symétrie – asymétrie
Je m'interroge sur le choix de la graphie. Y a-t-il une raison pour laquelle on le note « symétrie - asymétrie » plutôt qu'avec une barre oblique?
symétrie – asymétrie
Je m'interroge sur le choix de la graphie. Y a-t-il une raison pour laquelle on le note « symétrie - asymétrie » plutôt qu'avec une barre oblique?
Document de Synthèse : Lancement du Cours en Ligne "Qui S'en Soucie ?" : Un Guide ARAO pour le Soutien à la Santé Mentale des Jeunes
Introduction
1. Contexte et Objectifs du Projet
La Fondation Canadienne des Femmes est un leader national dans le mouvement pour l'égalité des genres au Canada, œuvrant depuis 1991 au changement systémique.
Le projet "Qui S'en Soucie ?" est le résultat d'un financement de la Fondation Tree of Life, visant à créer un outil utile pour les bénéficiaires du Fonds pour les Filles (Girls Fund) de la FCF.
2. Phase de Recherche : Constats et Recommandations Clés
La phase de recherche a été menée par Taylor Newberry Consulting, combinant une revue de littérature et des consultations (entretiens et groupes de discussion) avec des jeunes et des organisations jeunesse.
L'objectif était de comprendre les défis rencontrés par les jeunes racisé.e.s et de diverses identités de genre dans l'accès aux services de santé mentale, ainsi que le rôle des organisations jeunesse.
3. Phases de Conception et de Pilotage du Cours en Ligne
Le développement du cours a été un processus intensif de 10 mois, impliquant New Room et Pure and Applied, ainsi que les bénéficiaires du Fonds pour les Filles.
4. Public Cible et Application Pratique
Le cours est principalement destiné aux travailleurs jeunesse, mais sa portée est plus large :
5. Représentation et Perspectives Futures
La question de la représentation au sein des présentateurs a été soulevée et adressée.
Bien que les présentatrices du webinaire soient toutes blanches, l'équipe de développement du cours était diverse, incluant la partenaire d'affaires d'Erin Willlet, Danny Joe, qui est noire.
Les intervenantes ont reconnu l'importance de cette préoccupation et ont remercié la participante de l'avoir soulevée, promettant de faire mieux à l'avenir en termes de représentation lors des présentations publiques.
En conclusion, le cours "Qui S'en Soucie ?" est une ressource complète et soigneusement élaborée, ancrée dans la recherche et la co-création, visant à renforcer la capacité des travailleurs jeunesse à soutenir la santé mentale des jeunes marginalisés à travers une lentille antiraciste, anti-oppressive et axée sur la communauté.
Compte rendu : Le Recueil de la Parole des Enfants Victimes et le Défi des Enquêtes sur les Agressions Sexuelles
Ce document examine les méthodes et les défis rencontrés par la police, en particulier la brigade des mineurs, lors du recueil de la parole des enfants victimes de violences, notamment sexuelles.
Il met en lumière l'évolution des pratiques d'audition, l'importance des environnements adaptés et la complexité de l'enquête judiciaire face à des témoignages d'enfants.
1. L'Évolution du Recueil de la Parole des Enfants : Les "Salles Mélanie"
Historiquement, l'audition des enfants victimes se faisait dans des "bureaux ordinaires, des endroits où les conditions de confort et d'écoute n'étaient pas réunies pour avoir leur témoignage souvent si douloureux."
Conscients de cette lacune, de nouvelles approches ont été mises en place, notamment la création de "salles Mélanie".
L'objectif est de créer un environnement qui "ressemble à une école, ça ressemble à ce qu'ils ont d'habitude, ça ressemble à une chambre d'enfance, ça ressemble pas à un bureau", afin de les "sécuriser" et qu'ils se "sentent en sécurité ici."
Les poupées anatomiques, par exemple, sont essentielles : "Il y a un garçon et une fille et on peut complètement les déshabiller de des chaussures jusqu'au vêtements...
Ce qui fait que ça permet à l'enfant de matérialiser s'il souhaite pas parler, ben finalement il peut nous expliquer à travers les les poupons en fait."
Des dessins ou l'utilisation d'objets comme des stylos peuvent également aider l'enfant à "montrer ce qu'il a demandé de faire".
Un policier souligne que l'enfant "va révéler le traumatisme qu'il a pu vivre."
2. La Spécificité de l'Audition des Mineurs
L'audition d'un enfant diffère grandement de celle d'un adulte ou même d'un adolescent, nécessitant des compétences et des approches spécifiques.
Ils peuvent bloquer, avoir peur, ou manquer de vocabulaire pour exprimer ce qu'ils ont vécu.
Certains "font que des signes de la tête qui veulent pas parler mais par contre qui vont nous répondre à travers ce gestuel surtout les petits parce qu'ils ont peut-être moins de vocabulaire moins la facilité de s'exprimer."
Auparavant, les questions étaient "dirigées" ("il a mis son zizi dans ton sexe"), ce qui pouvait influencer l'enfant.
Désormais, la technique consiste à aborder le sujet "du général" et d'y aller "petit à petit", pour que l'enfant "puisse dire ce qui s'est passé exactement mais de façon moins brutale et moins l'influencer aussi."
L'enquêteur doit s'adapter au "terme" propre à l'enfant pour désigner les parties intimes ou les actes.
La Vérité et le Mensonge : Une règle fondamentale est de "dire que la vérité et puis jamais de mensonge."
L'enfant est invité à donner un exemple de mensonge, comme "Il m'a rien fait" si un problème est survenu.
Cependant, la déstabilisation de l'enquêteur face à un enfant qui ne parle pas révèle la complexité de cette approche.
3. La Charge Émotionnelle et Psychologique des Enquêteurs
Le travail à la brigade des mineurs est humainement très exigeant et impacte profondément les policiers.
Un enquêteur confie que cela "nous bouleverse", car ils ne sont "pas dénué du tout de sentiments.
Bien au contraire." Ils doivent cependant "essayer de mettre de la distance parce que sinon c'est trop ça serait trop compliqué au quotidien."
Le fait d'en "parler aussi, c'est aussi une façon de de se libérer quoi, de passer à autre chose, de pas garder tout pour nous."
L'humour peut parfois être utilisé comme "une façon de détourner en fait."
Certains dossiers restent cependant "ancrés dans un coin du cerveau et c'est vrai que parfois c'est difficile de faire le vide en rentrant à la maison."
Donc c'est vrai que c'est important de pouvoir l'aider à notre niveau si on si on peut essayer de comprendre et puis de lui trouver une solution."
4. Le Processus Judiciaire et ses Limites
Le recueil de la parole de l'enfant n'est que le début d'un processus judiciaire complexe où la preuve est primordiale.
"C'est difficile de dire au départ si la parole de l'enfant est crédible ou pas."
L'enquête vise à "vérifier les paroles que l'enfant a pu dénoncer à travers des auditions de témoin, à travers des examens médicaux, à travers des choses comme ça.
Parce que justement, il faut bah il faut étayer les paroles de l'enfant."
L'Absence de Preuves Matérielles : L'un des cas présentés illustre la difficulté lorsque le mis en cause "nie la totalité des faits" et qu'il n'y a "aucun autre élément matériel" pour corroborer la version de l'enfant, y compris le certificat médical. Dans ce cas, "c'est une parole, contre une autre" et "on n'a rien de plus."
Le Classement Sans Suite : Face à l'insuffisance de preuves, le parquet peut décider un "classement 21" (classement sans suite).
Cette décision est une source de "frustration" pour les enquêteurs car, même si cela ne signifie pas que la parole de l'enfant est mise en doute, "on n'a pas assez d'éléments pour vous confronter." C'est une application du principe "le doute profite à l'accusé."
L'exemple de Zoé, toujours placée en famille d'accueil et ne vivant plus avec son beau-père, montre que des mesures de protection sont prises indépendamment de l'issue judiciaire.
En conclusion, si la création des salles Mélanie et l'évolution des techniques d'audition représentent une avancée majeure pour mieux recueillir la parole des enfants victimes, la phase d'enquête et la confrontation aux limites de la preuve matérielle restent un défi constant pour la justice et un lourd fardeau pour les enquêteurs.
Document d'information détaillé : Violence des mineurs et culture du couteau en France
Ce document analyse les thèmes principaux, les faits marquants et les idées essentielles concernant l'augmentation de la violence chez les mineurs en France, en particulier l'usage des armes blanches.
Il s'appuie sur les extraits de l'émission "Envoyé spécial - Coups de couteaux chez les ados".
Thèmes principaux
Faits et idées les plus importants
Le père de la victime témoigne de l'horreur : "Je jamais vu ça. Même moi quand j'allais quand j'étais jeune et j'allais ici à l'école non je suis bagarré à coup de point et tout.
Bon après le lendemain j'étais réglé on était pote. C'est bon mais jamais coup de de couteau."
Prévalence du port de couteaux :
Il ajoute : "Si le mec il a un couteau, je préfère moi aussi en avoir un. Je préfère le planter qui me plante. C'est logique ça."
Il dissimule son couteau "dans les parties intimes soit dans la sacoche soit là je le mets dans la chaussette".
Les couteaux Opinel sont "le couteau à la mode.
C'est plus facile d'acheter un couteau que d'acheter un filet au fich à Mcdo. Mettre moins de temps." * Chiffres alarmants : "Rien qu'en 2024, 10400 agressions à l'arme blanche ont été perpétrées en France." Cependant, il est "difficile de savoir précisément combien concernent les mineurs. Faute de statistique." * Facilité d'acquisition des couteaux pour les mineurs : * La loi est claire : "Un couteau à la fixe comme un couteau de bouché, un poignard ou un cran d'arrêt sont des armes de catégorie D.
Leur détention et leur transport sont formellement interdits aux mineurs." * Pourtant, un adolescent de 17 ans a pu acheter un couteau dans un supermarché discount et une quincaillerie sans vérification d'âge.
Aucune vérification de l'âge ou de l'identité de l'acheteur n'est effectuée. 48 heures plus tard, nous recevons le couteau à notre domicile. Bref, acheter un couteau, rien de plus facile pour un mineur."
Causes et facteurs aggravants :
"L'adolescent agressé serait une victime de ce que les bandes appellent un match retour."
Ces drames font le tour des réseaux sociaux et la une des journaux."
Les jeunes se donnent rendez-vous pour se battre via des applications comme Snap, et les vidéos d'agressions sont partagées sur des boucles cryptées (Telegram) : "Avec les outils numériques, l'embrouille s'accélère."
C'est une compétition symbolique."
La rue offre "des gratifications immédiates et des gratifications puissantes" aux jeunes en échec scolaire ou sans emploi. * Musique : La "culture du couteau est présente dans de nombreux morceaux de rap comme celui du chanteur Jul paru il y a quelques années. dans lequel il fait l'apologie d'un célèbre couteau avec son manche en bois."
Réponse de la justice et de la prévention : * Contrôles policiers : Les forces de l'ordre effectuent des contrôles de sacs aux abords des lycées. "94 armes blanches ont été saisies à l'occasion de près de 1000 opérations de contrôle entre les mois de mars et d'avril."
Ces actions visent la "prévention" et "une présence dissuasive", mais sont limitées car les policiers "n'ont pas le droit d'effectuer des fouilles au corps".
Elle insiste sur le rôle "nouveau" et problématique des réseaux sociaux.
"À partir de 13 ans, on est responsable pénalement. Vous avez l'âge d'aller en prison."
"nous avons 200 mesures éducatives ou mesures de contrôle judiciaire qui ont été confiées à la protection judiciaire de la jeunesse et qui ne sont pas exécutées faute d'effectif éducatif."
On a une proportion de jeunes qui sont en échec scolaire et en désertion scolaire, en décrochage scolaire qui est extrêmement importante et les difficultés familiales globales".
Actions de sensibilisation :
Il fait de la prévention dans les collèges et lycées, soulignant les conséquences de la violence : "les embrouilles s'amèent à deux choses, voire trois. La mort, la prison, le handicap."
Il insiste : "arrêter de marcher avec des couteaux. On se protège pas avec un couteau. [...] Quand tu marches avec un couteau dans la poche, tu marches avec l'arme du crime."
"Ils apprennent à se défendre, à se débrouiller surtout dans la vie au quotidien parce que ce sont des situations qui se passent au quotidien."
Conclusion
Le reportage brosse un tableau préoccupant de la violence à l'arme blanche chez les mineurs en France.
Il met en lumière non seulement la gravité des actes et leurs conséquences dramatiques, mais aussi la complexité des facteurs sous-jacents (rivalités de quartiers, influence des réseaux sociaux, facilité d'accès aux armes, quête de réputation).
Si la justice des mineurs se veut plus rapide et non laxiste, elle est freinée par un manque criant de moyens pour l'application des mesures éducatives et un système de prévention en amont qui semble défaillant.
Les initiatives individuelles de sensibilisation et de self-défense tentent de combler ces lacunes face à un phénomène qui bouleverse les familles et les communautés éducatives.
Note d'information détaillée : Le marché du contenu pour adultes en ligne (OnlyFans & MYM)
Thèmes principaux et idées clés :
Ce document résume les informations clés concernant l'évolution du marché de la pornographie et de l'érotisme, avec une focalisation sur les plateformes en ligne comme OnlyFans et MYM.
Il explore les motivations des créateurs de contenu, le modèle économique de ces plateformes, leurs dérives (notamment la présence de mineurs et le rôle des agents), et le vide juridique actuel en France.
1. La transformation du marché du contenu pour adultes : De la rue à internet.
2. Le modèle économique des plateformes (OnlyFans et MYM).
3. Les motivations des créateurs de contenu.
4. Les dérives du marché : Mineurs et manque de régulation.
5. Le rôle croissant des "managers fan" et ses dérives.
L'avocat Raphaël Molina évoque le "proxénétisme 2.0" mais souligne l'absence de "relation physique" nécessaire pour la qualification de proxénétisme actuel.
6. Perspectives de régulation.
Idées de régulation :
Mettre en place une prévention et une sensibilisation "auprès des jeunes et auprès des parents sur les risques" liés à cette activité.
Conscience des parlementaires : Une commission d'enquête parlementaire a commencé à s'intéresser au sujet, montrant une prise de conscience, même si les députés n'étaient "pas du tout" au courant du fonctionnement de ces plateformes au départ.
Synthèse du Briefing : PFAS - La Grande Intox de l'Industrie
Il met en lumière la dangerosité de ces molécules, les efforts de l'industrie pour dissimuler leurs effets et retarder la réglementation, et les conséquences sanitaires et environnementales.
1. Les PFAS : Molécules "Miracles" devenues "Polluants Éternels"
Les PFAS sont des molécules chimiques synthétiques, plus de 10 000 recensées, créées dans les années 1930.
Elles ont révolutionné de nombreux secteurs grâce à leurs propriétés uniques : "ultra résistantes à la chaleur, imperméables à l'eau." Initialement utilisées pour la fabrication de la bombe atomique, leurs applications industrielles se sont multipliées : "poêles anti-adhésives, batteries électriques, vêtements imperméables, cosmétiques."
Le problème majeur des PFAS est leur persistance : "Indestructibles, ils s'accumulent dans l'environnement. On les appelle les polluants éternels."
On les retrouve "dans le corps des gens, dans la faune et la flore, dans l'air, dans l'eau. Toute notre planète est contaminée."
2. Dangers pour la Santé et l'Environnement
L'enquête révèle que la dangerosité de certaines de ces substances est connue depuis des décennies par les industriels, qui auraient "dissimulé pendant très longtemps ce qu'ils savaient sur ces molécules."
Le PFOA (acide perfluorooctanoïque), un PFAS particulièrement nocif utilisé par Tefal jusqu'en 2012, a été classé "cancérogène pour l'homme en 2023" par le Centre international de recherche sur le cancer (OMS).
L'exposition aux PFAS est associée à de nombreux effets sur la santé :
Cholestérol et diabète.
Un ancien salarié de Tefal témoigne de la détérioration de son foie avec des enzymes hépatiques "jusqu'à deux fois supérieures à la normale."
3. Dissimulation et Stratégies Industrielles
L'enquête dénonce les décennies d'efforts des industriels pour "tromper les pouvoirs publics, tromper la communauté scientifique à propos de leur impact réel sur la santé."
Tefal/Groupe Seb à Rumilly (Haute-Savoie) :
Pourtant, un email confidentiel de 2024 évoque des "campagnes de surveillance biologique" montrant une "corrélation entre la suppression du PFOA dans les matières premières et la diminution des valeurs biologiques mesurées," suggérant une présence antérieure dans le sang des ouvriers.
Les habitants ont bu de l'eau contaminée pendant des décennies, certains affichant des taux de PFOA "cinq fois plus que la moyenne française" dans le sang.
Arkema (Sud de Lyon) :
Un journaliste dénonce : "Ce sont des industriels qui fabriquent en permanence ce doute et qui n'ont pas peur de tronquer leur rapport."
4. Lobbying et Influence Politique
Les industriels déploient des efforts considérables pour "empêcher leur interdiction" et "retarder, retarder, retarder pour repousser le plus loin possible toute réglementation qui pourrait affecter ses produits, ses procédés et au final ses profits."
L'entreprise a fourni aux parlementaires des "éléments de langage" défendant les fluoropolymères, un type de PFAS, comme étant "peu préoccupant aux impacts insignifiants sur la santé et sur l'environnement" et "incapable de traverser la barrière gastro-intestinale."
L'OCDE réfute avoir "finalisé de telles conclusions," et le directeur de recherche au CNRS, Pierre Labadi, déclare que cette présentation est "tronquée" car "le caractère extrêmement persistant de l'ensemble d'épiface" est "avéré."
