the study period and, if so, how this was handled in the analysis, repeat hospitalizations are often not random and could influence the estimated effect of vaccination.

2. As the study includes children from 3 different hospitals, it would be helpful to clarify whether admission practices and discharge criteria were comparable across sites, and whether differences were considered in the analysis, as these could influence time to hospital discharge.

R0:

Reviewer #1:

What are “cover clothes “ ? It’s interesting that the majority state that they would take their child to a clinic for symptoms of scabies . Does this include taking to children to traditional healers as well as a government or private medical clinic? I am trying to reconcile this finding with the section that shows support for traditional treatments rather than orthodox treatments. Likewise does this question “Seeking medical help for scabies is unnecessary” include traditional healers ? “Scabies treatment is too expensive for most families in our community” . It would be informative to explain this further in the discussion – roughly how much does the treatment cost in this setting It is interesting that mothers achieving a higher level of education were also amongst those with poorer practices. Have you any explanation for this ? Is there a role for the “expert” patient in prevention ?

Reviewer #2:

The manuscript meets the publication criteria for PLOS Global Public Health. It contributes to existing knowledge gaps, and the results align with and support the stated research objectives.

Regarding the methodology, the author presents the overall approach clearly; however, a few areas require clarification:

1. In the Materials and Methods of the manuscript, the author provided that all mothers of under-five children who consented to participate were included in the study. However, the sampling technique used is not clearly described. It is unclear whether the author employed simple random sampling, purposive sampling, or another approach. Clarifying the sampling method is important for understanding the representativeness of the study population and the generalizability of the findings. So, there is a need for clarity.

2. In the manuscript, under study variables, it states that participants 0–5 were classified as having poor knowledge and those scoring 6–9 as having good knowledge. However, the results table presents only item-level frequencies and percentages for the nine knowledge questions. While this shows awareness of individual items, it does not allow readers to determine overall knowledge levels. The table does not reflect the good/poor knowledge classification described in the methods, making it difficult to assess the overall knowledge gap on scabies. I recommend revising the table or adding a separate summary table that reports the proportion of participants with good versus poor knowledge to ensure clarity and consistency between the analysis and the results. I suggest the author extends this modification to attitude and practice.

For the statistical analysis section, it clearly presents frequencies and percentages and attempts to identify socio-demographic variables associated with KAP. However, additional clarification is needed regarding the analysis of the attitude score. In the manuscript, under the study variable, it states that attitude was measured using a 14-item Likert scale, with total scores of 0–8 categorized as negative attitude and 9–14 as positive attitude. Since Likert responses are non-dichotomous, it is unclear how individual item responses were coded to produce these totals. Clarifying the scoring method used to convert the Likert responses into these two attitude categories is necessary, as it may have influenced the study’s findings.

For the availability data underlying the findings in the manuscript, they were fully available and easy to follow.

Reviewer #3:

This paper examines the knowledge, attitudes, and preventative practices toward scabies infections among mothers (N = 320) of children under the age of five in Ibadan, Nigeria. The findings are nuanced and show that knowledge about the infection is mixed, and practices are generally poor. The results will be useful to conduct targeted educational campaigns to improve mothers’ knowledge, attitudes, and practices toward scabies infections in the region. Overall, I feel positive about this paper, but I have several issues that need to be addressed before publication.

Major points: - Attitudes scale: what is the meaning of “traditional” vs. “orthodox” treatments? Do participants know the difference? To me, they sound the same. - The Attitudes question about the effectiveness of traditional vs. orthodox treatments seems rather a knowledge question because it seems to have a clear True/False answer. - “Once a child has had scabies, they become immune to future infections.” This is a knowledge question with a clear True/False answer, not an Attitudes question. - Why are the parts of the questionnaire in the Appendix E and F not reported in the Results? The questions look interesting.

Minor points: English: - In the title (and throughout the paper): write “attitudes” instead of “attitude.” - Throughout the paper, instead of “Majority of the” write “Most.” - Whenever you report results, you need to put the numbers and percentages in parentheses. For example, “A large number of respondents (254, or 79.3%),” and so on.

Regression tables: - Instead of including the baseline category under the list of coefficients with a 0 next to it, remove each baseline category and instead write a note at the bottom of the table where you indicate the baseline category for each variable.

Academic Editor:

Thank you for sharing this interesting manuscript. Kindly carefully review suggestions from our reviewers. One of the main issues is the English and academic writing that should be revised throughout the manuscript. In addition, please provide point-by-point information to indicate your revisions per individual comments, including line numbers and pages.

We hope to receive your revision soon.

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eLife Assessment

This is an important study that identifies the developmental time window during which re-expression of TCF4 mutated in Pitt-Hopkins syndrome, can rescue phenotypic features of brain function in a TCF4 knockout mouse. The study presents compelling data using a viral transgenic intersection approach to show that TCF4 expression is required early in perinatal life. These findings have implications for the timing of possible gene therapy in people with Pitt-Hopkins-associated TCF4 mutations.

Reviewer #1 (Public review):

Summary:

This manuscript follows up previous work from this group using a conditional TCF4 mouse where Cre-expression turns "on" expression of TCF4 to investigate whether postnatal re-expression of TCF4 is effective to correct phenotypes related to Pitt-Hopkins Syndrome (PTHS) in humans. Results may inform gene therapy human PTHS gene therapy efforts on effective developmental windows for gene therapy. The authors demonstrate that re-expression of TCF4, induced by retro-orbital (RO) AAV-PHP.eB-Cre, during 2-4th postnatal week, does not rescue brain or body weight, anxiety-like or nest-building behaviors, but rescues an object location memory task, a measure of cognition. These results are novel and interesting in that they reveal distinct developmental roles for TCF4 in distinct behaviors and suggest that TCF4 plays a role in the mature brain in hippocampal and memory-related plasticity. Results may inform gene therapy design in PTHS.