Résultat du lobbying : Les ustensiles de cuisine ont été "exclus du champ d'application de la loi" en première lecture, un député écologique dénonçant une "grosse pression au niveau de Ber."
Lobbying européen : Au niveau européen, l'industrie a inondé les autorités de "milliers de mails d'études socio-économiques, de rapports scientifiques" avec les mêmes arguments.
5. Coût de la Dépollution et Responsabilité
La dépollution des sites contaminés est un défi "titanesque."
Le coût estimé de la dépollution de toute l'Europe est de "100 milliards d'euros par an, plus de la moitié du budget annuel de l'Union européenne."
Conclusion
Le scandale des PFAS est symptomatique d'une industrie chimique qui a sciemment dissimulé les dangers de ses produits pendant des décennies, privilégiant les profits.
Les conséquences sanitaires et environnementales sont massives et durables, avec un coût de dépollution faramineux qui soulève la question de la responsabilité des industriels.
La bataille pour une réglementation stricte et l'indemnisation des victimes est loin d'être terminée, face à un lobbying industriel puissant.
Comme le souligne un militant, "Chaque mois, chaque année qui passe, ça se compte en vie humaine.
Donc c'est absolument inacceptable."
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Dossier d'information détaillé : Mieux dans ma tête - Parlons santé mentale
Introduction
Ce dossier vise à synthétiser les thèmes principaux et les informations cruciales abordées lors de l'émission "Mieux dans ma tête - Parlons santé mentale".
L'objectif central de cette émission est de libérer la parole autour de la santé mentale et de démystifier les troubles psychiques, souvent entourés de tabous et de stigmatisation en France.
Comme le souligne Eddie Riner, parrain de cette initiative, "La santé mentale, c'est une chose dont tout le monde devrait [se] soucier en France."
1. La Santé Mentale, un Sujet Universel et encore Tabou
Une préoccupation pour tous : L'émission insiste sur le fait que la santé mentale concerne chacun d'entre nous.
"Au cours de notre vie, nous allons tous connaître des moments où notre santé mentale sera altérée. Ça peut commencer avec un simple stress, mais aussi des tâches quotidiennes qui nous dépassent avec la fameuse charge mentale."
Les troubles psychiques : Au-delà du stress quotidien, des troubles plus complexes comme la dépression, la bipolarité et la schizophrénie sont évoqués, pour lesquels "on ose moins parler".
Le tabou persistant : Un sondage Ipsos révèle que "près d'un Français sur deux" trouve qu'il est difficile de parler de santé mentale, démontrant à quel point le sujet reste un tabou dans le pays.
2. Démystification et Lutte contre la Stigmatisation
L'émission met en lumière des témoignages poignants pour changer la perception des troubles psychiques.
"Au début de la maladie, j'associais la schizophrénie avec une forme de débilité mentale. C'est-à-dire que je m'interdisais de vivre puisque je me dis... de toutes les façons c'était vrai l'échec puisque j'étais plus bête que la moyenne."
"Absolument. Ça n'a rien à voir avec l'intelligence en fait. Les troubles psychiques, ce sont des maladies qui perturbent les émotions, la perception du monde... mais pas l'intelligence."
L'exemple de "grands personnages" comme Einstein ou Léonard de Vinci, ayant connu des épisodes psychiques, a aidé Florent à déclencher une prise de conscience et à croire en une vie heureuse malgré la maladie.
Bruno Guillon témoigne de son changement de perception après avoir rencontré des personnes atteintes de schizophrénie :
"Pour moi, quelqu'un qui était atteint de de schizophrénie, c'était un fou. C'est-à-dire c'était l'iconographie de la chambre capitonnée avec la camisole de force et quelqu'un qui a aucun discernement."
Son reportage à la radio "Les Antoonoirs" a prouvé le contraire.
Ce projet leur permet de "ne pas être dans la solitude parce que la solitude elle tue plus qu'une maladie" et de "diminuer le traitement médicamenteux".
"Moi, j'espère qu'une chose, c'est qu'on nous voit vraiment comme ce qu'on est, des êtres humain à part entière. Euh sur un même plan d'égalité, ça c'est important."
Bipolarité : Un chemin de "rétablissement" continu.Noël, atteint de troubles bipolaires, insiste sur la distinction fondamentale : "on ne vous qualifie pas comme patient bipolaire mais quelqu'un qui souffre de trouble bipolaire car la distinction est très importante." Il préfère le terme "en rétablissement" car "c'est un chemin qui se poursuit et qui comporte ce long périple."
Les tatouages de Noël symbolisent son parcours et sa volonté de vivre, comme le montre l'inscription "No way you kill yourself".
Le rôle de la famille : La mère de Noël, Monique, exprime le soulagement d'avoir enfin un diagnostic : "D'avoir un mot, je pense que ça a aidé parce que jusqu'ici on est bon, on savait que Noël était pas bien...
Mais le fait qu'il y a un nom euh sur la maladie, c'est quand même quelque chose, c'est quand même positif parce que ça ça nous dit bah finalement depuis son enfance, depuis tout le temps, il était pas bien mais on sait pourquoi et c'était peut-être pas de notre faute, c'était comme ça quoi."
3. Comprendre et Gérer la Dépression
La dépression est présentée comme la maladie psychiatrique la plus fréquente en France, touchant "12,5 % des Français âgés de 18 à 85 ans" chaque année.
Distinction déprime/dépression : La déprime est passagère, tandis que la dépression est "beaucoup plus intense" et "nous terrasse".
Symptômes de la dépression : Antoine Pellissolo décrit la dépression comme "une rupture avec sa vie habituelle", incluant désespoir, douleur et "dérèglements du corps" (perte d'appétit, troubles du sommeil, fatigue).
Ce qui se passe dans le cerveau : Une animation explique la baisse des neurotransmetteurs (sérotonine, dopamine, noradrénaline) entraînant moins de plaisir et de motivation.
Le cortex préfrontal, responsable des décisions rationnelles, n'est plus en mesure de prendre du recul, et l'amygdale cérébrale "surinterprète des messages négatifs".
Une "tempête de molécules inflammatoires" (cytokines) aggrave les symptômes.
Son mari, Michel, a été un aidant malgré lui, soulignant la difficulté de la famille à savoir comment aider et l'absence de soutien pour eux.
4. Le Burnout : Une Maladie des "Forts"
Christophe partage son expérience du burnout, soulignant que "beaucoup de gens considèrent que c'est la [maladie] des faibles et au contraire, c'est plutôt la maladie des forts, des personnes qui se sont pas écoutées, qui se sont pas arrêtées à temps."
Les signaux faibles : Douleurs physiques inexpliquées ("le corps parlait"), arrêt des activités plaisantes ("j'ai pas le temps d'aller au sport en ce moment").
Le soutien de l'entourage : La femme de Christophe témoigne de son désarroi face à son mari "sur son canapé toute la journée à se morfondre", sans savoir comment agir. Il est souligné que les aidants souffrent aussi et ont besoin de soutien.
Prévention en entreprise : L'importance de poser des questions sur le bien-être au travail et à la maison est mise en avant.
La durée de l'arrêt de travail : Christophe n'a été arrêté que 3 mois, ce qui est considéré comme très court.
En Belgique, où l'épuisement professionnel est reconnu, la moyenne est de 14 mois. L'idée est qu'il faut "sortir du milieu hostile" et revenir "outillé et différent".
5. L'Anxiété Généralisée et les Crises d'Angoisse
Clara souffre de troubles anxieux généralisés, ayant connu de "terribles crises d'angoisse" et des difficultés à sortir de chez elle.
Symptômes : Cœur qui bat à fond, pertes d'équilibre, sentiment de déréalisation ("on est plus ancré dans la réalité").
Le cercle vicieux : La peur de refaire des crises d'angoisse alimente les crises elles-mêmes : "on a peur d'avoir peur et plus on a peur d'angoisser, plus on fait des crises d'angoisse."
Thérapie et soutien : Clara a suivi une "thérapie comportementale et cognitive" pendant deux ans. Son compagnon l'a soutenue en étant à l'écoute et en contactant sa psychologue pour obtenir des outils.
La reconquête de soi : Clara a réalisé son rêve de voyager seule pendant un mois, défi qu'elle ne pouvait pas relever auparavant, montrant ainsi son rétablissement.
6. Le Rôle des Professionnels de la Santé Mentale Une clarification est apportée sur les différents rôles :
Médecin généraliste : Premier recours pour un diagnostic initial et une orientation.
Psychiatre : Médecin spécialiste des troubles psychiques (8-9 ans d'études de médecine). Ils peuvent prescrire des médicaments et certains sont également formés aux psychothérapies. Leurs consultations sont remboursées.
Psychologue : Ont un master de psychologie clinique et des stages. Ils sont décrits comme des "rééducateurs de la psychée". Depuis peu, "12 séances chez un psychologue sont remboursé[e]s par l'assurance maladie" sans prescription médicale.
Psychothérapeute/Psychopraticien : Des distinctions sont nécessaires pour s'y retrouver.
L'importance du bon "feeling" : Il est crucial de trouver un professionnel avec qui le courant passe. "C'est une histoire de rencontre."
7. L'Impact de l'Hygiène de Vie sur la Santé Mentale
L'émission met en évidence trois piliers essentiels pour le bien-être mental :
L'Activité Physique : Le sport a un impact direct sur le cerveau.
Production de dopamine (motivation, plaisir) et de sérotonine (humeur), "le même que celui visé par certains antidépresseurs."
Stimulation de la "neurogénèse" (création de nouveaux neurones) dans l'hippocampe, essentielle contre la dépression.
Témoignages : Marise a vaincu sa dépression grâce au football en rejoignant une équipe de femmes. Clara, Noël, Florent et Christophe témoignent tous des bienfaits de la danse, du vélo, de la musculation ou de la marche.
La "surf thérapie" : Des psychiatres utilisent le surf comme outil thérapeutique pour des patients atteints de troubles psychiques, permettant une "resocialisation" et une reconquête de la confiance en soi.
Virginie témoigne : "Ça m'a apporté ce côté-là. Ouais. de reprendre confiance en moi, de retrouver une certaine fierté de ce que je fais parce qu'au départ, je ne pensais pas y arriver."
L'Alimentation : Le régime méditerranéen est fortement recommandé.
Impact : Une étude montre qu'un régime de type méditerranéen a conduit à "une rémission pour un tiers des participants de la dépression en moins de 3 mois."
Composition : Principalement végétal, local, de saison, coloré, riche en antioxydants, polyphénols, oléagineux, céréales complètes, bonnes matières grasses (huile d'olive) et poissons gras (oméga-3).
Méfiance envers les aliments ultra-transformés : Ils "sont faits pour être irrésistibles" mais leur consommation excessive augmente le risque "d'anxiété, de dépression, de trouble du sommeil" en créant une inflammation "persistante" dans le corps.
L'équilibre : Les plaisirs occasionnels ("junk food") sont acceptables, mais ne doivent pas devenir la norme.
8. L'Importance de la Prévention et du Soutien
Le défi "Courir pour toi" : Louis, dont le frère Simon s'est suicidé après des années de lutte contre la dépression, a lancé un défi sportif (130 marathons en 150 jours) pour sensibiliser et lever des fonds pour la "Maison Perchée", une association qui aide les personnes confrontées à des troubles psychiques.
Détecter les signaux et agir : En cas d'idées noires ou suicidaires, il est crucial de "saisir la perche", d'être à l'écoute et d'engager le dialogue. Il ne faut jamais banaliser ces propos.
Appeler à l'aide : Si la situation est inquiétante, il faut contacter le médecin traitant ou, en urgence, le SAMU (15).
Différences hommes/femmes face à la dépression :
Les femmes sont plus souvent diagnostiquées (2 pour 1 homme), peut-être parce que la société leur laisse plus d'espace pour exprimer la tristesse, tandis que les hommes peuvent avoir plus de mal à parler de leur souffrance.
Conclusion
Elle démontre que les troubles psychiques sont des maladies réelles et complexes, qui ne doivent plus être stigmatisées.
Grâce aux témoignages et aux éclaircissements des professionnels, elle offre de l'espoir en montrant que des solutions existent, qu'il s'agisse de thérapies, de médicaments ou d'une meilleure hygiène de vie.
Le message clé est clair : oser en parler, demander de l'aide et reconnaître l'importance d'une approche globale pour la santé mentale.
Comme le dit un participant des "Antoonoirs", il s'agit de voir les personnes atteintes de troubles psychiques comme des "êtres humains à part entière" sur un pied d'égalité.
Compte Rendu Détaillé : Sommes-nous tous racistes ?
Ce document synthétise les thèmes principaux, les idées essentielles et les faits marquants tirés de l'émission "Sommes-nous tous racistes ?".
Il met en lumière les mécanismes inconscients des préjugés et de la discrimination à travers diverses expériences scientifiques.
Introduction : Les Préjugés Universels et la Question du Racisme
L'émission s'ouvre sur une interrogation fondamentale : "Vous êtes raciste, vous et moi ?
Est-ce que je suis raciste ?" (Lucien Jean-Baptiste).
Elle pose l'idée que, quelles que soient nos origines ou caractéristiques, "nous avons tous des idées reçues, des a prioris, des préjugés sur tout ce qui ne nous ressemble pas, que nous ne connaissons pas."
L'objectif de l'émission est d'explorer ces mécanismes inconscients.
Pour ce faire, 50 volontaires participent à des "expériences étonnantes" sous le faux titre "Les mystères de notre cerveau", afin de ne pas biaiser leurs réactions.
Le psychosociologue Sylvain De Louvet, expert scientifique, décode les résultats des expériences.
Marie Drucker et Lucien Jean-Baptiste, réalisateur et comédien engagé, commentent les comportements observés.
L'émission révèle que le racisme, la misogynie, le sexisme, l'antisémitisme, l'homophobie et la grossophobie s'appuient sur les "mêmes mécanismes" inconscients et documentés scientifiquement.
Thèmes et Idées Clés : Les Mécanismes Inconscients des Préjugés
1. La Recherche de Similarité et ses Conséquences (Expérience de la Salle d'Attente)
Description de l'expérience : Des participants sont invités à s'asseoir dans une salle d'attente où deux chaises sont disponibles, une à côté d'un homme blanc et l'autre à côté d'un homme noir. La position des acteurs est inversée à mi-parcours.
Observations et conclusions :
Ce qui s'explique très facilement, c'est l'idée que on cherche la similarité. On va chercher les gens qui nous ressemblent." * Cette tendance est qualifiée de "reptilien[ne]", certains thèse évolutionnistes suggérant que "les tribus primitives déjà avaient tendance à se méfier de la différence de l'autre et à plutôt chercher la similitude, la similarité."
2. L'Influence des Préjugés sur le Jugement (Expérience du Jury)
Description de l'expérience : Les participants jouent le rôle de jurés et doivent attribuer une peine de prison à un accusé pour le même crime (coups et blessures volontaires ayant entraîné la mort).
Deux profils sont présentés : un homme blanc et un homme d'origine maghrébine.
Observations et conclusions :
3. La Catégorisation Sociale, Racine des Stéréotypes (Explication et Expérience du Vol de Vélo)
Explication théorique :
Observations et conclusions :
4. Le Biais du Tireur et ses Implications (Expérience du Laser Game)
Description de l'expérience : Les participants, pensant tester leurs réflexes, doivent tirer avec un pistolet laser sur des figures armées et éviter celles désarmées.
Les figures sont de différentes origines ethniques (blanches, maghrébines, noires).
Observations et conclusions :
Les participants tirent "près de quatre fois plus sur les figurants désarmés noirs ou d'origine maghrébine que sur les figurants désarmés blancs."
Cette expérience s'inspire de recherches américaines sur le "biais du tireur", montrant que les policiers sont inconsciemment "plus enclins à tirer sur les citoyens noirs que sur les blancs, même quand ceux-ci sont désarmés."
5. L'Internalisation des Stéréotypes dès l'Enfance (Expérience des Marionnettes et des Poupées)
Expérience des marionnettes : Des enfants doivent désigner le voleur du goûter entre un petit garçon blanc et un petit garçon noir, tous deux clamant leur innocence.
Observations : Les enfants désignent "spontanément plus nombreux à désigner Mousa [le garçon noir] comme le voleur le plus probable." La révélation finale est que c'était un oiseau.
Expérience des poupées (tirée du documentaire "Noir en France") : Des enfants choisissent des poupées et expliquent leurs préférences.
Observations : Des enfants noirs préfèrent les poupées blanches, certaines petites filles noires exprimant le désir de devenir blanches. Une enfant dit préférer la poupée noire "parce que tu es mon préféré."
Il insiste sur la responsabilité de l'éducation : "les enfants, ils sont sensibles aux normes sociales.
Les enfants, ils observent ils observent qui ?
Nous, les adultes. [...] Et ils vont incorporer les stéréotypes, les préjugés de leur entourage."
6. Le Contexte Modifie la Perception des Stéréotypes (Expérience de la Photo de Femme Asiatique)
Description de l'expérience :
Les participants voient des photos, dont une femme d'origine asiatique. Ils doivent donner le premier mot qui leur vient à l'esprit.
La photo est présentée dans trois contextes différents : mangeant avec des baguettes, se maquillant, en blouse blanche de médecin.
Observations et conclusions :
7. Les Stéréotypes d'Accent et de Compétence (Expérience du Conférencier)
Description de l'expérience : Un acteur présente la même conférence sur l'IA et la finance, mais avec trois accents différents : allemand, marseillais, et un accent "africain" pour un faux professeur africain (en réalité le vrai professeur Diallo).
Observations et conclusions :
8. Les Préjugés Positifs et la Déconstruction (Expérience des Sprinters)
Description de l'expérience : Les participants doivent deviner quel sprinter (blanc ou noir) a le plus de chances de gagner une course.