Strengths:

The results are rigorous and high quality. Multiple methods are used to assess AAV-mediated re-expression of Cre, reactivation of TCF4, and the developmental time course of expression. Multiple behavioral phenotypes and molecular rescue are assessed. Most behavioral phenotypes are reproducible and robust, and it is clear whether a rescue was observed.

Weaknesses:

(1) Although the authors demonstrate the time course and spatial extent of Cre and a Cre-reporter (TdTom) in the brain with the AAV-Cre, it is unclear how many cells are transduced. Similarly, the authors do not measure TCF4 levels with immunohistochemistry or western blot. So the level of protein reactivation is unknown. A possible reason the rescue is incomplete is that the TCF4 protein is not induced in a large % of neurons in specific brain regions that mediate specific behaviors, such as the hippocampus vs. the striatum.

(2) The authors perform bulk qPCR to demonstrate a 20% increase in TCF4 RNA with Cre-mediated activation. It is unclear why the full gene reactivation is not observed. An alternative interpretation of the incomplete rescue of the phenotypes is that full TCF4 expression is required at later developmental time points.

Reviewer #2 (Public review):

Summary:

The basic helix-loop-helix transcription factor TCF4 (also known, as ITF2, SEF2, or E2-2) is a protein involved in the development and functioning of many different cell types. TCF4 plays important roles in the nervous system, both in health and disease. Its importance in the nervous system is underlined by its association with common and rare cognitive disorders. Specifically, variants of the TCF4 gene are implicated in increased susceptibility to schizophrenia, and mutations in the TCF4 gene cause Pitt-Hopkins syndrome (PTHS) or mild to moderate non-syndromic intellectual disability.

In this manuscript, the authors have studied whether reinstating TCF4 later in postnatal development in juvenile PTHS model mice could reverse behavioral phenotypes, thereby simulating gene therapy. Previous research by the same group has demonstrated that restoring TCF4 during embryonic or neonatal stages, corresponding to prenatal or neonatal periods in humans, improved phenotypes in a PTHS mouse model. In the current study, a conditional TCF4 reinstatement mouse model, Tcf4-lox-stop-lox (Tcf4-LSL), previously developed and characterized by their lab, where Cre-mediated recombination removes a floxed transcriptional stop cassette downstream of exon 17, leading to reinstatement of all TCF4 isoforms at appropriate levels in neurons, was used. The study showed that this later intervention failed to correct most phenotypes, suggesting that perinatal reinstatement of TCF4 holds the greatest potential to treat behavioral symptoms of PTHS. However, the study also suggests that some cognitive behaviors may still be responsive to TCF4 reinstatement later in life.

Strengths:

This is a very important study aimed at developing gene therapy for PTHS. The study is technically very well performed and written.

Weaknesses:

The only weakness is that a human disease is modelled in a mouse, which is evolutionarily not the closest mammal to humans. Hopefully, in the future, similar studies will also be performed in a nonhuman primate model, for example rhesus macaque.

Repérer et accompagner les vulnérabilités pour soutenir la persévérance scolaire : Document d'information

Résumé Exécutif

Ce document synthétise les enjeux de la vulnérabilité des élèves comme levier fondamental de la persévérance scolaire.

Loin d'être un simple concept sociologique, la vulnérabilité agit comme un « analyseur » permettant de comprendre la réalité vécue par les élèves, souvent masquée par des biais de désirabilité sociale dans les enquêtes officielles.

Le décrochage scolaire est présenté non comme un événement soudain, mais comme un processus multifactoriel où des vulnérabilités internes (personnelles, familiales) croisent des vulnérabilités scolaires (pédagogies, interactions).

Les points clés identifiés sont :

• L'écart entre perception et réalité : Alors que 92 % des élèves déclarent se sentir bien, des études approfondies révèlent qu'environ la moitié d'entre eux souffrent de mal-être (maux physiques, angoisse de l'évaluation).

• La vulnérabilité comme dénominateur commun : Les problématiques de violence, de harcèlement et de radicalisation sont des manifestations de vulnérabilités sous-jacentes.

• Le concept de « masque social » : Les élèves développent un « faux self » pour survivre à l'environnement scolaire, au prix d'une consommation d'énergie massive et d'un déni de soi.

• Le levier des Compétences Psychosociales (CPS) :

Le développement de l'autonomie et du bien-être passe par l'acquisition de compétences cognitives, émotionnelles et sociales, soutenues par une pédagogie de la bienveillance et de la réussite.

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1. La Réalité de la Vulnérabilité : Au-delà du Déni

L'analyse souligne un déni systémique de la vulnérabilité, masqué par des enquêtes nationales (ADEP) montrant un taux de bien-être de 92 à 94 %. Toutefois, les recherches en sciences sociales révèlent une réalité plus nuancée et inquiétante.

Données de recherche contrastées

| Source / Chercheur | Constat clé | | --- | --- | | Sébastien Rocher | Seuls 2/3 des élèves disent aimer l'école. | | Béatrice Mabillon Bonfils | 50 % des élèves de Première signalent un mal-être (maux de ventre, larmes, oppressions). | | Agnès Florin / Philippe Guimard | 2/3 des élèves ont peur d'avoir une mauvaise note. | | UNICEF | 45 à 55 % des élèves sont angoissés le matin à l'idée d'être évalués. | | Enquête de terrain | 1/6 des élèves (17,1 %) se trouve en situation de véritable souffrance. |

2. Le Processus de Décrochage et les Problématiques Sociales

Le décrochage n'est pas une fatalité mais une combinaison de facteurs singuliers (internes et externes au système scolaire).