Observations et conclusions :
9. Les Préjugés Annulent l'Empathie (Expérience de la Main Piquée)
Description de l'expérience : Des sujets (blancs ou noirs) regardent des mains (blanche, noire, violette) se faire piquer par une aiguille, tandis que l'activité cérébrale liée à la douleur est mesurée.
Observations et conclusions :
10. Les Préjugés Déforment la Réalité (Expérience de la Photo du Mendiant)
Description de l'expérience : Les participants observent une photo pendant 10 secondes, puis la décrivent de mémoire. La photo montre un homme d'origine maghrébine donnant une pièce à un homme blanc mendiant.
Observations et conclusions :
Près de la moitié des participants décrivent l'homme d'origine maghrébine comme le SDF mendiant et l'homme blanc comme le généreux.
Impact : Lucien Jean-Baptiste partage une anecdote où il a lui-même appliqué un cliché en Afrique : "Ça voulait bien dire que j'étais enfermé par des clichés venant de France enfin de mon éducation à me dire en Afrique les noirs sont pauvres et les blanc sont riches."
La Révélation et le Message Final : Un Appel à la Déconstruction
À la fin de l'émission, le véritable objectif est révélé aux participants : déconstruire "les mécanismes inconscients qui nous conduisent à avoir des préjugés, des préjugés qui eux-mêmes nous amènent à avoir des comportements discriminatoire."
Le titre "Sommes-nous tous racistes ?" est dévoilé.
Les animateurs rassurent les participants : "il ne s'agissait pas de pointer du doigt un tel ou un tel. Le véritable objectif de ces expériences c'est de démontrer que nous avons toutes et tous [...] les mêmes mécanismes qui se déclenchent dans nos têtes et c'est en apprenant à mieux nous connaître que l'on peut lutter contre ces mécanismes."
L'ultime expérience :
Les participants sont répartis en groupes par couleur.
Ils avancent vers un cercle central s'ils sont concernés par une question posée (peur du noir, revente de cadeaux, amour en voiture, sentiment de solitude, etc.).
Cette expérience vise à montrer que "nous avons tous des points communs au-delà de nos différences."
Des moments d'émotion intense sont partagés, soulignant que "On est plus seul."
Conclusion Générale :
Bien que le racisme soit "multifactoriel" (causes économiques, historiques, sociales), le cerveau est "extrêmement plastique".
La lutte contre le racisme et les préjugés passe par "l'éducation, par l'exposition culturelle, le fait de rencontrer, de se mettre en face de personnes différentes de nous.
Et c'est cette exposition là, c'est cette éducation, c'est cette familiarisation avec celles et ceux qui ne nous ressemblent pas qui va permettre aussi au cerveau d'être plus empathique."
L'émission conclut sur l'idée que "Tous les humains, ils partent avec 100 points" et que notre responsabilité est de reconnaître l'égalité de l'autre.
Author response:
Reviewer 1:
Concern 1: Figures 1I, 1J, and the whole of Figure 2 could be placed as supplementary figures. Also, for Figure 3E, it would be preferable to show the percentage of cells expressing cytokines rather than their absolute numbers. In fact, the drop in the numbers of cytokine-producing cells is probably due solely to the drop in total cell numbers and not to a decrease in the proportion of cells expressing cytokines. If this is the case, these data should be shown in supplementary figures. Finally, Figures 4 and 5 could be merged.
We thank you for your recommendations. As rearranging figures is not critical to convey the data, we have decided to keep the figures and supplemental figures as they are currently presented.
Concern 2a: It would be important to show the proportion of Treg, Tconv, and CD8 expressing Layilin in healthy skin and in patients developing psoriasis, as well as in the blood of healthy subjects.
This data is published in a previous manuscript from our group. Please see Figure 1 in “Layilin Anchors Regulatory T Cells in Skin” (PMID: 34470859)
Concern 2b: We lack information to be convinced that there is enrichment for migration and adhesion genes in Layilin+ Tregs in the GSEA data. The authors should indicate what geneset libraries they used. Indeed, it is tempting to show only the genesets that give results in line with the message you want to get across. If these genesets come from public banks, the bank used should be indicated, and the results of all gene sets shown in an unbiased way. In addition, it should be indicated whether the analyses were performed on untransformed or pseudobulk scRNAseq data analyses. Finally, it would be preferable to confirm the GSEA data with z-score analyses, as Ingenuity does, for example. Indeed, in GSEA-type analyses, there are genes that have activating but also inhibiting effects on a pathway in a given gene set.
Given that we have already shown that layilin plays a major role in Treg and CD8+ T cell adhesion in tissues, we used a candidate approach for our GSEA. We tested the hypothesis that adhesion and motility pathways are enriched in Layilin-expressing Tregs. There was a statistically significant enrichment for these genes in Layilin+ Tregs compared to Layilin- Tregs, which we feel adequately tests our hypothesis.
Concern 2c: For all FACS data, the raw data should be shown as histograms or dot plots for representative samples.
We respect this concern. We omit these secondary to space constraints.
Concern 2d: For Figure 5B, the number of samples analyzed is insufficient to draw clear conclusions.
We respectfully disagree. Three doners were used in a paired fashion (internally controlled) achieving statistical significance.
Concern 3: For Figs. 4 and 5, the design of the experiment poses a problem. Indeed, the comparison between Layn+ and Layn- cells may, in part, not be directly linked to the expression or absence of expression of this protein. Indeed, Layn+ and Layn- Tregs may constitute populations with different biological properties, beyond the expression of Layn. However, in the experiment design used here, a significant fraction of the sorted Layn- Tregs will be cells belonging to the population that has never expressed this protein. It would have been preferable to sort first the Layn+ Tregs, then knock down this protein and re-sort the Layn- Tregs and Layn+ Tregs. If this experiment is too cumbersome to perform, I agree that the authors should not do it. However, it would be important to mention the point I have just made in the text.
We agree. However, as the reviewer points out, these experiments are not logistically and practically feasible at this point. We do perform several experiments in this manuscript in which layilin is reduced via gene editing with results supporting our hypotheses.
Reviewer 2:
Some of the conclusions drawn by the authors must be treated with caution, as the experimental conditions were not always appropriate, leading to a risk of misinterpretation.
We have been transparent with all our methods and data. We will leave this to the reader to determine level of rigor and the robustness of the data.
Reviewer 3:
Weaknesses:
It is not clear that the assays used for functional analysis of the patient samples were optimal. (2) Several conclusions are not fully substantiated. (3) The report is lacking some experimental details.
We have tried to be as comprehensive and thorough as possible. We feel that the data supports our conclusions. We will leave this to the reader to interpret and conclude.
45 media formulations (Table S2) had high absorbance (≥2.0) by SRB assay for all replicate wells at 48-72h of incubation (Fig S4), with four media leading to mean absorbance values ≥2.5 at 72 hours of incubation (Fig 1A).
It would be helpful to add a short line here like "..indicating more successful encystment"
Finally, MEA recordings revealed functional responses to histamine and hIL-31
I'm not sure if these data are available, but is it possible to compare the increase in mean firing rate with HA or IL-31 treatment in iPruriceptors vs primary human pruriceptors?
Author response:
The following is the authors’ response to the original reviews
Public Reviews:
Reviewer #1 (Public review):
Summary:
Aicardi-Goutières Syndrome (AGS) is a genetic disorder that primarily affects the brain and immune system through excessive interferon production. The authors sought to investigate the role of microglia in AGS by first developing bone-marrow-derived progenitors in vitro that carry the estrogen-regulated (ER) Hoxb8 cassette, allowing them to expand indefinitely in the presence of estrogen and differentiate into macrophages when estrogen is removed. When injected into the brains of Csf1r-/- mice, which lack microglia, these cells engraft and resemble wild-type (WT) microglia in transcriptional and morphological characteristics, although they lack Sall1 expression. The authors then generated CRISPR-Cas9 Adar1 knockout (KO) ER-Hoxb8 macrophages, which exhibited increased production of inflammatory cytokines and upregulation of interferon-related genes. This phenotype could be rescued using a Jak-Stat inhibitor or by concurrently mutating Ifih1 (Mda5). However, these Adar1-KO macrophages fail to successfully engraft in the brain of both Csf1r-/- and Cx3cr1-creERT2:Csf1rfl/fl mice. To overcome this, the authors used a mouse model with a patient-specific Adar1 mutation (Adar1 D1113H) to derive ER-Hoxb8 bone marrow progenitors and macrophages. They discovered that Adar1 D1113H ER-Hoxb8 macrophages successfully engraft the brain, although at lower levels than WT-derived ER-Hoxb8 macrophages, leading to increased production of Isg15 by neighboring cells. These findings shed new light on the role of microglia in AGS pathology.
Strengths:
The authors convincingly demonstrate that ER-Hoxb8 differentiated macrophages are transcriptionally and morphologically similar to bone marrow-derived macrophages. They also show evidence that when engrafted in vivo, ER-Hoxb8 microglia are transcriptomically similar to WT microglia. Furthermore, ER-Hoxb8 macrophages engraft the Csf1r-/- brain with high efficiency and rapidly (2 weeks), showing a homogenous distribution. The authors also effectively use CRISPR-Cas9 to knock out TLR4 in these cells with little to no effect on their engraftment in vivo, confirming their potential as a model for genetic manipulation and in vivo microglia replacement.
Weaknesses:
The robust data showing the quality of this model at the transcriptomic level can be strengthened with confirmation at protein and functional levels. The authors were unable to investigate the effects of Adar1-KO using ER-Hoxb8 cells and instead had to rely on a mouse model with a patient-specific Adar1 mutation (Adar1 D1113H). Additionally, ER-Hoxb8-derived microglia do not express Sall1, a key marker of microglia, which limits their fidelity as a full microglial replacement, as has been rightfully pointed out in the discussion.
Overall, this paper demonstrates an innovative approach to manipulating microglia using ER-Hoxb8 cells as surrogates. The authors present convincing evidence of the model's efficacy and potential for broader application in microglial research, given its ease of production and rapid brain engraftment potential in microglia-deficient mice. While Adar1-KO macrophages do not engraft well, the success of TLR4-KO line highlights the model's potential for investigating other genes. Using mouse-derived cells for transplantation reduces complications that can come with the use of human cell lines, highlighting the utility of this system for research in mouse models.
Thank you for this thoughtful and balanced assessment. The major suggestion from Reviewer 1 was that confirmation of RNAseq data with protein or functional studies would add strength. We provided protein staining by IHC for IBA1 in vivo, as well as protein staining by FACS for CD11B, CD45, and TMEM119 in vitro and in vivo. For TLR4, we showed successful protein KO and blunted response to LPS (a TLR4 ligand) challenge, which we believe provides some protein and functional data to support the approach. To bolster these data, we added staining for P2RY12 on brain-engrafted ER-Hoxb8s.
Regarding the Adar1 KO phenotypes showing non-engraftment. Because ADAR1 KO mice are embryonically lethal due to hematopoietic failure, we see the health impacts of Adar1 KO on ER-Hoxb8s as a strength of the transplantation model, enabling the assessment of ADAR1 global function in macrophages and microglia-like cells without generation of a transgenic mouse line. In addition, it was a surprise that the health impact occurs at the macrophage and not the progenitor stage, perhaps providing insight for future studies of ADAR1’s role in hematopoiesis. Instead, we were able to show a significant impact of complete loss of Adar1 on survival and engraftment, suggesting an important biological function of ADAR1. Macrophage-specific D1113H mutation, which affects part of the deaminase domain, shows that when the RNA deamination (but not the RNA binding) function of ADAR1 is disrupted, we find brain-wide interferonopathy. This is very exciting to our group and hopefully the community as astrocytes are thought to be a major driver of brain interferonopathy in patients with ADAR1 mutations. Instead, this suggests that disruption of brain macrophages is also a major contributor.
Reviewer #2 (Public review):
Summary:
Microglia have been implicated in brain development, homeostasis, and diseases. "Microglia replacement" has gained traction in recent years, using primary microglia, bone marrow or blood-derived myeloid cells, or human iPSC-induced microglia. Here, the authors extended their previous work in the area and provided evidence to support: (1)
Estrogen-regulated (ER) homeobox B8 (Hoxb8) conditionally immortalized macrophages from bone marrow can serve as stable, genetically manipulated cell lines. These cells are highly comparable to primary bone marrow-derived (BMD) macrophages in vitro, and, when transplanted into a microglia-free brain, engraft the parenchyma and differentiate into microglia-like cells (MLCs). Taking advantage of this model system, the authors created stable, Adar1-mutated ER-Hoxb8 lines using CRISPR-Cas9 to study the intrinsic contribution of macrophages to the Aicardi-Goutières Syndrome (AGS) disease mechanism.
Strengths:
The studies are carefully designed and well-conducted. The imaging data and gene expression analysis are carried out at a high level of technical competence and the studies provide strong evidence that ER-Hoxb8 immortalized macrophages from bone marrow are a reasonable source for "microglia replacement" exercise. The findings are clearly presented, and the main message will be of general interest to the neuroscience and microglia communities.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
This is an elegant study, demonstrating both the utility and limitations of ER-Hoxb8 technology as a surrogate model for microglia in vivo. The manuscript is well-designed and clearly written, but authors should consider the following suggestions:
(1) Validation of RNA hits at the protein level: To strengthen the comparison between ER-Hoxb8 macrophages and WT bone marrow-derived macrophages, validating several RNA hits at the protein level would be beneficial. As many of these hits are surface markers, flow cytometry could be employed for confirmation (e.g., Figure 1D, Figure 3E).
In vitro, we show protein levels by flow cytometry for CD11B (ITGAM) and CD45 (PTPRC; Figure 1C), as well as TMEM119 (Supplemental Figure 2A) and TLR4 (Supplemental Figure 3C/D). In vivo, we show TMEM119 protein levels by flow cytometry (Figure 3A), as well as their CD11B/CD45 pregates (Supplemental Figure 2C), plus immunostaining for IBA1 (AIF1; Figure 2D). We now provide additional data showing P2RY12 immunostaining in brain-engrafted cells (Supplemental Figure 2B).
(2) The authors should consider testing the phagocytic capacity of ER-Hoxb8-derived macrophages to further validate their functionality.
Thank you for the suggestion. We measured ER-Hoxb8 macrophage ability to engulf phosphatidylserine-coated beads that mimic apoptotic cells, compared with phosphatidylcholine-coated beads, now as new Supplemental Figure 1C/D. This agrees with existing literature showing efficient engulfment/phagocytosis by ER-Hoxb8-derived cells (Elhag et al., 2021).
(3) For Figure 3E, incorporating a wild-type (WT) microglia reference would be beneficial to establish a baseline for comparison (e.g. including WT microglia data in the graph or performing a ratio analysis against WT expression levels).
We agree - we now include bars representing our sequenced primary microglia data in Figure 3E as a comparison.
(4) Some statistical analyses may require refinement. Specifically, for Figure 4J, where the effects of Adar1 KO and Adar1 KO with Bari are compared, it would be more appropriate to use a two-way ANOVA.
Thank you for noting it. We have now done more appropriate two-way ANOVA and included the updated results in Figure 4J and the corresponding Supplemental Figure 4G. Errors in figure legend texts have also been corrected to reflect the statistical tests used.
(5) Cx3cr1-creERT2 pups injected with tamoxifen: The authors could clarify the depletion ratio in these experiments before the engraftment and assess whether the depletion is global or regional. In comparison to Csf1r-/-, where TLR4-KO ER-Hoxb8 engraft globally, in Cx3cr1-creERT2, the engraftment seems more regional (Figure 5A vs Supplementary Figure 5B); is this due to the differences in depletion efficiency?
This is an excellent question and observation, and one that we are very interested in, though that finding does not change the conclusions of this particular study. We find some region-specific differences in depletion early after tamoxifen injection, but that all brain regions are >95% depleted by P7. For instance, in a recently published manuscript (Bastos et al., 2025) we find some differences in the depletion kinetics in the genetic model. By P3, we find 90% depletion in cortex with 50-60% in thalamus and hippocampus. In other studies, we typically deliver primary monocytes, and this is the first study where we report engraftment of ER-Hoxb8 cells in the inducible model. In this sense, it is possible that depletion kinetics may regionally affect engraftment, but future studies are required to more finely assess this point with ER-Hoxb8s, as it may change how these models are used in the future.
Bastos et al., Monocytes can efficiently replace all brain macrophages and fetal liver monocytes can generate bonafide SALL1+ microglia, Immunity (2025), https://doi.org/10.1016/j.immuni.2025.04.006
(6) It would be helpful for the authors to clarify whether Adar1 is predominantly expressed by microglia, especially since the study aims to show its role in dampening the interferon response.
That’s a wonderful point. Adar1 is expressed by all brain cells, with highest transcript level in some neurons, astrocytes, and oligodendrocytes. It is an interferon-stimulated gene, and mutation itself leads to interferonopathy, we believe, due to poor RNA editing and detection of endogenous RNA as non-self by MDA5. We hope it can dampen the interferon response, but in the case of mutation, Adar1 is probably causal of interferonopathy. It is induced in microglia upon systemic inflammatory challenge (LPS). We have edited the text to highlight its expression pattern. See BrainRNAseq.org (Zhang*, Chen*, Sloan*, et al., 2014 and Bennett et al., 2016)
Reviewer #2 (Recommendations for the authors):
(1) There appears to be a morphological difference between wt and Adar1/Ifih1 double KO (dKO) cells in the engrafted brains (Figure 5). It would be good if the authors could systematically compare the morphology (e.g., soma size, number, and length of branches) of the engrafted MLCs between the wt and mutant cells.