• Facteurs Externes : Climat familial, environnement social, parcours d'immigration (processus particulièrement vulnérabilisant).

• Facteurs Internes : Rapport aux professeurs, relations entre pairs, sentiment d'injustice.

• Symptomatologie comportementale : Il convient de requalifier les « élèves perturbateurs » en élèves dont le « comportement est perturbé ».

Les incivilités, l'absentéisme et même la radicalisation sont à interpréter comme des symptômes de vulnérabilités intrafamiliales ou communautaires.

• Répartition géographique : Bien que les problématiques soient plus denses en éducation prioritaire, 74 % des élèves en grande difficulté sont répartis hors de ces zones.

3. Typologie Multidimensionnelle des Vulnérabilités

La vulnérabilité est définie comme une « blessure » touchant les besoins psychologiques fondamentaux. Elle se décline en plusieurs formes qui s'accumulent.

Les vulnérabilités de base

1. Physique et Sexuelle : Inclut le manque de sommeil, la malnutrition et les violences sexuelles (estimées à 1 élève sur 10, soit environ 3 par classe). Ces dernières peuvent mener à l'amnésie traumatique.

2. Psychologique et Affective : Menaces, humiliations, chantage, rejet ou manque de lien sécurisant à la maison (Violence Éducative Ordinaire - VEO).

3. Cognitive : Difficultés liées au jugement de valeur en classe, obstacles à l'apprentissage et au discernement.

Les vulnérabilités émergentes et sociétales

• Climatique (Éco-anxiété) : Inquiétude face à l'avenir de la planète.

• Économique : Impact de la pauvreté et de la précarité résidentielle.

• Numérique : Exposition à la cyberviolence et à la désinformation sur les réseaux sociaux.

• Médias : Sentiment de fragilité accru par la dramatisation médiatique des conflits mondiaux.

4. Le Masque Social et le "Faux Self"

Pour s'adapter à l'école, lieu décrit comme symboliquement et factuellement violent (classement, comparaison, pédagogie magistrale), l'élève adopte un « masque social ».

• Mécanisme de survie : Le masque (élève parfait, élève anesthésié, élève autonome à l'excès) permet de sauver les apparences mais empêche l'accomplissement authentique de la personne.

• Conséquences : Ce « faux self » est un grand consommateur d'énergie et peut entraîner un sentiment de ne jamais se réaliser, persistant jusqu'à l'âge adulte.

• Double peine : L'élève vulnérable qui n'est pas compris par l'adulte subit une stigmatisation supplémentaire, ce qui accroît son mal-être et bloque sa résilience.

5. Les Compétences Psychosociales (CPS) comme Solution

Les CPS sont définies par Santé Publique France comme un ensemble de capacités psychologiques permettant de maintenir un état de bien-être et de faire face aux difficultés de la vie.

Catégories de CPS

• Cognitives : Conscience de soi, contrôle des impulsions, prise de décisions constructives.

• Émotionnelles : Identification et gestion des émotions, capacité de « coping » (adaptation).

• Sociales : Communication positive, écoute empathique, résolution de conflits de manière prosociale.

L'Universalisme Proportionné : Cette approche consiste à proposer le développement des CPS à tous les élèves, tout en intensifiant l'accompagnement pour les plus fragiles.

6. Leviers pour la Persévérance Scolaire

Le document identifie des pratiques concrètes pour transformer la vulnérabilité en force.

Posture de l'adulte et climat scolaire

• Qualité relationnelle : L'enseignant doit être authentique, disponible et manifester une confiance sincère.

La relation « académique » suffit aux élèves favorisés, mais les plus fragiles ont besoin d'une relation humaine profonde.

• Cadrage bienveillant : Un environnement sécurisant où les problèmes sont discutés collectivement plutôt que niés.

• Reconnaissance des besoins fondamentaux : Sécurité, appartenance, justice et estime de soi.

Stratégies didactiques

• Pédagogie active et différenciée : Favoriser la réussite dans la zone proximale de développement pour restaurer l'estime de soi.

• Droit à l'erreur : Utiliser les feedbacks positifs centrés sur la tâche.

• Espaces de parole : Créer des lieux de dialogue authentique (ex: dispositif Prodas) où l'élève peut s'exprimer sans crainte du jugement de ses pairs (60 % des adolescents craignent la moquerie en allant voir un professionnel de l'établissement).

Formation des personnels

Le texte conclut sur la nécessité pour les adultes de travailler sur leurs propres compétences psychosociales et leurs propres masques sociaux.

La formation continue doit inclure des moments d'analyse de la vulnérabilité des enseignants pour améliorer la relation pédagogique et réduire les tensions en classe.

eLife Assessment

This study proposes a cross-species transcriptomic framework to predict vaccine reactogenicity, with implications for preclinical vaccine safety assessment. The findings show that mouse muscle transcriptomic signatures capture conserved inflammatory programs and can identify highly reactogenic formulations, with supportive but limited evidence for finer discrimination among licensed human vaccines. Overall, the work establishes a valuable foundation for translational biomarkers of reactogenicity, although the strength of evidence for broad cross-species predictive performance remains incomplete and would benefit from further validation.

Reviewer #1 (Public review):

Summary:

The authors aimed to develop a translational framework for predicting vaccine reactogenicity by training a penalized ordinal regression model on mouse muscle transcriptomics and applying it across tissues and species to rank human vaccines by their inflammatory potential.

Strengths:

The study addresses an important gap in preclinical vaccine safety assessment. The identification of IL6/JAK/STAT3 signaling as a key pathway implicated in reactogenicity is biologically plausible, and the observation of coordinated changes between muscle and blood compartments supports the biological relevance of the signature. The model achieves near-perfect classification in mouse muscle tissue and successfully identifies Fluad (MF59-adjuvanted) as the most reactogenic among licensed human vaccines, consistent with clinical safety data.

Weaknesses:

The methodological foundation has several concerns. The reactogenicity class definitions rely on PC1 scores with modest variance explained, yet no sensitivity analyses demonstrate robustness to different normalization strategies, feature selection approaches, or dimensionality reduction methods. I suggest performing sensitivity analyses demonstrating that reactogenicity class definitions are robust to alternative normalization methods, feature selection criteria, and dimensionality reduction approaches.

The combined mouse analysis reveals that tissue effects dominate over vaccine-induced variation, and no explicit batch or compartment correction was reported. The authors can apply batch/compartment correction (e.g., SVA) when analyzing combined mouse muscle and blood data, then recompute PCA and downstream analyses.

The central claim regarding cross-species ranking capability is not fully supported. In human blood, the model largely distinguishes Fluad from other vaccines but shows limited separation among non-Fluad formulations, with many pairwise comparisons yielding non-significant adjusted p-values. This pattern suggests the model may be tuned to detect large inflammatory magnitudes-likely a consequence of training on extreme stimuli such as LPS and whole-cell pertussis-rather than capturing the finer gradations relevant for distinguishing licensed vaccines with moderate reactogenicity profiles. I highly suggest retraining the model, excluding extreme stimuli (LPS, Pentavac), to evaluate whether mid-range separations among licensed vaccines can be recovered.

Impact:

While the conceptual framework is promising, the current evidence does not convincingly demonstrate that the model can rank vaccines beyond identifying highly inflammatory outliers. The utility for preclinical assessment of novel vaccine candidates with moderate reactogenicity profiles remains uncertain.

Reviewer #2 (Public review):

Summary:

The authors derived a time-specific signature of reactogenicity from mouse muscle following exposure to vaccines /TLRs for capturing the reactogenicity patterns. They tested this reactogenicity signature in mouse blood, and then they applied the reactogenicity signature to human blood from subjects having received different vaccines. They identified biomarkers in mouse muscle which are also observed in mouse and human blood and could be used as a reactogenicity signature in mice, instead of CRP.

Strengths:

(1) The authors used transcriptomic response following vaccination and used common genes to human and mice for defining a reactogenic signature.

(2) As the authors used different formulations in mice, the model was trained across a broad reactogenicity spectrum, which has the advantage of being used for evaluating new vaccines/vaccine platforms.

Weaknesses:

(1) The muscle gene signature reflects local reactogenicity. Systemic reactogenicity is not specifically addressed, except where overlapping gene signatures are observed for both local and systemic reactogenicity.

(2) In the same logic, could we find additional genes in the blood which are not captured in the muscle?

(3) The peak of the reactogenicity is usually 24h; it is not certain that additional TPs have helped the findings. If they have, the authors should explain.

eLife Assessment

The study presents a valuable finding on the ubiquitin-dependent regulatory loop in which proapoptotic Bim is targeted to the E3 ubiquitin ligase Cul5-Wsb2-mediated degradation through its sequestration by BCL2 proteins. The conclusions are supported by incomplete evidence and would benefit from additional experiments addressing both the mechanistic understanding and the physiological/cancer-related significance of the study.

Reviewer #1 (Public review):

This manuscript by Toczyski and colleagues explores the role of ubiquitin-dependent degradation in the co-regulation between pro- and anti-apoptotic proteins. The binding of the pro-apoptotic sensor Bim to BCL2 anti-apoptotic proteins sequesters it into inactive complexes, inhibiting BCL2 members but also preventing Bim from activating the apoptotic executors BAX and BAK. The authors now suggest that the E3 ubiquitin ligase Cul5-Wsb2 targets Bim turnover while in complex with BLC2 members. The authors reveal the importance of WSB2 in apoptosis of neuroblastoma cell lines, highlighting the importance of Wsb2 as a cancer biomarker. In sum, this study identifies Bim as a novel Wsb2 target and suggests a novel co-receptor mechanism using BCL-2 members as bridging factors, thus adding a novel mechanistic layer to the apoptosis repressor role of Wsb2. Their experimental approach is sound, and in most cases, the conclusions are justified. However, whether Cul5-Wsb2 targets Bim via BLC2 anti-apoptotic members would require further analysis.

Major comments:

(1) They find that Wsb2 or Cul5 downregulation increases the levels of Puma and Bim isoforms, and that Wsb2 strongly interacts with all Bim isoforms. Moreover, Wsb2 regulates Bim turnover, especially visible for Bim-EL, and controls Bim-L ubiquitylation. Finally, Figure 2E suggests that Wsb2-Bim interaction is bridged by Bcl-xL, and they identify the domain in Bcl-xL/Wsb2 responsible for their binding in Figure 4A-E. However, Figure 4F shows only a mild decrease between Bim-EL and HA-Wsb2EEE, which is inconsistent with their model. This important gap should be backed up by further experimental evidence. For example, by performing (a) coIP studies between Bim and Wsb2 in the presence of Bcl-xlAAA and (b) Bim stability and ubiquitylation analysis in the presence of either Bcl-xlAAA or Wsb2EEE.

(2) The manuscript lacks quantifications and statistical analysis in most figures, which are particularly important for Figure 1D - especially regarding the upregulation of Puma and Bim isoforms upon downregulation of Cul5 and Wsb2, for Fig 3A - also including statistical analyses of Bim1 stability in presence or absence of proteasomal inhibitors, and for Figure 4D, F, especially regarding the interaction of Bim-EL- with WT and mutant Bcl-xL in 4D and with WT and mutant Wsb2 in 4F.