We agree. While cells did not differ in branch number or length, engrafted dKO cells had significantly larger somas compared with controls, which we now present in Figure S5A.
(2) To fully appreciate the extent of how those engrafted ER-Hoxb8 immortalized macrophages resemble primary, engrafted yolk sac-myeloid cells, vs engrafted iPSC-induced microglia, it would be informative to provide a comparison of their RNAseq data derived from the engrafted ER-Hoxb8 immortalized macrophages with published data transcriptomic data sets (e.g. Bennett et al. Neuron 2018; Chadarevian et al. Neuron 2024; Schafer et al. Cell 2023).
Thank you for this suggestion. To address this, we provide our full dataset for additional experiments. To compare with a similar non-immortalized model, we compared top up- and down-regulated genes from our data to those of ICT yolk sac progenitor cells from our previous work (Bennett et al., 2018). We find overlap between brain-engrafted ER-Hoxb8-, bone marrow-, and yolk sac-derived cells (Supplemental Figure 2F, Supplemental Table 3).
Minor comments:
Figure 6C: red arrow showing zoom in regions are not matchable. It might be beneficial to provide bigger images with each channel for C and D as a Supplemental Figure.
We fixed this in Figure 6C to show areas of interest in the cortex for both conditions. Figure S7A shows intermediate power images to aid in interpretation.
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eLife Assessment
This valuable work proposes a novel, rapid S. aureus entry mechanism via Ca²⁺-dependent lysosomal exocytosis and acid sphingomyelinase release, which influences bacterial sub-cellular fate. However, reliance on chemical inhibitors and the absence of a knockout phenotype weakens the overall impact, making the study incomplete.
Reviewer #3 (Public review):
This revised paper develops and characterizes a new approach for screening drugs for epilepsy. The idea is to increase the ability to study seizures in animals with epilepsy because most animal models have rare seizures. Thus, the authors use the existing intrahippocampal kainic acid (IHKA) mouse model, which can have very unpredictable seizures with long periods of time between seizures. This approach is of clear utility to researchers who may need to observe many seizure events per mouse during screening of antiseizure medications. A key strength is also that more utility can be derived from each individual mouse. The authors modified the IHKA model to inject KA into CA3 instead of CA1 in order to preserve the CA1 pyramidal cells that they will later stimulate. To express the excitatory opsin channelrhodopsin (ChR2) in area CA1, they use a virus that expresses ChR2 in cells that express the Thy-1 promoter. The authors demonstrate that CA3 delivery of KA can induce a very similar chronic epilepsy phenotype to the injection of KA in CA1 and show that optical excitation of CA1 can reliably induce seizures. The authors evaluate the impact of repeated stimulation on the reliability of seizure induction and show that seizures can be reliably induced by CA1 stimulation, at least for the short term (up to 16 days). These are strengths of the study.
However, there are several limitations: the seizures are evoked, not spontaneous. It is not clear how induced seizures can be used to investigate if antiseizure medication can reduce spontaneous seizures. Although seizure inducibility and severity can be assessed, the lack of spontaneous seizures is a limitation. To their credit, the authors show that electrophysiological signatures of induced vs spontaneous seizures are similar in many ways, but the authors also show several differences. Notably, the induced seizures are robustly inhibited by the antiseizure medication levetiracetam and variably but significantly inhibited by diazepam, similar to many mouse models with chronic recurrent seizure activity. One also wonders if using a mouse model with numerous seizures (such as the pilocarpine model) might be more efficient than using a modified IHKA protocol.
In this revised manuscript, the authors address some previous concerns related to definitions of seizures and events that are trains of spikes, sex as a biological variable, and present new images of ChR2 expression (but these images could be improved to see the cells more clearly). A few key concerns remain unaddressed, however. For example, it is still not clear that evoked seizures triggered by stimulating CA1 are similar to spontaneous seizures, regardless of the idea that CA1 plays a role in seizure disorders. It also remains unclear whether repeated activation of the hippocampal circuit will result in additional alterations to this circuit that affect the seizure phenotype over prolonged intervals (after 16 days). Furthermore, the use of SVM with the number of seizures being used as replicates (instead of number of mice) is inappropriate. Another theoretical concern is whether the authors are correct in suggesting that one will be able to re-use the mice for screening multiple drugs in a row.
Strengths:<br /> - The authors show that the IHKA model of chronic epilepsy can be modified to preserve CA1 pyramidal cells, allowing optogenetic stimulation of CA1 to trigger seizures.<br /> - The authors show that repeated optogenetic stimulation of CA1 in untreated mice can promote kindling and induce seizures, indeed generating two mouse models in total.<br /> - Many electrophysiological signatures are similar between the induced and spontaneous seizures, and induced seizures reliably respond to treatment with antiseizure medications.<br /> - Given that more seizures can be observed per mouse using on-demand optogenetics, this model enhances the utility of each individual mouse.<br /> - Mice of each sex were used.
Weaknesses:<br /> - Evaluation of seizure similarity using the SVM modeling and clustering is not sufficiently justified when using number of seizures as the statistical replicate (vs mice).<br /> - Related to the first concern, the utility of increasing number of seizures for enhancing statistical power is limited because standard practice is for sample size to be numbers of mice.<br /> - The term "seizure burden" usually refers to the number of spontaneous seizures per day, not the severity of the seizures themselves. Because the authors are evoking the seizures being studied, this study design precludes assessment of seizure burden.<br /> - It seems likely that repeatedly inducing seizures will have a long-term effect, especially in light of the downward slope at day 13-16 for induced seizures seen in Figure 4C. A duration of evaluation that is longer than 16 days is warranted.<br /> - Human epilepsy is extensively heterogeneous in both etiology and individual phenotype, and it may be hard to generalize the approach.
Author response:
The following is the authors’ response to the original reviews
Reviewer 1 (Public review):
Weaknesses:
While the data generally supports the authors' conclusions, a weakness of this manuscript lies in their analytical approach where EEG feature-space comparisons used the number of spontaneous or evoked seizures as their replicates as opposed to the number of IHK mice; these large data sets tend to identify relatively small effects of uncertain biological significance as being highly statistically significant. Furthermore, the clinical relevance of similarly small differences in EEG feature space measurements between seizure-naïve and epileptic mice is also uncertain.
In this work, we used linear mixed effect model to address two levels of variability –between animals and within animals. The interactive linear mixed effect model shows that most (~90%) of the variability in our data comes from within animals (Residual), the random effect that the model accounts for, rather than between animals. Since variability between animals are low, the model identifies common changes in seizure propagation across animals, while accounting for the variability in seizures within each animal. Therefore, the results we find are of changes that happen across animals, not of individual seizures. We made text edits to clarify the use of the linear mixed effect model. (page6, second paragraph and page 11, first paragraph)
Finally, the multiple surgeries and long timetable to generate these mice may limit the value compared to existing models in drug-testing paradigms.
Thank you for the suggestion. We added a discussion in the ‘Comparison to other seizure models…’ section on pages 15 and 16. In an existing model investigating spontaneous tonic-clonic seizures (such as the intra-amygdala kainate injection model), the time investment is back-loaded, requiring two to three weeks per condition while counting spontaneous seizures, which may occur only once a day. In contrast, our model requires a front-loaded time investment. Once the animals are set up, we can test multiple drugs within a few weeks, providing significant time savings. Additionally, we did not pre-screen animals in our study. Existing models often pre-select mice with high rates of spontaneous seizures, whereas in our model, seizures can be induced even in animals with few spontaneous seizures. We believe that bypassing the need for pre-screening also is a key advantage of our induced seizure model.
Reviewer 1 (Recommendations for the authors):
(1) Address why the EEG data comparisons were performed between seizures and not between animals (as explicitly described in the public review). Further, a discussion of the biological significance (or lack thereof) of the effect size differences observed is warranted. This is especially concerning when the authors make the claim that spontaneous and induced seizures are essentially the same while their analysis shows all evaluated feature space parameters were significantly difference in the initial 1/3 of the EEG waveforms.
We made text edits to clarify the use of the linear mixed effects model (page 6, second paragraph, and page 11, first paragraph)
(2) The authors place great emphasis on the use of clinically/etiologically relevant epilepsy models in drug discovery research. There is discussion criticizing the time points required to enact kindling and the artificial nature of acute seizure induction methods. However, the combination IHK-opto seizure induction model also requires a lengthy timeline. A more tempered discussion of this novel model's strengths may benefit readers.
Thank you for the suggestion. We added a discussion in the ‘Comparison to other seizure models…’ section on pages 15 and 16.
(3) The authors should further emphasize the benefit of having an inducible seizure model of focal epilepsy since other mouse models (e.g., genetic or TBI models) may have superior etiological relevance (construct and face validity) but may not be amenable to their optogenetic stimulation approach.
Thank you for the suggestion. We revised the manuscript to better emphasize the potential significance of our approach. We added a discussion in the 'Application of Models...' section on page 15, second paragraph. The on-demand seizure model can be applied to address biologically and clinically relevant questions beyond its utility in drug screening. For example, crossing the Thy1-ChR2 mouse line with genetic epilepsy models, such as Scn1a mutants, could reveal how optogenetic stimulation differentially induces seizures in mutant versus non-mutant mice, providing insights into seizure generation and propagation in Dravet syndrome. Due to the cellular specificity of optogenetics, we also envision this approach being used to study circuit-specific mechanisms of seizure generation and propagation.
(4) Suggestion: Provide immunolabeled imagery demonstrating ChR2 presence in Thy1 cells.
Thank you for the suggestion. We added a fluorescence image showing ChR2 expression in Fig. 2A
(5) It might be prudent to mention any potential effects of laser heat on hippocampal cell damage, although the 10 Hz, ~10 mW, and 6 s stim is unlikely to cause any substantial burns. Without knowing the diameter and material of the optic fiber, this is left up to some interpretation.
Thank you for the comments. In the Methods section, we listed the optical fiber diameter as 400 microns (page 17, EEG and Fiber Implantation section). Using 5–18 mW laser power with a relatively large fiber diameter of 400 microns, the power density falls within the range of commonly employed channelrhodopsin activation conditions in vivo. That said, we would like to investigate potential heat effects or cell damage in a follow-up study.
(6) There are instances in the manuscript where the authors describe experimental and analytical parameters vaguely (e.g. "Seizures were induced several times a day", "stimulation was performed every 1 - 3 hours over many days"). These descriptions can and should be more precise.
Thank you for the comments. To enhance clarity, we added the stimulation protocol in a flowchart format in Fig. S2A, describing how we determined the threshold and proceeded to the drug test. Following this protocol, there was variability in the number of stimulations per day.
(7) In the second to last paragraph of the discussion, the authors state "However, HPDs are not generalizable across species - they are specific to the mouse model (55)." This statement is inaccurate. The paper cited comes from Dr. Corrine Roucard's lab at Synapcell. In fact, Dr. Rouchard argues the opposite (See Neurochem Res (2017) 42:1919-1925).
Thank you for pointing out the mistake. On page 16, in the first paragraph, reference 55 (now 58 in the revised version) was intended to refer to 'quickly produce dose-response curves with high confidence.' In the revision, we cited another paper reporting that hippocampal spikes were not reproduced in the rat IHK model. R. Klee, C. Brandt, K. Töllner, W. Löscher, Various modifications of the intrahippocampal kainate model of mesial temporal lobe epilepsy in rats fail to resolve the marked rat-to-mouse differences in type and frequency of spontaneous seizures in this model. Epilepsy Behav. 68, 129–140 (2017).
(8) In the discussion, Levetiracetam is highlighted as an ASM that would not be detected in acute induced seizure models; the authors point out its lack of effect in MES and PTZ. However, LEV is effective in the 6Hz test (also an acute-induced seizure model). This should be stated.
Thank you for the comments. We highlighted the discussion on LEV in the 'Application of Model to Testing Multiple Classes of ASMs...' section on page 14.
(9) The results text indicates that 9 epileptic mice were used to test LEV and DZP. However, the individual data points illustrated in Figure 5B show N=8 mice. Please correct.
Thank you for the comments. A total of nine epileptic mice were used to assess two drugs, with the animals being re-used as indicated in the schematic. A total of eight assessments were conducted for DZP with six mice and eight assessments for LEV with five mice. Each assessment included hourly ChR2 activations without an ASM and hourly ChR2 activations after ASM injection.
(10) Figure 4D: Naïve mice are labeled as solid blue circles in the legend while the data points are solid blue triangles. Please correct.
Thank you. We corrected the marker in Fig.4D.
Reviewer 2 (Public Review):
Weaknesses:
(1) Although the figures provide excellent examples of individual electrographic seizures and compare induced seizures in epileptic and naïve animals, it is unclear which criteria were used to identify an actual seizure induced by the optogenetic stimulus, versus a hippocampal paroxysmal discharge (HPD), an "afterdischarge", an "electrophysiological epileptiform event" (EEE, Ref #36, D'Ambrosio et al., 2010 Epilepsy Currents), or a so-called "spike-wave-discharge" (SWD). Were HPDs or these other non-seizure events ever induced using stimulation in animals with IH-KA? A critical issue is that these other electrical events are not actual seizures, and it is unclear whether they were included in the column showing data on "electrographic afterdischarges" in Figure 5 for the studies on ASDs. This seems to be a problem in other areas of the paper, also.
Thank you for pointing out the unclear definition of the seizures analyzed. We added sentences at the beginning of the Results section (page 3) to clarify the terminology we used. We analyzed animal behavior during evoked events, and a high percentage of induced electrographic events were accompanied by behavioral seizures with a Racine scale of three or above. We added Supplemental Figure S9, which shows behavioral seizure severity scores observed before and during ASM testing. We hope these changes address the reviewer’s concern and improve the clarity of the manuscript.
(2) The differences between the optogenetically evoked seizures in IH-KA vs naïve mice are interpreted to be due to the "epileptogenesis" that had occurred, but the lesion from the KA-induced injury would be expected to cause differences in the electrically and behaviorally recorded seizures - even if epileptogenesis had not occurred. This is not adequately addressed.
Thank you for the comments. IHK-injected mice had spontaneous tonic-clonic seizures before the start of optical stimulation, as shown in Figure S1.
(3) The authors offer little mention of other research using animal models of TLE to screen ASDs, of which there are many published studies - many of them with other strengths and/or weaknesses. For example, although Grabenstatter and Dudek (2019, Epilepsia) used a version of the systemic KA model to obtain dose-response data on the effects of carbamazepine on spontaneous seizures, that work required use of KA-treated rats selected to have very high rates of spontaneous seizures, which requires careful and tedious selection of animals. The ETSP has published studies with an intra-amygdala kainic acid (IA-KA) model (West et al., 2022, Exp Neurol), where the authors claim that they can use spontaneous seizures to identify ASDs for DRE; however, their lack of a drug effect of carbamazepine may have been a false negative secondary to low seizure rates. The approach described in this paper may help with confounds caused by low or variable seizure rates. These types of issues should be discussed, along with others.
We appreciate the reviewer’s insights. We added a discussion comparing our model with other existing models in the Discussion section (pages 15 and 16, 'Comparison to Other Seizure Models Used in Pharmacologic Screening' section). In an existing model investigating spontaneous tonic-clonic seizures (such as the intra-amygdala kainate injection model), the time investment is back-loaded, requiring two to three weeks per condition while counting spontaneous seizures, which may occur only once a day. In contrast, our model requires a front-loaded time investment. Once the animals are set up, we can test multiple drugs within a few weeks, providing significant time savings. Additionally, we did not pre-screen animals in our study. Existing models often pre-select mice with high rates of spontaneous seizures, whereas in our model, seizures can be induced even in animals with few spontaneous seizures. We believe that bypassing the need for pre-screening is a key advantage of our induced seizure model.
(4) The outcome measure for testing LEV and DZP on seizures was essentially the fraction of unsuccessful or successful activations of seizures, where high ASD efficacy is based on showing that the optogenetic stimulation causes fewer seizures when the drug is present. The final outcome measure is thus a percentage, which would still lead to a large number of tests to be assured of adequate statistical power. Thus, there is a concern about whether this proposed approach will have high enough resolution to be more useful than conventional screening methods so that one can obtain actual dose-response data on ASDs.
Thank you for the comments. In this revision, we added Supplemental Figure S9, showing the severity of behavioral seizures observed before and during ASM testing for each animal. We observed a reduction in behavioral seizure severity for each subject. We would like to explore using behavioral severity as an outcome measure in a follow-up study.
(5) The authors state that this approach should be used to test for and discover new ASDs for DRE, and also used for various open/closed loop protocols with deep-brain stimulation; however, the paper does not actually discuss rigorously or critically the background literature on other published studies in these areas or how this approach will improve future research for a broader audience than the ETSP and CROs. Thus, it is not clear whether the utility will apply more widely and how extensive a readership will be attracted to this work.
We appreciate the reviewer’s insights. We revised the manuscript to better emphasize the potential significance of our approach (page 15, second paragraph). The on-demand seizure model can be applied to address biologically and clinically relevant questions beyond its utility in drug screening. For example, crossing the Thy1-ChR2 mouse line with genetic epilepsy models, such as Scn1a mutants, could reveal how optogenetic stimulation differentially induces seizures in mutant versus non-mutant mice, providing insights into seizure generation and propagation in Dravet syndrome. Due to the cellular specificity of optogenetics, we also envision this approach being used to study circuit-specific mechanisms of seizure generation and propagation. Regarding drug-resistant epilepsy (DRE) and anti-seizure drug (ASD) screening, we agree with the reviewer that probing new classes of ASDs for DRE represents a critical goal. However, we believe that a full exploration of additional ASD classes and/or modeling DRE lies outside the scope of this manuscript, and we would like to explore it in a follow-up study.