(3) The localization of BCL2 family members at the mitochondrial outer membrane is a crucial step in the implementation of apoptosis, and BCL2 members recruit Bim to the OM. Despite their finding suggesting that Bim insertion into the OM might be dispensable for interaction with Bim, the interaction was abolished by BH3-mimetics that disrupt Bcl-xL interaction with BIM. This suggests that Wsb2 interacts with Bim at the mitochondrial surface. Therefore, it would be interesting to investigate the sub-cellular localization Bim and WSB2 with and without ABT-263.

(4) Wsb2 mildly interacts with Bcl-xL and with Mcl1, but does not interact with Bcl-w or Bcl2. However, they show that Wsb2 recognizes Bcl-xl through a motif conserved between Bcl-xl, Bcl-w and Bcl2. Therefore, it would be helpful to precipitate Bcl-w or Bcl2 and check interaction with Wsb2.

Reviewer #2 (Public review):

Summary

This manuscript proposes an original and conceptually interesting model in which anti-apoptotic BCL-2 family proteins, particularly BCL-XL and MCL-1, not only sequester BIM but also act as adaptor "co-receptors" that recruit BIM to the CUL5-WSB2 ubiquitin ligase complex for degradation. The authors present a mechanistic framework supported by structure-guided mutagenesis, BH3 mimetic perturbations and co-immunoprecipitation assays performed in RPE1 cells. In parallel, the study shows that neuroblastoma cell lines are highly dependent on WSB2 for survival. These observations give the work both conceptual and translational relevance.

Strengths

The principal strength of the study lies in its conceptual novelty. Reframing BCL-XL and MCL-1 not only as sequestration factors but also as adaptors that facilitate substrate engagement by an E3 ligase substantially extends current models of apoptotic regulation. The mechanistic narrative developed in RPE1 cells is clear and internally consistent: the combination of AlphaFold-guided motif identification with complementary mutagenesis provides a persuasive framework for how WSB2 associates with anti-apoptotic BCL-2 family members and promotes BIM turnover. The definition of a BCL-XL/MCL-1 co-receptor mechanism for WSB2-mediated BIM degradation is therefore both intuitive and mechanistically appealing. In parallel, the authors present a distinct experimental series showing that neuroblastoma cells exhibit pronounced sensitivity to WSB2 loss, undergo apoptosis upon its depletion and display reduced competitiveness in mixed-culture assays. Although the mechanistic connection between these observations requires further clarification, the convergence of a well-defined biochemical model with a clear cancer-relevant phenotype enhances the potential biological significance of WSB2 and raises the possibility that its regulation may hold therapeutic relevance.

Weaknesses

There are several limitations that readers should consider when interpreting the study. The most fundamental issue is the disconnect between the mechanistic model established in RPE1 cells and the apoptotic phenotype observed in neuroblastoma. Although the manuscript convincingly demonstrates the WSB2-BCL-XL/MCL-1-BIM axis in RPE1 cells and independently shows that WSB2 loss compromises neuroblastoma viability, it does not examine whether BIM levels are elevated upon WSB2 depletion in neuroblastoma, nor whether apoptosis in these cells requires BIM. Without demonstrating WSB2-BCL-2-BIM complex formation or BIM dependence in the disease-relevant context, it remains unclear whether the co-receptor mechanism characterised in RPE1 cells explains the phenotype. This gap is compounded by the observation that PUMA, another potent pro-apoptotic factor, also increases following WSB2 loss, raising the possibility that multiple death pathways contribute to the outcome. The absence of a genetic rescue experiment, such as re-expression of an shRNA-resistant WSB2 restoring viability and suppressing apoptosis, further limits causal inference regarding WSB2's role in neuroblastoma.

Many central claims rely on single Western blots and pulldown assays without quantification or assessment of reproducibility. This complicates the interpretation of CHX chase experiments (where initial steady-state levels differ between samples) and limits confidence in BH3 mimetic experiments, which use a single concentration and short exposure time. Without dose-response curves, time-course analyses, caspase inhibition, or orthogonal genetic perturbation of BCL-XL or MCL-1, indirect or off-target drug effects cannot be excluded. Reduced co-IP signals in these assays could therefore reflect early apoptotic events or compound instability rather than specific disruption of protein-protein interactions.

A further limitation concerns the inference of a direct WSB2-BCL-XL interaction. The mutagenesis analyses are performed in lysates that contain endogenous or overexpressed BIM, and BH3 mimetics disrupt the WSB2 interaction only when the BCL-XL-BIM heterodimer is dismantled. The study thus cannot distinguish whether the mapped WSB2 motifs mediate direct contact with BCL-XL or whether they influence the architecture or stability of the BCL-XL-BIM complex. Because no purified protein reconstitution or biophysical binding assays are presented, the evidence for direct binding remains suggestive rather than conclusive.

The ubiquitination data also remain incomplete. Although the WSB2 mutation reduces the ubiquitin smear on BIM, the assay does not demonstrate dependence on CUL5, RBX2 or ARIH2, leaving open which ligase components are directly responsible. MLN4924 implicates CRLs more broadly, but the ubiquitination assay itself does not assign activity to the CUL5-WSB2 module.

Finally, several methodological details are insufficiently described, including the generation and validation of the doxycycline-inducible WSB2 and HA-WSB2 lines and the suitability of the WSB2-overexpressing control line used in immunoprecipitations.

Collectively, these issues do not undermine the conceptual interest of the proposed co-receptor model, but they do limit the strength of the mechanistic claims and weaken the connection between the defined mechanism and the neuroblastoma phenotype.

eLife Assessment

This study is a valuable contribution that comprehensively identifies and characterizes LC3B-binding peptides through a bacterial cell-surface display screen covering approximately 500,000 human peptides. The data presented are solid, although this approach has limitations (e.g., it cannot assess the effects of post-translational modifications, which are often important for LIR-mediated interactions). Validation of the newly identified binding peptides by demonstrating their interactions with full-length proteins in cells would further strengthen this manuscript.

Reviewer #1 (Public review):

Summary:

This study uses high-throughput bacterial cell-surface display to identify LC3B-interacting peptides in the human proteome. The screen is unbiased, and this type of assay has not previously been used for selecting LC3B-interacting peptides. The screen was done with a library of 500,000 peptides, and they ended up with 427 peptides that they scored as high-confidence LC3B binders. The experiments performed are solid, and data are analyzed using well-documented methods and statistics.

The aim of the authors was to isolate LC3B-interacting peptides from the human proteome, and the screen succeeded in doing so. The selected set of peptides included several previously reported LIR motifs, but also many novel LC3B binding peptides that either contained or did not contain the canonical core LIR motif [WFY]xx[LVI].

Another aim was to identify binding determinants important for the LC3B interaction, and they made an interesting sequence logo based on selected LIR-containing peptides. However, this study does not really extend our knowledge related to binding determinants essential for LIR motifs in LC3B binding. They basically verify known characteristics, including the importance of varied types of electrostatic interactions supporting the docking of the core LIR into the LDS of LC3B.

Strengths:

The approach used here (high-throughput bacterial-surface-display) is new. The screen is unbiased, and the fact that peptides are directly tested for LC3B binding may facilitate the discovery of non-canonical LIR motifs. The screen appears to be highly selective and manages to distinguish between peptides that interact with LC3B and peptides that do not interact.

Weaknesses:

It is a limitation that no proteins are analyzed in this study. Further work is therefore needed to verify that identified LIR motifs are functional in full-length proteins and in cells.

Reviewer #2 (Public review):

Summary:

To discover peptides that interact with autophagy-related protein LC3B and profile the key binding determinants, the authors screened a library of ~500,000 36-residue peptides derived from the human proteome using bacterial cell-surface display. Analysis of the screening data revealed exceptions to the reported LIR motif and a strong preference for negatively charged residues adjacent to the LIR.<br /> These results support a refinement of the LIR motif definition and expand the network of candidate LC3B interaction partners.

Strengths:

High-throughput approach.

Weaknesses:

Lack of in vitro data and molecular dynamics simulations.

Reviewer #3 (Public review):

Summary:

The LC3 family of proteins, which includes LC3B, are ubiquitin-like proteins that are covalently linked to phosphatidylethanolamine in the expanding autophagosomal membrane during autophagy. LC3 family members bind to short sequences of amino acids that reside within dynamic regions in a wide variety of proteins. These sequences, termed LC3 Interacting Regions (LIRs), were initially thought to function primarily to link LIR-containing autophagy cargo receptors to LC3 family members to help facilitate their capture during autophagy. However, the functional importance of LIRs in autophagy has broadened to include more general functions in autophagy as well. While a general consensus for LIR sequences has been described as [FWY]0-X1-X2-[LVI]3, recent work has suggested that additional sequences outside of the canonical LIR sequence can bind LC3 family members and play important roles in autophagy. In this manuscript by Kosmatka et al, the authors perform a high-throughput screen using bacterial surface display coupled with fluorescence-associated cell sorting to identify which human sequences can bind to LC3B. They identify a variety of peptides capable of binding LC3B, including peptides from proteins that have not previously been described as LC3B-binding proteins. The results from the bacterial surface display were then used to guide sequence analysis, mutational analysis, and structural studies to further characterize the range of LIR sequences that are capable of binding LC3B. Taken together, this work adds to the growing knowledge of how LIR sequences interact with LC3 family members and demonstrates which amino acids both inside and outside of the LIR sequence aid in binding. This work also identifies new potential LC3 binding proteins, which may play unknown roles in autophagy regulation. Lastly, this work reinforces the importance of alternative LIR sequences such as the [WFY]0-X1-X2-[WFY]3 sequence, which the authors have dubbed the LIR+ sequence.

Strengths:

The manuscript uses a robust approach to identify and characterize different peptide sequences that can interact with LC3B. They validate a large number of sequences using biolayer interferometry (BLI) and attempt to correlate different amino acids with their binding affinity for LC3B. The large number of LC3B binding sequences and their dissociation constants adds significant new information to the field that will help others understand what sequences can bind to LC3B. The authors are also very careful to accurately report on their data and not overly interpret their findings.

Weaknesses:

After the authors identify proteins from their bacterial display assay, the remainder of the manuscript is focused on characterizing the different types of sequences that are identified in addition to validating the LC3B-LIR interactions using biochemical approaches, including BLI and X-ray crystallography. However, it's not entirely clear if the screen identified novel LC3B binders that interact with LC3B in cells. While I acknowledge that the focus of the manuscript is on the characterization of LIR sequences that can bind LC3B, it seems like a missed opportunity not to validate a few of the novel LC3B binders in vivo. This may result in the demonstration of novel binders of LC3B in cells and may further demonstrate the strength of this approach for identifying LC3 family member binding partners. Therefore, it would be helpful to look at a few proteins identified in the HC set that have not previously been identified as LC3B binders in cells to determine if they CO-IP with LC3B or interact with LC3B using a different approach.