Reviewer 2 (Recommendations for the authors):
(1) The authors should explain why 10 Hz was chosen as the stimulation frequency.
Thank you for the comment. A frequency of 10 Hz was determined based on previous work using anesthetized animals prepared in an acute in vivo setting. To simplify the paper and avoid confusion, we did not include a discussion on how we determined the frequency. Instead, we added a detailed description of how we optimized the power in a flowchart format in Supplemental Figure S2. We hope this improves reproducibility.
(2) After micro-injection of KA, morphological changes were observed in the hippocampus, but no comparison of Chr2 expression was made in naïve animals vs KA-injected animals. Presumably, the Thy1-Chr2 mouse expresses GFP in cells that express Chr2. Thus, it may be useful to show the expression of Chr2 in animals with hippocampal sclerosis. This may explain the lack of dramatic difference between stimulation parameters in naïve vs epileptic animals, as shown in supplemental Figure S2.
Thank you for the suggestion. We added a fluorescence image of ChR2 expression in CA1, ipsilateral to the KA-injected site, in Fig. 2A.
(3) The authors state that "During epileptogenesis, neural networks in the brain undergo various changes ranging from modification of membrane receptors to the formation of new synapses" and that these changes are critical for successful "on-demand" seizure induction. However, it is not clear or well-discussed whether changes in neuronal cell densities that occur during sclerosis are important for "on-demand" seizure induction as well. Also, the authors showed that naïve animals exhibit a kindling-like effect, but it was unclear whether a similar effect was present in epileptic animals (i.e. do stimulation thresholds to seizure induction change as the animal gets more induction stimulations)? If present, would the secondary kindling affect drug-testing studies (e.g., would the drug effect be different on induced seizure #2 vs induced seizure #20)?
Thank you for the suggestion. Since this is an important aspect of the model, we would like to address the kindling effect, the secondary kindling effect, and histopathology in a longer-term setting (several weeks) in a follow-up study.
(4) The authors show that in their model, LEV and DZP were both efficacious. The authors do not seem to mention that, over 25 years ago, LEV was originally missed in the standard ETSP screens; and, it was only discovered outside of the ETSP with the kindling model. The kindling model is now used to screen ASDs. The authors should consider adding this point to the Discussion. It remains unclear, however, if the author's screening strategy shows advantages over kindling and other such approaches in the field.
Thank you for the suggestion. We added a discussion on LEV in the 'Application of Model to Testing Multiple Classes of ASMs...' section on page 14.
(5) P8 paragraph 2. The authors state values for naïve animals, but they should also provide values for epileptic animals since they state that the groups were not significantly different (p>0.05). It would be useful to show values for both and state the actual p-value from the test. This issue of stating mean/median values with SD and sample size should be addressed for all data throughout the paper. Additionally, Figure S2 should be added to the manuscript and discussed, as it has data that may be valuable for the reproducibility of the paper.
Thank you for the suggestion. Figure S2 shows the threshold power required to induce electrographic activity for n = 10 epileptic animals (9.14 ± 4.75 mW) and n = 6 naïve animals (6.17 ± 1.58 mW) (Wilcoxon rank-sum test, p = 0.137). The threshold duration was comparable between the same epileptic animals (6.30 ± 1.64 s) and naïve animals (5.67 ± 1.03 s) (Wilcoxon rank-sum test, p = 0.7133).
(6) In addition to the other stated references on synaptic reorganization in the CA1 area, the authors should mention similar studies from Esclapez et al. (1999, J Comp Neurol).
Thank you. We have included the reference in the revision.
(7) All of the raw EEG data on the seizures should be accessible to the readers.
Thank you for the suggestion. We will consider depositing EEG data in a publicly accessible site.
Reviewer 3 (Public review):
Weaknesses:
(1) Evaluation of seizure similarity using the SVM modeling and clustering is not sufficiently explained to show if there are meaningful differences between induced and spontaneous seizures. SVM modeling did not include analysis to assess the overfitting of each classifier since mice were modeled individually for classification.”
Thank you for the comment. We made text edits to clarify the purpose of the SVM analysis. It was not intended to identify meaningful differences between induced and spontaneous seizures. Rather, it was used to classify EEG epochs as 'seizures' based on spontaneous seizures as the training set, demonstrating the gross similarity between induced and spontaneous seizures.
(2) The difference between seizures and epileptiform discharges or trains of spikes (which are not seizures) is not made clear.
Thank you for pointing out the unclear definition of the seizures analyzed. We added sentences at the beginning of the Results section (page 3) to clarify the terminology we used. We analyzed animal behavior during evoked events, and a high percentage of induced electrographic events were accompanied by behavioral seizures with a Racine scale of three or above. We added Supplemental Figure S9 to show the types of seizures observed before and during ASM testing. We hope these changes address the reviewer’s concern and improve the clarity of the manuscript.
(3) The utility of increasing the number of seizures for enhancing statistical power is limited unless the sample size under evaluation is the number of seizures. However, the standard practice is for the sample size to be the number of mice.
In this work, we used a linear mixed-effects model to address two levels of variability—between animals and within animals. The interactive linear mixed-effects model shows that most (~90%) of the variability in our data comes from within animals (residual), the random effect that the model accounts for, rather than between animals. Since variability between animals is low, the model identifies common changes in seizure propagation across animals while accounting for the variability in seizures within each animal. Therefore, the results we find reflect changes that occur across animals, not individual seizures. We made text edits to clarify the use of the linear mixed-effects model.
(4) Seizure burden is not easily tested.
Thank you for the comment. We added Supplemental Figure S9 to summarize the severity of behavioral seizures before and during ASM testing. This addresses the reviewer’s comment on seizure burden. In a follow-up study, we would like to explore this type of outcome measure for drug screening.
Reviewer 3 (Recommendations for the authors):
(1) Provide a stronger rationale to use area CA1. For example, the authors mention that CA1 is active during seizure activity, but can seizures originate from CA1? That would make the approach logical and also explain why induced and spontaneous seizures are similar.
Thank you for the comment. We discussed it in the Discussion section (page 14, first and second paragraphs).
(2) Explain the use of SVM classifiers so it is more convincing that induced and spontaneous seizures are similar. Or, if they are not similar, explain that this is a limitation.
We made text edits to clarify the purpose of the SVM analysis. It was not intended to identify meaningful differences between induced and spontaneous seizures. Rather, it was used to classify EEG epochs as 'seizures' based on spontaneous seizures as the training set, demonstrating the gross similarity between induced and spontaneous seizures.
(3)If feasible, extend the duration over which seizure induction reliability is assessed so that the long-term utility of the model can be demonstrated.
Thank you for the suggestion. We would like to assess long-term utility in a follow-up study.
(4) The GitHub link is not yet active. The authors will be required to supply their relevant code for peer evaluation as well as publication.
Thank you. The GitHub repository is now active.
(5) State and assess the impacts of sex as a biological variable.
Thank you for pointing this out. Both female and male animals were included in this study: Epileptic cohort: 7 males, 3 females; Naïve cohort: 3 males, 4 females.
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Reviewer #1 (Public review):
Strengths:
This work adds another mouse model for LAMA2-MD that re-iterates the phenotype of previously published models. Such as dy3K/dy3K; dy/dy and dyW/dyW mice. The phenotype is fully consistent with the data from others.
One of the major weaknesses of the manuscript initially submitted was the overinterpretation and the overstatements. The revised version is clearly improved as the authors toned-down their interpretation and now also cite the relevant literature of previous work.
Comments on revisions:
This is the second revision of a paper focusing on the generation of a CRISPR/Cas9-engineered mouse model for LAMA2-MD. I have reviewed the initial submission, the first revision, and now this second revision. While there have been improvements, several issues still need to be addressed by the authors. I will outline these points without dividing them into major and minor categories:
Introduction:
The statement regarding existing mouse models requires correction: The claim, "They were established in the pre-gene therapy era, leaving trace of engineering, such as bacterial elements in the Lama2 gene locus, thus unsuitable for testing various gene therapy strategies," is inaccurate. Current mouse models can indeed be used for testing gene therapy strategies, regardless of whether they contain elements in the Lama2 locus. The primary consideration is whether or not they express laminin-alpha2. Please revise this statement.<br /> Results Section:
scRNA-seq:
The authors note that they analyzed "a total of 8,111 cells from the dyH/dyH mouse brain and 8,127 cells from the WT mouse brain were captured using the 10X Genomics platform (Figure supplement 4A, B)." This is too few cells to support firm conclusions. Furthermore, there is a discrepancy in the referred figure S4, which indicates that 10,094 cells were analyzed for dyH/dyH mice and 10,496 for wild-type mice. Please correct this inconsistency.
Figure 5C displays differences in cell populations between wild-type and dyH/dyH mice. Given the low number of cells analyzed and the lack of replicates, these differences cannot be considered reliable. More samples should be analyzed to support these findings.
The data suggest a defect in the BBB for dyH/dyH mice, but this conclusion is based on minimal cell counts and remains purely correlative. If BBB issues exist, experimental validation is necessary, such as injecting dyes into the bloodstream to detect any leakage. I have previously highlighted this in my comments on earlier manuscript versions.
Bulk RNA-seq:
The number of samples analyzed here is substantial, making the data potentially more robust. These data could serve as a valuable resource for other researchers. However, it is important to note that all data are correlative and do not provide functional insights.
Overall:
The manuscript still lacks significant insights, partly because existing mouse models for LAMA2-MD have been extensively analyzed. While the bulk RNA-seq data offer some value as a resource, I recommend that the authors re-assess their writing and further temper their interpretations of the findings.
Author response:
The following is the authors’ response to the original reviews
We thank the Reviewers for their constructive comments and the Editor for the possibility to address the Reviewers’ points in this rebuttal. We
(1) Conducted new experiments with NP6510-Gal4 and TH-Gal4 lines to address potential behavioral differences due to targeting dopaminergic vs. both dopaminergic and serotonergic neurons
(2) Conducted novel data analyses to emphasize the strength of sampling distributions of behavioral parameters across trials and individual flies
(3) Provided Supplementary Movies
(4) Calculated additional statistics
(5) Edited and added text to address all points of the Reviewers.
Please see our point-by-point responses below.
Public Reviews:
Reviewer #1 (Public Review):
Summary:
Translating discoveries from model organisms to humans is often challenging, especially in neuropsychiatric diseases, due to the vast gaps in the circuit complexities and cognitive capabilities. Kajtor et al. propose to bridge this gap in the fly models of Parkinson's disease (PD) by developing a new behavioral assay where flies respond to a moving shadow by modifying their locomotor activities. The authors believe the flies' response to the shadow approximates their escape response to an approaching predator. To validate this argument, they tested several PD-relevant transgenic fly lines and showed that some of them indeed have altered responses in their assay.
Strengths:
This single-fly-based assay is easy and inexpensive to set up, scalable, and provides sensitive, quantitative estimates to probe flies' optomotor acuity. The behavioral data is detailed, and the analysis parameters are well-explained.
We thank the Reviewer for the positive assessment of our study.
Weaknesses:
While the abstract promises to give us an assay to accelerate fly-to-human translation, the authors need to provide evidence to show that this is indeed the case. They have used PD lines extensively characterized by other groups, often with cheaper and easier-to-setup assays like negative geotaxis, and do not offer any new insights into them. The conceptual leap from a low-level behavioral phenotype, e.g. changes in walking speed, to recapitulating human PD progression is enormous, and the paper does not make any attempt to bridge it. It needs to be clarified how this assay provides a new understanding of the fly PD models, as the authors do not explore the cellular/circuit basis of the phenotypes. Similarly, they have assumed that the behavior they are looking at is an escape-from-predator response modulated by the central complex- is there any evidence to support these assumptions? Because of their rather superficial approach, the paper does not go beyond providing us with a collection of interesting but preliminary observations.
We thank the Reviewer for pointing out some limitations of our study. We would like to emphasize that what we perceive as the main advantage of performing single-fly and single-trial analyses is the access to rich data distributions that provide more fine-scale information compared to bulk assays. We think that this is exactly going one step closer to ‘bridging the enormous conceptual leap from a low-level behavioral phenotype, e.g. changes in walking speed, to recapitulating human PD progression’, and we showcase this in our study by comparing the distributions over the entire repertoire of behavioral responses across fly mutants. Nevertheless, we agree with the Reviewer that many more steps in this direction are needed to improve translatability. Therefore, we toned down the corresponding statements in the Abstract and in the Introduction. Moreover, to further emphasize the strength of sampling distributions of behavioral parameters across trials and individual flies, we complemented our comparisons of central tendencies with testing for potential differences in data dispersion, demonstrated in the novel Supplementary Figure S4.
Looming stimuli have been used to characterize flies’ escape behaviors. These studies uncovered a surprisingly rich behavioral repertoire (Zacarias et al., 2018), which was modulated by both sensory and motor context, e.g. walking speed at time of stimulus presentation (Card and Dickinson, 2008; Oram and Card, 2022; Zacarias et al., 2018). The neural basis of these behaviors was also investigated, revealing loom-sensitive neurons in the optic lobe and the giant fiber escape pathway (Ache et al., 2019; de Vries and Clandinin, 2012). Although less frequently, passing shadows were also employed as threat-inducing stimuli in flies (Gibson et al., 2015). We opted for this variant of the stimulus so that we could ensure that the shadow reached the same coordinates in all linear track concurrently, aiding data analysis and scalability. Similar to the cited study, we found the same behavioral repertoire as in studies with looming stimuli, with an equivalent dependence on walking speed, confirming that looming stimuli and passing shadows can both be considered as threat-inducing visual stimuli. We added a discussion on this topic to the main text.
Reviewer #2 (Public Review):
In this study, Kajtor et al investigated the use of a single-animal trial-based behavioral assay for the assessment of subtle changes in the locomotor behavior of different genetic models of Parkinson's disease of Drosophila. Different genotypes used in this study were Ddc-GAL4>UASParkin-275W and UAS- α-Syn-A53T. The authors measured Drosophila's response to predatormimicking passing shadow as a threatening stimulus. Along with these, various dopamine (DA) receptor mutants, Dop1R1, Dop1R2 and DopEcR were also tested.
The behavior was measured in a custom-designed apparatus that allows simultaneous testing of 13 individual flies in a plexiglass arena. The inter-trial intervals were randomized for 40 trials within 40 minutes duration and fly responses were defined into freezing, slowing down, and running by hierarchical clustering. Most of the mutant flies showed decreased reactivity to threatening stimuli, but the speed-response behavior was genotype invariant.
These data nicely show that measuring responses to the predator-mimicking passing shadows could be used to assess the subtle differences in the locomotion parameters in various genetic models of Drosophila.
The understanding of the manifestation of various neuronal disorders is a topic of active research. Many of the neuronal disorders start by presenting subtle changes in neuronal circuits and quantification and measurement of these subtle behavior responses could help one delineate the mechanisms involved. The data from the present study nicely uses the behavioral response to predator-mimicking passing shadows to measure subtle changes in behavior. However, there are a few important points that would help establish the robustness of this study.
We thank the Reviewer for the constructive comments and the positive assessment of our study.
(1) The visual threat stimulus for measuring response behavior in Drosophila is previously established for both single and multiple flies in an arena. A comparative analysis of data and the pros and cons of the previously established techniques (for example, Gibson et al., 2015) with the technique presented in this study would be important to establish the current assay as an important advancement.
We thank the Reviewer for this suggestion. We included the following discussion on measuring response behavior to visual threat stimuli in the revised manuscript.
Many earlier studies used looming stimulus, that is, a concentrically expanding shadow, mimicking the approach of a predator from above, to study escape responses in flies (Ache et al., 2019; Card and Dickinson, 2008; de Vries and Clandinin, 2012; Oram and Card, 2022; Zacarias et al., 2018) as well as rodents (Braine and Georges, 2023; Heinemans and Moita, 2024; Lecca et al., 2017). These assays have the advantage of closely resembling naturalistic, ecologically relevant threatinducing stimuli, and allow a relatively complete characterization of the fly escape behavior repertoire. As a flip side of their large degree of freedom, they do not lend themselves easily to provide a fully standardized, scalable behavioral assay. Therefore, Gibson et al. suggested a novel threat-inducing assay operating with moving overhead translational stimuli, that is, passing shadows, and demonstrated that they induce escape behaviors in flies akin to looming discs (Gibson et al., 2015). This assay, coined ReVSA (repetitive visual stimulus-induced arousal) by the authors, had the advantage of scalability, while constraining flies to a walking arena that somewhat restricted the remarkably rich escape types flies otherwise exhibit. Here we carried this idea one step further by using a screen to present the shadows instead of a physically moving paddle and putting individual flies to linear corridors instead of the common circular fly arena. This ensured that the shadow reached the same coordinates in all linear tracks concurrently and made it easy to accurately determine when individual flies encountered the stimulus, aiding data analysis and scalability. We found the same escape behavioral repertoire as in studies with looming stimuli and ReVSA (Gibson et al., 2015; Zacarias et al., 2018), with a similar dependence on walking speed (Oram and Card, 2022; Zacarias et al., 2018), confirming that looming stimuli and passing shadows can both be considered as threat-inducing visual stimuli.
(2) Parkinson's disease mutants should be validated with other GAL-4 drivers along with DdcGAL4, such as NP6510-Gal4 (Riemensperger et al., 2013). This would be important to delineate the behavioral differences due to dopaminergic neurons and serotonergic neurons and establish the Parkinson's disease phenotype robustly.