P持有G的上下文，M运行G需通过P分配（同意）

G是groutine, P负责调度，P的数量与内核线程数相等？M又是什么

eLife Assessment

This important work substantially advances our understanding of the role of synaptotagmin-7 (Syt7) in short-term plasticity at cortical glutamatergic synapses. The evidence supporting the conclusions is convincing, with rigorous and elegant quantal-level iGluSnFR imaging and failure-based analyses at single boutons. The work will be of broad interest to synaptic physiologists and molecular biologists.

Reviewer #1 (Public review):

Kotzadimitriou et al. investigate how synaptotagmin-7 (syt7) contributes to short-term plasticity at cortical glutamatergic synapses. Using quantal-level iGluSnFR imaging and failure-based analyses at single boutons, the authors distinguish between synchronous and asynchronous glutamate release across boutons with differing baseline efficacy. They show that knocking out syt7 abolishes facilitation of synchronous release while leaving asynchronous facilitation largely intact, although reduced in magnitude. Furthermore, they argue that synchronous and asynchronous events arise from functionally distinct vesicle pools. The manuscript concludes that syt7 is essential for the facilitation of synchronous release, while other calcium sensors govern asynchronous release.

Strengths:

(1) The use of iGluSnFR provides a robust readout of single-synapse activity. Unlike traditional ephys methods that average the activity of thousands of synapses (which may mask the facilitation of low Pr synapses), the authors employ quantal imaging to analyze thousands of individual boutons and stratify them by efficacy. The representative images and traces in Figure 1 are of high quality, and the quantal analysis demonstrating multiple quantal peaks aligns well with previously published work (Mendonca et al., 2022; Wang et al., 2022).

(2) The failure-based analysis is thoughtfully implemented. By isolating trials in which no release occurred, the authors effectively separate facilitation from depletion, strengthening their central argument that syt7 is required for facilitation independent of vesicle depletion.

(3) The proposed model (depicted in Figure 7) is interesting and may reconcile the contradictory roles attributed to syt7, as described by others in the field. Specifically, the authors provide data to address syt7's potential function in facilitation, asynchronous release, and replenishment. However, to further support their model, which argues that "multiple Ca2+ sensors have both unique and overlapping roles in regulating synaptic plasticity," additional experiments are needed (see point 2 below).

Weaknesses:

(1) While the authors use cultures from syt7 knockout mice (and wild-type controls), there are no acute rescue experiments (e.g., syt7 viral transduction in KO cultures) or checks for compensatory changes in other proteins. Previous studies (Bacaj et al., 2013; Jackman et al., 2016) have utilized viral rescues to confirm specificity. Without such experiments, it remains theoretically possible that the chronic loss of syt7 leads to downregulation of another protein essential for facilitation. At a minimum, the authors should perform rescue experiments for at least some of their findings. Additionally, western blots for syt1 and syt7 should be conducted to confirm that their knockout is specific to syt7.

(2) The manuscript acknowledges the possible roles of Doc2a and syt3 but fails to address them experimentally. Recent work (Wu et al., 2024; Weingarten et al., 2024) has identified Doc2a as the primary sensor for asynchronous release. Even if its expression in cortical cultures remains unconfirmed (as claimed by the authors), they should, at the very least, perform Western blots for Doc2a and syt3 in both wild-type (to determine basal expression levels) and syt7 knockout cultures. Without analyzing the levels of these proteins, the mechanism/model behind the "remaining" asynchronous release remains speculative. Is it possible that these other calcium sensors are upregulated in their syt7 KO cultures and could instead explain their results?

Reviewer #2 (Public review):

Summary:

In this elegant study, the authors employ live iGluSnFR-based imaging of glutamate release from cortical boutons to dissect the distinct roles of the Ca²⁺ sensor synaptotagmin-7 (Syt7) in synaptic transmission. Although multiple functions have been attributed to Syt7 over the years, the field remains conflicted. The authors argue that one major obstacle for resolving some of these discrepancies lies in a fundamental limitation of electrophysiological recordings, which aggregate signals across all synapses to yield averaged readouts, dominated by strong, high-release-probability synapses. By using a live glutamate imaging approach combined with sensitive detection of action potential-evoked activity across different stimulation regimes, and a dedicated analysis pipeline, the authors confirm a role for Syt7 in facilitating synchronous release and in regulating the magnitude of asynchronous release. In contrast, they find no evidence that Syt7 contributes to the facilitation of asynchronous release, do not find evidence for a role for Syt7 in synaptic vesicle replenishment during AP trains, and provide evidence suggesting that the maintenance of facilitation by Syt7 may occur independently of vesicle depletion.

Strengths:

This study offers a fresh perspective on a debated issue, using a new experimental approach that the authors previously explored in the context of Synaptotagmin 1 (Mendonca et al. 2022). The authors record the response to a series of pair-pulse stimulations, followed by an AP train. By carefully quantifying individual events and by sorting events based on their efficacy, the authors extract quantitative information that they assign to different properties of synaptic function. They also devised an interesting approach for monitoring aspects of facilitation, in which they isolate PPR events where the first response did not elicit detectable release (thus regarding the release in response to the second AP as facilitating), and compare them with successful events. Together, the authors provide semi-quantitative descriptions of synchronous and asynchronous release during single, paired, and AP trains, yielding a weighted estimate of Syt7's contribution to distinct features of synaptic vesicle release that are independent of postsynaptic readouts. A major strength of the study is the confirmation of two principal proposed functions of Syt7: facilitation of synchronous release and regulation of the magnitude of asynchronous release.

Weaknesses:

The experimental approach presented here is elegant and well-executed. However, a principal limitation lies in translating electrophysiological terminology to imaging-based measurements. For instance, interpreting signals persisting beyond 10 ms as a proxy for asynchronous release relies on assumptions that would be good to experimentally justify. Could such signals arise from iGluSnFR saturation, or be affected by desensitization?. Moreover, the quantification of asynchronous release is based on very small signals that represent only a fraction of the already small synchronous release component, raising concerns about signal-to-noise limitations. A key issue is that failures to evoke glutamate release may arise from AP failures, such that the second response in a PPR does not necessarily represent facilitation. Given that many of the findings largely confirm existing literature, the study might have benefited from a different framing, for example, as an additional validation of the correspondence between electrophysiological measures and the authors' imaging-based readouts. Another point concerns the analysis of synaptic vesicle replenishment following depletion, which would ideally be addressed using alternative stimulation protocols, such as quantifying the response/success rate to single APs at varying time points after a train. Although the authors are appropriately cautious in their conclusions (e.g., with respect to Figure 5b), this limitation remains. Finally, the use of heterogeneous cortical neuronal cultures is likely to introduce substantial variability, as the authors themselves acknowledge, which may arise from the co-expression of multiple Ca²⁺ sensors across diverse cell types.

In summary, the authors were able to confirm previously-described changes in neurotransmission properties upon the loss of Syt7 using live imaging of glutamate release at the level of single boutons. They also present preliminary evidence for the interdependence of Syt7 function, synaptic vesicle replenishment, and the facilitation of asynchronous release, although these results will need to be substantiated in future studies using alternative stimulation protocols and complementary methodologies. Taken together with the group's prior work on synaptotagmin-1, this study illustrates that live imaging of glutamate release offers an alternative approach that recapitulates some elements detectable via electrophysiological analysis, while possibly revealing new insights into the function of synaptic proteins. As a whole, taking a live imaging approach may be a broadly accessible way forward to analyze synaptic function. The potential of studying synaptic proteins in diverse cell types that are difficult to access with patch-clamp electrophysiology is particularly compelling.

Reviewer #3 (Public review):

In this manuscript, the authors examine the role of Syt7 in the plasticity of synchronous and asynchronous release in cultured neurons. The experimental approach is the imaging of SF-iGluSnFR.A184V expressed in cultured neurons while delivering stimulation through whole-cell patch clamping of single neurons in the culture. In this manner, they could examine the optical signature of glutamate release in single presynaptic terminals, while separating the release events into synchronous (<10ms) and asynchronous (>10ms) while delivering both paired pulses or trains of stimuli (at 20 Hz, 50 ms between stimuli).

This manuscript employs techniques previously reported by the research group in their Mendoca et al., Nat Comms 2022 paper. This paper uses this approach to specifically examine the role of Syt7. The use of iGluSnFR in this manner provides significant rigor to the paper. The most significant weakness is that some of the events the authors discuss in this manuscript are rare, and the strength of the conclusions regarding those is somewhat unclear.

The main novel contribution of this manuscript is that single-bouton high-frequency imaging allowed them to examine paired-pulse plasticity in boutons that had not released neurotransmitter during the first pulse (failure-based analysis), thus separating between the effects of vesicle depletion and facilitation of the release machinery. This approach also allowed them to segregate their observations according to bouton-specific release efficacy. Both examinations are unavailable when performing cell-level analysis of neurotransmitter release, as is reported by most electrophysiological approaches.

The authors conclude that Syt7 contributes specifically to facilitation of synchronous release, not asynchronous release, while reducing the magnitude of the asynchronous component. Finally, the authors suggest segregation of synchronous and asynchronous release, either by differential use of calcium sensors or spatial segregation of the vesicles contributing to both modes of release.

This report contributes significantly to the discussion of the control of synaptic plasticity by different molecular players. It is not the first to examine Syt7, but its contribution to the examination of this protein is significant.

I find this report to be well executed and reasoned. In my opinion, the authors could improve the manuscript by clarifying the description of several methodological and experimental sections. Furthermore, in my opinion, some of the conclusions are overstated.

The authors state: "Because boutons along a single axon originate from the same presynaptic neuron, they are expected to share broadly similar molecular components of the vesicular release machinery and experience comparable presynaptic action potential waveforms." The authors should address the idea that presynaptic terminals from the same neuron on different postsynaptic targets can differ in the molecular components, as well as in the presynaptic side. There is ample evidence for differences between synapses onto glutamatergic and GABAergic neurons of the same neuron.

The authors used 4ms-long frames, but the stimuli are delivered at 20Hz (50ms apart). Therefore, in paired pulse stimulation, isn't there going to be a difference between the first and second stimuli regarding the timing of the frames relative to the stimulus? Isn't the deconvolution sensitive to such an offset?

A 10ms threshold for defining synchronous vs. asynchronous release full in-between frames. Doesn't this increase the chance of assigning borderline events to the wrong category?

On page 11 of the conclusion, the authors state that "Our data indicate that in our conditions during paired-pulse protocol Syt7 primarily enhances release probability rather than increasing the RRP size." While I understand the reasoning behind this statement, it should be toned down. The authors did not directly address the RRP size.

In failure-based analysis, the number of failure events in high-efficiency boutons is expected to be low. How does this affect the conclusions of the authors concerning the effects of Syt7 deletion on facilitation in high-efficiency boutons?<br /> SourceData.xlsx was not available to me, as far as I could tell.

How can the conclusions of the authors on the differential molecular composition of vesicles contributing to synchronous and asynchronous release be related to the reported effect of strontium on the nature of release? (see 10.1523/JNEUROSCI.20-12-04414.2000)

Is this the first use of failure-based analysis? If not, the authors should cite precedents. In 10.1016/s0896-6273(00)80338-4, failure of release during the 1st AP was presented, with facilitation during the 2nd, although no formal analysis was performed.

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