We thank the Reviewer for point out this limitation. To address this, we repeated our key experiments in Fig.3. with both TH-Gal4 and NP6510-Gal4 lines, and their respective controls. These yielded largely similar results to the Ddc-Gal4 lines reported in Fig.3., reproducing the decreased speed and decreased overall reactivity of PD-model flies. Nevertheless, TH-Gal4 and NP6510-Gal4 mutants showed an increased propensity to stop. Stop duration showed a significant increase not only in α-Syn but also in Parkin fruit flies. These novel results have been added to the text and are demonstrated in Supplementary Figure S3.
(3) The DopEcR mutant genotype used for behavior analysis is w1118; PBac{PB}DopEcRc02142TM6B, Tb1. Balancer chromosomes, such as TM6B,Tb can have undesirable and uncharacterised behavioral effects. This could be addressed by removing the balancer and testing the DopEcR mutant in homozygous (if viable) or heterozygous conditions.
We appreciate the Reviewer's comment and acknowledge the potential for the DopEcR balancer chromosome to produce unintended behavioral effects. However, given that this mutant was not essential to our main conclusions, we opted not to repeat the experiment. Nevertheless, we now discuss the possible confounds associated with using the PBac{PB}DopEcRc02142 mutant allele over the balancer chromosome. “We recognize a limitation in using PBac{PB}DopEcRc02142 over the TM6B, Tb<sup>1</sup> balancer chromosome, as the balancer itself may induce behavioral deficits in flies. We consider this unlikely, as the PBac{PB}DopEcRc02142 mutation demonstrates behavioral effects even in heterozygotes (Ishimoto et al., 2013). Additionally, to our knowledge, no studies have reported behavioral deficits in flies carrying the TM6B, Tb<sup>1</sup> balancer chromosome over a wild-type chromosome.”
(4) The height of the arena is restricted to 1mm. However, for the wild-type flies (Canton-S) and many other mutants, the height is usually more than 1mm. Also, a 1 mm height could restrict the fly movement. For example, it might not allow the flies to flip upside down in the arena easily. This could introduce some unwanted behavioral changes. A simple experiment with an arena of height at least 2.5mm could be used to verify the effect of 1mm height.
We thank the Reviewer for this comment, which prompted us to reassess the dimensions of the apparatus. The height of the arena was 1.5 mm, which we corrected now in the text. We observed that the arena did not restrict the flies walking and that flies could flip in the arena. We now include two Supplementary Movies to demonstrate this.
(5) The detailed model for Monte Carlo simulation for speed-response simulation is not described. The simulation model and its hyperparameters need to be described in more depth and with proper justification.
We thank the Reviewer for pointing out a lack of details with respect to Monte Carlo simulations. We used a nested model built from actual data distributions, without any assumptions. Accordingly, the stimulation did not have hyperparameters typical in machine learning applications, the only external parameter being the number of resamplings (3000 for each draw). We made these modeling choices clearer and expanded this part as follows.
“The effect of movement speed on the distribution of behavioral response types was tested using a nested Monte Carlo simulation framework (Fig. S5). This simulation aimed to model how different movement speeds impact the probability distribution of response types, comparing these simulated outcomes to empirical data. This approach allowed us to determine whether observed differences in response distributions are solely due to speed variations across genotypes or if additional behavioral factors contribute to the differences. First, we calculated the probability of each response type at different specific speed values (outer model). These probabilities were derived from the grand average of all trials across each genotype, capturing the overall tendency at various speeds. Second, we simulated behavior of virtual flies (n = 3000 per genotypes, which falls within the same order of magnitude as the number of experimentally recorded trials from different genotypes) by drawing random velocity values from the empirical velocity distribution specific to the given genotype and then randomly selecting a reaction based on the reaction probabilities associated with the drawn velocity (inner model). Finally, we calculated reaction probabilities for the virtual flies and compared it with real data from animals of the same genotype.
Differences were statistically tested by Chi-squared test.”
(6) The statistical analysis in different experiments needs revisiting. It wasn't clear to me if the authors checked if the data is normally distributed. A simple remedy to this would be to check the normality of data using the Shapiro-Wilk test or Kolmogorov-Smirnov test. Based on the normality check, data should be further analyzed using either parametric or non-parametric statistical tests. Further, the statistical test for the age-dependent behavior response needs revisiting as well. Using two-way ANOVA is not justified given the complexity of the experimental design. Again, after checking for the normality of data, a more rigorous statistical test, such as split-plot ANOVA or a generalized linear model could be used.
We thank the Reviewer for this comment. We performed Kolmogorov-Smirnov test for normality on the data distributions underlying Figure 3, and normality was rejected for all data distributions at p = 0.05, which justifies the use of the non-parametric Mann-Whitney U-test. Regarding ANOVA, we would like to point out that the ANOVA hypothesis test design is robust to deviations from normality (Knief and Forstmeier, 2021; Mooi et al., 2018). While the Kruskal-Wallis test is considered a reasonable non-parametric alternative of one-way ANOVA, there is no clear consensus for a non-parametric alternative of two-way ANOVA. Therefore, we left the two-way ANOVA for Figure 5 in place; however, to increase the statistical confidence in our conclusions, we performed Kruskal-Wallis tests for the main effect of age and found significant effects in all genotypes in accordance with the ANOVA, confirming the results (Stop frequency, DopEcR p = 0.0007; Dop1R1, p = 0.004; Dop1R2, p = 9.94 × 10<sup>-5</sup>; w<sup>1118</sup>, p = 9.89 × 10<sup>-13</sup>; y<sup>1</sup> w<sup>67</sup>c<sup>23</sup>, p = 2.54 × 10<sup>-5</sup>; Slowing down frequency, DopEcR, p = 0.0421; Dop1R1, p = 5.77 x 10<sup>-6</sup>; Dop1R2, p = 0.011; w<sup>1118</sup>, p = 2.62 x 10<sup>-5</sup>; y<sup>1</sup> w<sup>67</sup>c<sup>23</sup>, p = 0.0382; Speeding up frequency, DopEcR, p = 0.0003; Dop1R1, p = 2.06 x 10<sup>-7</sup>; Dop1R2, p = 2.19 x 10<sup>-6</sup>; w<sup>1118</sup>, p = 0.0044; y<sup>1</sup> w<sup>67</sup>c<sup>23</sup>, p = 1.36 x 10<sup>-5</sup>). We also changed the post hoc Tukey-tests to post hoc Mann-Whitney tests in the text to be consistent with the statistical analyses for Figure 3. These resulted in very similar results as the Tukey-tests. Of note, there isn’t a straightforward way of correcting for multiple comparisons in this case as opposed to the Tukey’s ‘honest significance’ approach, we thus report uncorrected p values and suggest considering them at p = 0.01, which minimizes type I errors. These notes have been added to the ‘Data analysis and statistics’ Methods section.
(7) The dopamine receptor mutants used in this study are well characterized for learning and memory deficits. In the Parkinson's disease model of Drosophila, there is a loss of DA neurons in specific pockets in the central brain. Hence, it would be apt to use whole animal DA receptor mutants as general DA mutants rather than the Parkinson's disease model. The authors may want to rework the title to reflect the same.
We thank the Reviewer for this comment, which suggests that we were not sufficiently clear on the Drosophila lines with DA receptor mutations. We used Mi{MIC} random insertion lines for dopamine receptor mutants, namely y<sup>1</sup> w<sup>*1</sup>; Mi{MIC}Dop1R1<sup>MI04437</sup> (BDSC 43773), y<sup>1</sup> w<sup>*1</sup>; Mi{MIC}Dop1R2<sup>MI08664</sup> (BDSC 51098) (Harbison et al., 2019; Pimentel et al., 2016), and w<sup>1118</sup>; PBac{PB}DopEcR<sup>c02142</sup>/TM6B, Tb<sup>1</sup> (BDSC 10847) (Ishimoto et al., 2013; Petruccelli et al., 2020, 2016). These lines carried reported mutations in dopamine receptors, most likely generating partial knock down of the respective receptors. We made this clearer by including the full names at the first occurrence of the lines in Results (beyond those in Methods) and adding references to each of the lines.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
(1) Please think about focusing the manuscript either on the escape response or the PD pathology and provide additional evidence to demonstrate that you indeed have a novel system to address open questions in the field.
As detailed above, we now emphasize more that the main advantage of our single-trial-based approach lies in the appropriate statistical comparison of rich distributions of behavioral data. Please see our response to the ‘Weaknesses’ section for more details.
(2) Please explain the rationale for choosing the genetic lines and provide appropriate genetic controls in the experiments, e.g. trans-heterozygotes. Why use Ddc-Gal4 instead of TH or other specific Split-Gal4 lines?
We thank the Reviewer for this suggestion. We repeated our key experiments with TH-Gal4 and NP6510-Gal4 lines. Please see our response to Point #2 of Reviewer #2 for details.
(3) Please proofread the manuscript for ommissions. e.g. there's no legend for Fig 4b.
We respectfully point out that the legend is there, and it reads “b, Proportion of a given response type as a function of average fly speed before the shadow presentation. Top, Parkin and α-Syn flies. Bottom, Dop1R1, Dop1R2 and DopEcR mutant flies.”
Reviewer #2 (Recommendations For The Authors):
(1) In figure 2(c), representing the average walking speed data for different mutants would be useful to visually correlate the walking differences.
We thank the Reviewer for this suggestion. The average walking speed was added in a scatter plot format, as suggested in the next point of the Reviewer.
(2) The data could be represented more clearly using scatter plots. Also, the color scheme could be more color-blindness friendly.
We thank the Reviewer for this suggestion. We added scatter plots to Fig.2c that indeed represent the distribution of behavioral responses better. We also changed the color scheme and removed red/green labeling.
(3) The manuscript should be checked for typos such as in line 252, 449, 484.
Thank you. We fixed the typos.
References
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Summary
The authors aim to understand the consequences of disrupting N6 methyladenosine, an abundant mRNA modification in plants and other organisms, in Arabidopsis. Genetic ablation of the N6 methyladenosine transferase complex is embryonic lethal in Arabidopsis. Therefore, the authors utilize a hypomorphic allele of VIRILIZER, a component of the complex, to examine gene expression changes and other phenotypes. The authors demonstrate that immune response pathway genes are misregulated in the vir mutant. This transcriptional phenotype is suppressed at higher temperatures, although developmental phenotypes are not. The manuscript provides strong evidence that reduced function of the m6A methyltransferase complex leads to upregulation of immune response genes, although a mechanistic connection between the immune response and m6A in mRNA is not discerned.
Major comments
The major claims of the manuscript are that disrupting the m6A writer complex triggers an autoimmune response that is present at 17C and suppressed at 27C (in line with known aspects of Arabidopsis immunity). Consistent with this, they also show that at 17C the vir-1 mutant has more cell death and is more resistant to infection by Pseudomonas syringae. All of these claims are well supported by the data. The authors also claim that polyA tail lengths are different between the two temperatures. They further speculate that mRNAs that lack m6A trigger immune signaling, but this is not directly tested in the study.
The conclusions about transcriptional activation of the immune response at lower temperatures are sufficiently supported by two types of mRNA sequencing data (direct RNA sequencing and short-read sequences) and appropriate biological replication. The initial profiling was at 22 C, later profiling was at 17C and 27 C. How similar/overlapping were the vir-1 misregulated genes at 17C and 22C? Is the immune response transcriptional signature stronger at 17C than at 22C? The authors sought to determine whether the vir-1 response at 17C was due to pathogen infection of those plants. They used their Illumina RNA-seq data to try and identify pathogen RNAs. They report that there was no significant enrichment of plant pathogen sequences (supplemental table 7). Significant compared to what? Supplemental Table 7 does not indicate that the WT data was assessed and there's no information on significance of enrichment (or nothing obvious, based on column titles). Did the Illumina library prep preparation rely on polyA tails? If so, this is not a sensitive assay to detect bacterial transcripts.
I found the last section on altered poly(A) tail length and site usage somewhat difficult to follow and the analysis rather cursory. The authors find no difference in polyA site usage in vir-1 at 17C or 27C (although both are different than WT). For Figure 7A, in addition to the histogram of poly A site shift, I would like to see a plot (heatmap?) that compares poly A sites shift for individual mRNAs across samples, instead of only aggregated data. Are there individual mRNAs that differ between 17 and 27C in vir-1?
A similar comment applies to the data in 7E. Please also compare individual mRNA polyA tail length across samples. What is the significance of the change in polyA tail length? The tails are shorter in vir-1 than Col at 27 C. But vir-1 has a very similar phenotype to WT at 27 C. At 17 C, vir-1 tails are longer than WT. Together, do these two results imply that polyA tail length is unlikely to be related to the observed phenotypes? In other words, if longer tails have no effect, do shorter tails? Is there any relationship between RNAs with altered polyA site usage or tail length and those mRNAs that are misexpressed in the mutant? Are immunity gene mRNAs more likely to be m6A modified than other mRNAs?
Minor comments
At times it felt like the authors were stretching to fill seven figure with data. For example, in Figure 1, it was not necessary to show the data on increased PR1 expression in 6 different sub panels (B-F) to convince the reader that PR1 expression was increased. A similar comment applies to Figure 3A-D. In Figure 3 please write the common gene names above the plots.
In Supplemental Table 3 the Enriched GO Terms tab is blank. Supplementary File 1 appeared to be missing from the submission, so I could not evaluate the sequencing statistics (# of reads per sample, mapping %, etc). Many of the Supplemental Tables would benefit from a readme that describes what analysis was performed and what the different columns mean.
The manuscript provides additional insight on the functional consequences of disrupting adenosine methylation in RNA, identifying features of an autoimmune response. Given the ubiquity of m6A in RNA across eukaryotes, this is a result that will be of interest to basic researchers in the plant RNA modification community and likely those working in other eukaryotes. However, the study is not able to connect the inappropriate expression of immune response genes back to the function m6A in RNA, and the effects might be indirect. Although there is speculation that RNA that lacks m6A might trigger autoimmunity, the presented experiments do not directly test that hypothesis (nor do the authors claim to).
Author response:
The following is the authors’ response to the previous reviews
Reviewer #1 (Recommendations for the authors):
The authors have done an impressive job in responding to the previous critique and even gone beyond what was asked. I have only very minor comments on this excellent manuscript. The manuscript also needs some light editing for grammar and readability.
We have worked to improve the grammar and readability of the manuscript.
Comments:
Lines 227-234: At what age was tamoxifen administered to the various CreERTM mice?
We have updated the ages of the mice used in this study in the methods sections.
UMAP in Figure 5A is missing label for cluster 19.
The UMAP in Figure 5A has the label for cluster 19 at the center-bottom of the image.
Supplement Figure 6: Cluster 10 seems to be separate from the other AdvC clusters, and it includes some expression of Myh11 and Notch3. Further, there is low expression of Pdgfra in this cluster, which can be seen in panel B and panels D-I. Are the Pdgfra negative cells in the pie charts from cluster 10? Could the cells in this cluster by more LMC like than AdvC like?
We agree with the reviewer that the subcluster 10 of the fibroblasts cells are intriguing if only a minor population. When assessing just this population of cells, which is 77 cells out of 2261 total, 40 of the 77 were Pdgfra+ and of the 37 remaining Pdgfra- but 11 of those were still CD34+. Thus at least half of these cells could be expected to have the PdgfraCreERTM. Only 8 of the 37 were Pdgfra-Notch3+ while 12 cells were Pdgfra+Notch3+, and only 3 were Pdgfra-Myh11+ while 3 were Pdgfra+Myh11+. 26 of 77 cells were Pdgfra+Pdgfrb+ double positive, while 12 of 37 Pdgfra- cells were still Pdgfrb+. Additionally, within the 77 cells of subcluster 10 17 were positive for Scn3a (Nav1.3), 21were positive for Kcnj8 (Kir6.1), and 33 were positive for Cacna1c (Cacna1c) which are typically LMC markers would support the reviewers thinking that this group contains a fibroblast-LMC transitional cell type. Only 2 of 77 cells were positive for the BK subunit (Kcnma1), which is a classic smooth muscle marker. Another possibility is this population represents the Pdgfra+Pdgfrb+ valve interstitial cells we identified in our IF staining and in our reporter mice. Of note almost all cells in this cluster were Col3a1+ and Vim+. Even though we performed QC analysis to remove doublets, it is also possible some of these cells could represent doublets or contaminants, however the low % of Myh11 expression, a very highly expressed gene in LMCs especially compared to ion channels, would suggest this is less likely. Assessing the presence of this particular cell cluster in future RNAseq or with spatial transcriptomics will be enlightening.
Line 360. Proofread section title.
We have simplified this title to read “Optogenetic Stimulation of iCre-driven Channel Rhodopsin 2”
Lines 370-371. Are the length units supposed to be microns or millimeters?
We have corrected this to microns as was intended. Thank you for catching this error.
The resolution for each UMAP analysis should be stated, particularly for the identification of subclusters. How was the resolution chosen?
To select the optimal cluster resolution, we used Clustree with various resolutions. We examined the resulting tree to identify a resolution where the clusters were well-separated and biologically meaningful, ensuring minimal merging or splitting at higher resolutions. Our goal was to find a resolution that captures relevant cell subpopulations while maintaining distinct clusters without excessive fragmentation. We have now stated the resolution for the subclustering of the LECs, LMCs, and fibroblasts. We have also added greater detail regarding the total number of cells, QC analysis, and the marker identification criteria used to the methods sections. We used resolution of 0.5 for sub-clustering LMCs, 0.87 for LECs, and 1.0 for fibroblasts. These details are now added to the manuscript.
Reviewer #1 (Public review):
Fombellida-Lopez and colleagues describe the results of an ART intensification trial in people with HIV infection (PWH) on suppressive ART to determine the effect of increasing the dose of one ART drug, dolutegravir, on viral reservoirs, immune activation, exhaustion, and circulating inflammatory markers. The authors hypothesize that ART intensification will provide clues about the degree to which low-level viral replication is occurring in circulation and in tissues despite ongoing ART, which could be identified if reservoirs decrease and/or if immune biomarkers change. The trial design is straightforward and well-described, and the intervention appears to have been well tolerated. The investigators observed an increase in dolutegravir concentrations in circulation, and to a lesser degree in tissues, in the intervention group, indicating that the intervention has functioned as expected (ART has been intensified in vivo). Several outcome measures changed during the trial period in the intervention group, leading the investigators to conclude that their results provide strong evidence of ongoing replication on standard ART. The results of this small trial are intriguing, and a few observations in particular are hypothesis-generating and potentially justify further clinical trials to explore them in depth. However, I am concerned about over-interpretation of results that do not fully justify the authors' conclusions.
(1) Trial objectives: What was the primary objective of the trial? This is not clearly stated. The authors describe changes in some reservoir parameters and no changes in others. Which of these was the primary outcome? No a priori hypothesis / primary objective is stated, nor is there explicit justification (power calculations, prior in vivo evidence) for the small n, unblinded design, and lack of placebo control. In the abstract (line 36, "significant decreases in total HIV DNA") and conclusion (lines 244-246), the authors state that total proviral DNA decreased as a result of ART intensification. However, in Figures 2A and 2E (and in line 251), the authors indicate that total proviral DNA did not change. These statements are confusing and appear to be contradictory. Regarding the decrease in total proviral DNA, I believe the authors may mean that they observed transient decrease in total proviral DNA during the intensification period (day 28 in particular, Figure 2A), however this level increases at Day 56 and then returns to baseline at Day 84, which is the source of the negative observation. Stating that total proviral DNA decreased as a result of the intervention when it ultimately did not is misleading, unless the investigators intended the day 28 timepoint as a primary endpoint for reservoir reduction - if so, this is never stated, and it is unclear why the intervention would then be continued until day 84? If, instead, reservoir reduction at the end of the intervention was the primary endpoint (again, unstated by the authors), then it is not appropriate to state that the total proviral reservoir decreased significantly when it did not.
(2) Intervention safety and tolerability: The results section lacks a specific heading for participant safety and tolerability of the intervention. I was wondering about clinically detectable viremia in the study. Were there any viral blips? Was the increased DTG well tolerated? This drug is known to cause myositis, headache, CPK elevation, hepatotoxicity, and headache. Were any of these observed? What is the authors' interpretation of the CD4:8 ratio change (line 198)? Is this a significant safety concern for a longer duration of intensification? Was there also a change in CD4% or only in absolute counts? Was there relative CD4 depletion observed in the rectal biopsy samples between days 0 and 84? Interestingly, T cells dropped at the same timepoints that reservoirs declined... how do the authors rule out that reservoir decline reflects transient T cell decline that is non-specific (not due to additional blockade of replication)?
(3) The investigators describe a decrease in intact proviral DNA after 84 days of ART intensification in circulating cells (Figure 2D), but no changes to total proviral DNA in blood or tissue (Figures 2A and 2E; IPDA does not appear to have been done on tissue samples). It is not clear why ART intensification would result in a selective decrease in intact proviruses and not in total proviruses if the source of these reservoir cells is due to ongoing replication. These reservoir results have multiple interpretations, including (but not limited to) the investigators' contention that this provides strong evidence of ongoing replication. However, ongoing replication results in the production of both intact and mutated/defective proviruses that both contribute to reservoir size (with defective proviruses vastly outnumbering intact proviruses). The small sample size and well-described heterogeneity of the HIV reservoir (with regard to overall size and composition) raise the possibility that the study was underpowered to detect differences over the 84-day intervention period. No power calculations or prior studies were described to justify the trial size or the duration of the intervention. Readers would benefit from a more nuanced discussion of reservoir changes observed here.
(4) While a few statistically significant changes occurred in immune activation markers, it is not clear that these are biologically significant. Lines 175-186 and Figure 3: The change in CD4 cells + for TIGIT looks as though it declined by only 1-2%, and at day 84, the confidence interval appears to widen significantly at this timepoint, spanning an interquartile range of 4%. The only other immune activation/exhaustion marker change that reached statistical significance appears to be CD8 cells + for CD38 and HLA-DR, however, the decline appears to be a fraction of a percent, with the control group trending in the same direction. Despite marginal statistical significance, it is not clear there is any biological significance to these findings; Figure S6 supports the contention that there is no significant change in these parameters over time or between groups. With most markers showing no change and these two showing very small changes (and the latter moving in the same direction as the control group), these results do not justify the statement that intensifying DTG decreases immune activation and exhaustion (lines 38-40 in the abstract and elsewhere).
(5) There are several limitations of the study design that deserve consideration beyond those discussed at line 327. The study was open-label and not placebo-controlled, which may have led to some medication adherence changes that confound results (authors describe one observation that may be evidence of this; lines 146-148). Randomized/blinded / cross-over design would be more robust and help determine signal from noise, given relatively small changes observed in the intervention arm. There does not seem to be a measurement of key outcome variables after treatment intensification ceased - evidence of an effect on replication through ART intensification would be enhanced by observing changes once intensification was stopped. Why was intensification maintained for 84 days? More information about the study duration would be helpful. Table 1 indicates that participants were 95% male. Sex is known to be a biological variable, particularly with regard to HIV reservoir size and chronic immune activation in PWH. Worldwide, 50% of PWH are women. Research into improving management/understanding of disease should reflect this, and equal participation should be sought in trials. Table 1 shows differing baseline reservoir sizes betweenthe control and intervention groups. This may have important implications, particularly for outcomes where reservoir size is used as the denominator.
(6) Figure 1: the increase in DTG levels is interesting - it is not uniform across participants. Several participants had lower levels of DTG at the end of the intervention. Though unlikely to be statistically significant, it would be interesting to evaluate if there is a correlation between change in DTG concentrations and virologic / reservoir / inflammatory parameters. A positive relationship between increasing DTG concentration and decreased cell-associated RNA, for example, would help support the hypothesis that ongoing replication is occurring.
(7) Figure 2: IPDA in tissue- was this done? scRNA in blood (single copy assay) - would this be expected to correlate with usCaRNA? The most unambiguous result is the decrease in cell-associated RNA - accompanying results using single-copy assay in plasma would be helpful to bolster this result. The use of the US RNA / Total DNA ratio is not helpful/difficult to interpret since the control and intervention arms were unmatched for total DNA reservoir size at study entry.
Reviewer #3 (Public review):
The introduction does a very good job of discussing the issue around whether there is ongoing replication in people with HIV on antiretroviral therapy. Sporadic, non-sustained replication likely occurs in many PWH on ART related to adherence, drug-drug interactions and possibly penetration of antivirals into sanctuary areas of replication and as the authors point out proving it does not occur is likely not possible and proving it does occur is likely very dependent on the population studied and the design of the intervention. Whether the consequences of this replication in the absence of evolution toward resistance have clinical significance challenging question to address.
It is important to note that INSTI-based therapy may have a different impact on HIV replication events that results in differences in virus release for specific cell type (those responsible for "second phase" decay) by blocking integration in cells that have completed reverse transcription prior to ART initiation but have yet to be fully activated. In a PI or NNRTI-based regimen, those cells will release virus, whereas with an INSTI-based regimen, they will not.
Given the very small sample size, there is a substantial risk of imbalance between the groups in important baseline measures. Unfortunately, with the small sample size, a non-significant P value is not helpful when comparing baseline measures between groups. One suggestion would be to provide the full range as opposed to the inter-quartile range (essentially only 5 or 6 values). The authors could also report the proportion of participants with baseline HIV RNA target not detected in the two groups.
A suggestion that there is a critical imbalance between groups is that the control group has significantly lower total HIV DNA in PBMC, despite the small sample size. The control group also has numerically longer time of continuous suppression, lower unspliced RNA, and lower intact proviral DNA. These differences may have biased the ability to see changes in DNA and US RNA in the control group. Notably, there was no significant difference in the change in US RNA/DNA between groups (Figure 2C). The fact that the median relative change appears very similar in Figure 2C, yet there is a substantial difference in P values, is also a comment on the limits of the current sample size. The text should report the median change in US RNA and US RNA/DNA when describing Figures 2A-2C. This statistical comparison of changes in IPDA results between groups should be reported. The presentation of the absolute values of all the comparisons in the supplemental figures is a strength of the manuscript.
In the assessment of ART intensification on immune activation and exhaustion, the fact that none of the comparisons between randomized groups were significant should be noted and discussed.
The changes in CD4:CD8 ratio and sCD14 levels appear counterintuitive to the hypothesis and are commented on in the discussion.
Overall, the discussion highlights the significant changes in the intensified group, which are suggestive. There is limited discussion of the comparisons between group,s where the results are less convincing.
The limitations of the study should be more clearly discussed. The small sample size raises the possibility of imbalance at baseline. The supplemental figures (S3-S5) are helpful in showing the differences between groups at baseline, and the variability of measurements is more apparent. The lack of blinding is also a weakness, though the PK assessments do help (note 3TC levels rise substantially in both groups for most of the time on study (Figure S2).
The many assays and comparisons are listed as a strength. The many comparisons raise the possibility of finding significance by chance. In addition, if there is an imbalance at baseline outcomes, measuring related parameters will move in the same direction.
The limited impact on activation and inflammation should be addressed in the discussion, as they are highlighted as a potentially important consequence of intermittent, not sustained replication in the introduction.
The study is provocative and well executed, with the limitations listed above. Pharmacokinetic analyses help mitigate the lack of blinding. The major impact of this work is if it leads to a much larger randomized, controlled, blinded study of a longer duration, as the authors point out.
Author response:
Reviewer #1 (Public Review):
Fombellida-Lopez and colleagues describe the results of an ART intensification trial in people with HIV infection (PWH) on suppressive ART to determine the effect of increasing the dose of one ART drug, dolutegravir, on viral reservoirs, immune activation, exhaustion, and circulating inflammatory markers. The authors hypothesize that ART intensification will provide clues about the degree to which low-level viral replication is occurring in circulation and in tissues despite ongoing ART, which could be identified if reservoirs decrease and/or if immune biomarkers change. The trial design is straightforward and well-described, and the intervention appears to have been well tolerated. The investigators observed an increase in dolutegravir concentrations in circulation, and to a lesser degree in tissues, in the intervention group, indicating that the intervention has functioned as expected (ART has been intensified in vivo). Several outcome measures changed during the trial period in the intervention group, leading the investigators to conclude that their results provide strong evidence of ongoing replication on standard ART. The results of this small trial are intriguing, and a few observations in particular are hypothesis-generating and potentially justify further clinical trials to explore them in depth. However, I am concerned about over-interpretation of results that do not fully justify the authors' conclusions.
We thank Reviewer #1 for their thoughtful and constructive comments, which will help us clarify and improve the manuscript. Below, we address each of the reviewer’s points and describe the changes that we intend to implement in the revised version. We acknowledge the reviewer’s concern regarding potential over-interpretation of certain findings, and we will take particular care to ensure that all conclusions are supported by the data and framed within the exploratory nature of the study.
(1) Trial objectives: What was the primary objective of the trial? This is not clearly stated. The authors describe changes in some reservoir parameters and no changes in others. Which of these was the primary outcome? No a priori hypothesis / primary objective is stated, nor is there explicit justification (power calculations, prior in vivo evidence) for the small n, unblinded design, and lack of placebo control. In the abstract (line 36, "significant decreases in total HIV DNA") and conclusion (lines 244-246), the authors state that total proviral DNA decreased as a result of ART intensification. However, in Figures 2A and 2E (and in line 251), the authors indicate that total proviral DNA did not change. These statements are confusing and appear to be contradictory. Regarding the decrease in total proviral DNA, I believe the authors may mean that they observed transient decrease in total proviral DNA during the intensification period (day 28 in particular, Figure 2A), however this level increases at Day 56 and then returns to baseline at Day 84, which is the source of the negative observation. Stating that total proviral DNA decreased as a result of the intervention when it ultimately did not is misleading, unless the investigators intended the day 28 timepoint as a primary endpoint for reservoir reduction - if so, this is never stated, and it is unclear why the intervention would then be continued until day 84? If, instead, reservoir reduction at the end of the intervention was the primary endpoint (again, unstated by the authors), then it is not appropriate to state that the total proviral reservoir decreased significantly when it did not.
We agree with the reviewer that the primary objective of the study was not explicitly stated in the submitted manuscript. We will clarify this in the revised manuscript. As registered on ClinicalTrials.gov (NCT05351684), the primary outcome was defined as “To evaluate the impact of treatment intensification at the level of total and replication-competent reservoir (RCR) in blood and in tissues”, with a time frame of 3 months. Accordingly, our aim was to explore whether any measurable reduction in the HIV reservoir (total or replication-competent) occurred during the intensification period, including at day 28, 56, or 84. The protocol did not prespecify a single time point for this effect to occur, and the exploratory design allowed for detection of transient or sustained changes within the intensification window.
We recognize that this scope was not clearly articulated in the original text and may have led to confusion in interpreting the transient drop in total HIV DNA observed at day 28. While total DNA ultimately returned to baseline by the end of intensification, the presence of a transient reduction during this 3-month window still fits within the framework of the study’s registered objective. Moreover, although the change in total HIV DNA was transient, it aligns with the consistent direction of changes observed across the multiple independent measures, including CA HIV RNA, RNA/DNA ratio and intact HIV DNA, collectively supporting a biological effect of intensification.
We would also like to stress that this is the first clinical trial ever, in which an ART intensification is performed not by adding an extra drug but by increasing the dosage of an existing drug. Therefore, we were more interested in the overall, cumulative, effect of intensification throughout the entire trial period, than in differences between groups at individual time points. We will clarify in the manuscript that this was a proof-of-concept phase 2 study, designed to generate biological signals rather than confirm efficacy in a powered comparison. The absence of a pre-specified statistical endpoint or sample size calculation reflects the exploratory nature of the trial.
(2) Intervention safety and tolerability: The results section lacks a specific heading for participant safety and tolerability of the intervention. I was wondering about clinically detectable viremia in the study. Were there any viral blips? Was the increased DTG well tolerated? This drug is known to cause myositis, headache, CPK elevation, hepatotoxicity, and headache. Were any of these observed? What is the authors' interpretation of the CD4:8 ratio change (line 198)? Is this a significant safety concern for a longer duration of intensification? Was there also a change in CD4% or only in absolute counts? Was there relative CD4 depletion observed in the rectal biopsy samples between days 0 and 84? Interestingly, T cells dropped at the same timepoints that reservoirs declined... how do the authors rule out that reservoir decline reflects transient T cell decline that is non-specific (not due to additional blockade of replication)?
We will improve the Methods section to clarify how safety and tolerability were assessed during the study. Safety evaluations were conducted on day 28 and day 84 and included a clinical examination and routine laboratory testing (liver function tests, kidney function, and complete blood count). Medication adherence was also monitored through pill counts performed by the study nurses.
No virological blips above 50 copies/mL were observed and no adverse events were reported by participants during the 3-month intensification period. Although CPK levels were not included in the routine biological monitoring, no participant reported muscle pain or other symptoms suggestive of muscle toxicity.
The CD4:CD8 ratio decrease noted during intensification was not associated with significant changes in absolute CD4 or CD8 counts, as shown in Figure 5. We interpret this ratio change as a transient redistribution rather than an immunological risk, therefore we do not consider it to represent a safety concern.
We would like to clarify that CD4<sup>+</sup> T-cell counts did not significantly decrease in any of the treatment groups, as shown in Figure 5. The apparent decline observed concerns the CD4/CD8 ratio, which transiently dropped, but not the absolute number of CD4<sup>+</sup> T cells.
(3) The investigators describe a decrease in intact proviral DNA after 84 days of ART intensification in circulating cells (Figure 2D), but no changes to total proviral DNA in blood or tissue (Figures 2A and 2E; IPDA does not appear to have been done on tissue samples). It is not clear why ART intensification would result in a selective decrease in intact proviruses and not in total proviruses if the source of these reservoir cells is due to ongoing replication. These reservoir results have multiple interpretations, including (but not limited to) the investigators' contention that this provides strong evidence of ongoing replication. However, ongoing replication results in the production of both intact and mutated/defective proviruses that both contribute to reservoir size (with defective proviruses vastly outnumbering intact proviruses). The small sample size and well-described heterogeneity of the HIV reservoir (with regard to overall size and composition) raise the possibility that the study was underpowered to detect differences over the 84-day intervention period. No power calculations or prior studies were described to justify the trial size or the duration of the intervention. Readers would benefit from a more nuanced discussion of reservoir changes observed here.
We sincerely thank the reviewer for this insightful comment. We fully agree that the reservoir dynamics observed in our study raise several possible interpretations, and that its complexity, resulting from continuous cycles of expansion and contraction, reflects the heterogeneity of the latent reservoir.
Total HIV DNA in PBMCs showed a transient decline during intensification (notably at day 28), ultimately returning to baseline by day 84. This biphasic pattern may reflect the combined effects of suppression of ongoing low-level replication by an increased DTG dosage, followed by the expansion of infected cell clones (mostly harboring defective proviruses). In other words, the transient decrease in total (intact + defective) DNA at day 28 may be due to an initial decrease in newly infected cells upon ART intensification, however at the subsequent time points this effect was masked by proliferation (clonal expansion) of infected cells with defective proviruses. This explains why the intact proviruses decreased, but the total proviruses did not change, between days 0 and 84.
Importantly, we observed a significant decrease in intact proviral DNA between day 0 and day 84 in the intensification group (Figure 2D). We will highlight this result more clearly in the revised manuscript, as it directly addresses the study’s primary objective: assessing the impact of intensification on the replication-competent reservoir. In comparison, as the reviewer rightly points out, total HIV DNA includes over 90% defective genomes, which limits its interpretability as a biomarker of biologically relevant reservoir changes.
In addition, other reservoir markers, such as cell-associated unspliced RNA and RNA/DNA ratios, also showed consistent trends supporting a modest but biologically relevant effect of intensification. Even in the absence of sustained changes in total HIV DNA, the coherence across these independent measures suggests a signal indicative of ongoing replication in at least some individuals, and at specific timepoints.
Regarding tissue reservoirs, the lack of substantial change in total HIV DNA between days 0 and 84 is also in line with the predominance of defective sequences in these compartments. Moreover, the limited increase in rectal tissue dolutegravir levels during intensification (from 16.7% to 20% of plasma concentrations) may have limited the efficacy of the intervention in this site.
As for the IPDA on rectal biopsies, we attempted the assay using two independent DNA extraction methods (Promega Reliaprep and Qiagen Puregene), but both yielded high DNA Shearing Index values, and intact proviral detection was successful in only 3 of 40 samples. Given the poor DNA integrity and weak signals, these results were not interpretable.
That said, we fully acknowledge the limitations of our study, especially the small sample size, and we agree with the reviewer that caution is needed when interpreting these findings. In the revised manuscript, we will adopt a more measured tone in the discussion, clearly stating that these observations are exploratory and hypothesis-generating, and require confirmation in larger, more powered studies. Nonetheless, we believe that the convergence of multiple reservoir markers pointing in the same direction constitutes a potentially meaningful biological signal that deserves further investigation.
(4) While a few statistically significant changes occurred in immune activation markers, it is not clear that these are biologically significant. Lines 175-186 and Figure 3: The change in CD4 cells + for TIGIT looks as though it declined by only 1-2%, and at day 84, the confidence interval appears to widen significantly at this timepoint, spanning an interquartile range of 4%. The only other immune activation/exhaustion marker change that reached statistical significance appears to be CD8 cells + for CD38 and HLA-DR, however, the decline appears to be a fraction of a percent, with the control group trending in the same direction. Despite marginal statistical significance, it is not clear there is any biological significance to these findings; Figure S6 supports the contention that there is no significant change in these parameters over time or between groups. With most markers showing no change and these two showing very small changes (and the latter moving in the same direction as the control group), these results do not justify the statement that intensifying DTG decreases immune activation and exhaustion (lines 38-40 in the abstract and elsewhere).
We agree with the reviewer that the observed changes in immune activation and exhaustion markers were modest. We will revise the manuscript to reflect this more accurately. We will also note that these differences, while statistically significant (e.g., in TIGIT+ CD4+ T cells and CD38+HLA-DR+ CD8+ T cells), were limited in magnitude. We will explicitly acknowledge these limitations and interpret the findings with appropriate caution.
(5) There are several limitations of the study design that deserve consideration beyond those discussed at line 327. The study was open-label and not placebo-controlled, which may have led to some medication adherence changes that confound results (authors describe one observation that may be evidence of this; lines 146-148). Randomized/blinded / cross-over design would be more robust and help determine signal from noise, given relatively small changes observed in the intervention arm. There does not seem to be a measurement of key outcome variables after treatment intensification ceased - evidence of an effect on replication through ART intensification would be enhanced by observing changes once intensification was stopped. Why was intensification maintained for 84 days? More information about the study duration would be helpful. Table 1 indicates that participants were 95% male. Sex is known to be a biological variable, particularly with regard to HIV reservoir size and chronic immune activation in PWH. Worldwide, 50% of PWH are women. Research into improving management/understanding of disease should reflect this, and equal participation should be sought in trials. Table 1 shows differing baseline reservoir sizes between the control and intervention groups. This may have important implications, particularly for outcomes where reservoir size is used as the denominator.
We will expand the limitations section to address several key aspects raised by the reviewer: the absence of blinding and placebo control, the predominantly male study population, and the lack of post-intervention follow-up. While we acknowledge that open-label designs can introduce behavioral biases, including potential changes in adherence, we will now explicitly state that placebo-controlled, blinded trials would provide a more robust assessment and are warranted in future research.
The 84-day duration of intensification was chosen based on previous studies and provided sufficient time for observing potential changes in viral transcription and reservoir dynamics. However, we agree that including post-intervention follow-up would have strengthened the conclusions, and we will highlight this limitation and future direction in the revised manuscript.
The sex imbalance is now clearly acknowledged as a limitation in the revised manuscript, and we fully support ongoing efforts to promote equitable recruitment in HIV research. We would like to add that, in our study, rectal biopsies were coupled with anal cancer screening through HPV testing. This screening is specifically recommended for younger men who have sex with men (MSM), as outlined in the current EACS guidelines (see: https://eacs.sanfordguide.com/eacs-part2/cancer/cancer-screening-methods). As a result, MSM participants had both a clinical incentive and medical interest to undergo this procedure, which likely contributed to the higher proportion of male participants in the study.
Lastly, although baseline total HIV DNA was higher in the intensified group, our statistical approach is based on a within-subject (repeated-measures) design, in which the longitudinal change of a parameter within the same participant during the study was the main outcome. In other words, we are not comparing absolute values of any marker between the groups, we are looking at changes of parameters from baseline within participants, and these are not expected to be affected by baseline imbalances.
(6) Figure 1: the increase in DTG levels is interesting - it is not uniform across participants. Several participants had lower levels of DTG at the end of the intervention. Though unlikely to be statistically significant, it would be interesting to evaluate if there is a correlation between change in DTG concentrations and virologic / reservoir / inflammatory parameters. A positive relationship between increasing DTG concentration and decreased cell-associated RNA, for example, would help support the hypothesis that ongoing replication is occurring.
We agree with the reviewer that assessing correlations between DTG concentrations and virological, immunological, or inflammatory markers would be highly informative. In fact, we initially explored this question in a preliminary way by examining whether individuals who showed a marked increase in DTG levels after intensification also demonstrated stronger changes in the viral reservoir. While this exploratory analysis did not reveal any clear associations, we would like to emphasize that correlating biological effects with DTG concentrations measured at a single timepoint may have limited interpretability. A more comprehensive understanding of the relationship between drug exposure and reservoir dynamics would ideally require multiple pharmacokinetic measurements over time, including pre-intensification baselines. This is particularly important given that DTG concentrations vary across individuals and over time, depending on adherence, metabolism, and other individual factors. We will clarify these points in the revised manuscript.
(7) Figure 2: IPDA in tissue- was this done? scRNA in blood (single copy assay) - would this be expected to correlate with usCaRNA? The most unambiguous result is the decrease in cell-associated RNA - accompanying results using single-copy assay in plasma would be helpful to bolster this result.
As mentioned in our response to point 3, we attempted IPDA on tissue samples, but technical limitations prevented reliable detection of intact proviruses. Regarding residual viremia, we did perform ultra-sensitive plasma HIV RNA quantification but due to a technical issue (an inadvertent PBMC contamination during plasma separation) that affected the reliability of the results we felt uncomfortable including these data in the manuscript.
The use of the US RNA / Total DNA ratio is not helpful/difficult to interpret since the control and intervention arms were unmatched for total DNA reservoir size at study entry.
We respectfully disagree with this comment. The US RNA / Total DNA ratio is commonly used to assess the relative transcriptional activity of the viral reservoir, rather than its absolute size. While we acknowledge that the total HIV-1 DNA levels differed at baseline between the two groups, the US RNA / Total DNA ratio specifically reflects the relationship between transcriptional activity and reservoir size within each individual, and is therefore not directly confounded by baseline differences in total DNA alone.
Moreover, our analyses focus on within-subject longitudinal changes from baseline, not on direct between-group comparisons of absolute marker values. As such, the observed changes in the US RNA / Total DNA ratio over time are interpreted relative to each participant's baseline, mitigating concerns related to baseline imbalances between groups.
Reviewer #2 (Public Review):
Summary:
An intensification study with a double dose of 2nd generation integrase inhibitor with a background of nucleoside analog inhibitors of the HIV retrotranscriptase in 2, and inflammation is associated with the development of co-morbidities in 20 individuals randomized with controls, with an impact on the levels of viral reservoirs and inflammation markers. Viral reservoirs in HIV are the main impediment to an HIV cure, and inflammation is associated with co-morbidities.
Strengths:
The intervention that leads to a decrease of viral reservoirs and inflammation is quite straightforward forward as a doubling of the INSTI is used in some individuals with INSTI resistance, with good tolerability.
This is a very well documented study, both in blood and tissues, which is a great achievement due to the difficulty of body sampling in well-controlled individuals on antiretroviral therapy. The laboratory assays are performed by specialists in the field with state-of-the art quantification assays. Both the introduction and the discussion are remarkably well presented and documented.
The findings also have a potential impact on the management of chronic HIV infection.
Weaknesses:
I do not think that the size of the study can be considered a weakness, nor the fact that it is open-label either.
We thank Reviewer #2 for their constructive and supportive comments. We appreciate their positive assessment of the study design, the translational relevance of the intervention, and the technical quality of the assays. We also take note of their perspective regarding sample size and study design, which supports our positioning of this trial as an exploratory, hypothesis-generating phase 2 study.
Reviewer #3 (Public Review):
The introduction does a very good job of discussing the issue around whether there is ongoing replication in people with HIV on antiretroviral therapy. Sporadic, non-sustained replication likely occurs in many PWH on ART related to adherence, drug interactions and possibly penetration of antivirals into sanctuary areas of replication and as the authors point out proving it does not occur is likely not possible and proving it does occur is likely very dependent on the population studied and the design of the intervention. Whether the consequences of this replication in the absence of evolution toward resistance have clinical significance challenging question to address.
It is important to note that INSTI-based therapy may have a different impact on HIV replication events that results in differences in virus release for specific cell type (those responsible for "second phase" decay) by blocking integration in cells that have completed reverse transcription prior to ART initiation but have yet to be fully activated. In a PI or NNRTI-based regimen, those cells will release virus, whereas with an INSTI-based regimen, they will not.
Given the very small sample size, there is a substantial risk of imbalance between the groups in important baseline measures. Unfortunately, with the small sample size, a non-significant P value is not helpful when comparing baseline measures between groups. One suggestion would be to provide the full range as opposed to the inter-quartile range (essentially only 5 or 6 values). The authors could also report the proportion of participants with baseline HIV RNA target not detected in the two groups.
We thank Reviewer #3 for their thoughtful and balanced review. We are grateful for the recognition of the strength of the Introduction, the complexity of evaluating residual replication, and the technical execution of the assays. We also appreciate the insightful suggestions for improving the clarity and transparency of our results and discussion.
We will revise the manuscript to address several of the reviewer’s key concerns. We agree that the small sample size increases the risk of baseline imbalances. We will acknowledge these limitations in the revised manuscript. We will provide both the full range and the IQR in Table 1 in the revised manuscript.
A suggestion that there is a critical imbalance between groups is that the control group has significantly lower total HIV DNA in PBMC, despite the small sample size. The control group also has numerically longer time of continuous suppression, lower unspliced RNA, and lower intact proviral DNA. These differences may have biased the ability to see changes in DNA and US RNA in the control group.
We acknowledge the significant baseline difference in total HIV DNA between groups, which we have clearly reported. However, the other variables mentioned, duration of continuous viral suppression, unspliced RNA levels, and intact proviral DNA, did not differ significantly between groups at baseline, despite differences in the median values. These numerical differences do not necessarily indicate a critical imbalance.
Notably, there was no significant difference in the change in US RNA/DNA between groups (Figure 2C).
The nonsignificant difference in the change in US RNA/DNA between groups is not unexpected, given the significant between-group differences for both US RNA and total DNA changes. Since the ratio combines both markers, it is likely to show attenuated between-group differences compared to the individual components. However, while the difference did not reach statistical significance (p = 0.09), we still observed a trend towards a greater reduction in the US RNA/Total DNA ratio in the intervention group.
The fact that the median relative change appears very similar in Figure 2C, yet there is a substantial difference in P values, is also a comment on the limits of the current sample size.
Although we surely agree that in general, the limited sample size impacts statistical power, we would like to point out that in Figure 2C, while the medians may appear similar, the ranges do differ between groups. At days 56 and 84, the median fold changes from baseline are indeed close but the full interquartile range in the DTG group stays below 1, while in the control group, the interquartile range is wider and covers approximately equal distance above and below 1. This explains the difference in p values between the groups.
The text should report the median change in US RNA and US RNA/DNA when describing Figures 2A-2C.
These data are already reported in the Results section (lines 164–166): "By day 84, US RNA and US RNA/total DNA ratio had decreased from day 0 by medians (IQRs) of 5.1 (3.3–6.4) and 4.6 (3.1–5.3) fold, respectively (p = 0.016 for both markers)."
This statistical comparison of changes in IPDA results between groups should be reported. The presentation of the absolute values of all the comparisons in the supplemental figures is a strength of the manuscript.
In the assessment of ART intensification on immune activation and exhaustion, the fact that none of the comparisons between randomized groups were significant should be noted and discussed.
We would like to point out that a statistically significant difference between the randomized groups was observed for the frequency of CD4<sup>+</sup> T cells expressing TIGIT, as shown in Figure 3A and reported in the Results section (p = 0.048).
The changes in CD4:CD8 ratio and sCD14 levels appear counterintuitive to the hypothesis and are commented on in the discussion.
Overall, the discussion highlights the significant changes in the intensified group, which are suggestive. There is limited discussion of the comparisons between groups where the results are less convincing.
We will temper the language accordingly and add commentary on the limited and modest nature of these changes. Similarly, we will expand our discussion of counterintuitive findings such as the CD4:CD8 ratio and sCD14 changes.
The limitations of the study should be more clearly discussed. The small sample size raises the possibility of imbalance at baseline. The supplemental figures (S3-S5) are helpful in showing the differences between groups at baseline, and the variability of measurements is more apparent. The lack of blinding is also a weakness, though the PK assessments do help (note 3TC levels rise substantially in both groups for most of the time on study (Figure S2).
The many assays and comparisons are listed as a strength. The many comparisons raise the possibility of finding significance by chance. In addition, if there is an imbalance at baseline outcomes, measuring related parameters will move in the same direction.
We agree that the multiple comparisons raise the possibility of chance findings but would like to stress that in an exploratory study like this it is very important to avoid a type II error. In addition, the consistent directionality of the most relevant outcomes (US RNA and intact DNA) lends biological plausibility to the observed effects.
The limited impact on activation and inflammation should be addressed in the discussion, as they are highlighted as a potentially important consequence of intermittent, not sustained replication in the introduction.
The study is provocative and well executed, with the limitations listed above. Pharmacokinetic analyses help mitigate the lack of blinding. The major impact of this work is if it leads to a much larger randomized, controlled, blinded study of a longer duration, as the authors point out.
Finally, we fully endorse the reviewer’s suggestion that the primary contribution of this study lies in its value as a proof-of-concept and foundation for future randomized, blinded trials of greater scale and duration. We will highlight this more clearly in the revised Discussion.
Reviewer #1 (Public review):
Summary:
The authors combine PSMC and habitat modeling to try to connect habitat change during the Last Glacial Period to changes in Ne.
Strengths:
Observing how tropical single-island endemic bird species responded to habitat change in the past may help inform conservation interventions for these particularly vulnerable species. The combination of genomics and habitat modeling is a good idea - this sort of interdisciplinary thinking is what is needed to tackle these complex questions. Additionally, the use of PSMC makes it possible to perform this analysis on poorly-studied species with only a single genome available.
Room for Improvement:
Why coalescent Ne is a better predictor of extinction risk than current genomic diversity, or current Ne, isn't explicitly explained. PSMC in particular has many caveats, and some are not acknowledged or adequately addressed by the authors. For example, the authors note that population structure is a confounding factor with PSMC, but that it is not a problem in this instance. They do not provide compelling evidence for why this would be the case, they simply state that the species studied are all single-island endemics. However, single-island endemic species are not necessarily panmictic; this is even less likely to be true for species studied here that inhabit a large geographic area (ie, Australian species). Differing PSMC parameters may also impact results: the differences between passerines and non-passerines were one of their main results, but they do not provide any analysis to show that this difference was not driven by the different mutation rates used for the two groups.
Parameters for many steps are not described, and choices that are described (such as the PSMC parameters) are not always fully explained. It is unclear why all data was mapped to the autosomes rather than removing reads that map to the sex chromosomes first. Using all the data, the reads belonging to the sex chromosomes could potentially map to other areas of the genome. It does not seem like a mapping quality filter was used, so these potential spurious alignments would not have been removed prior to analysis.
There are points where the results are described in ways that appear to potentially differ from the supplementary figures. The authors state that even for species where PSMC results differed between models, "trends of Ne increase or decrease from the LIG to LGM were robust across all three PSMC models considered." The figures in the supplement for Pachycephala philippinensis, Rhynochetos jubatus, and Zosterops hypoxanthus appear to potentially contradict this statement, but it is difficult to tell, as the time period observed is not clearly marked on the graphs. How this robustness of trends was determined is not explained, leaving the precision of the analysis unclear.
Table 1 also includes some information that contradicts what is in the Supplementary Tables, leading to a lack of clarity. Centropus unirufus, Chaetorhynchus papuensis, and Cnemophilus loriae are not included in Supplementary Table 4. Table 1 says Eulacestoma nigropectus, Paradisaea rubra, and Parotia lawesii did not undergo PSMC analysis, but Supplementary Table 4 says PSMC and modeling trends matched for these species. Table 1 says Rhagologus leucostigma underwent both PSMC and climate modeling, but Supplementary Table 4 says "NA" as if it was missing one of these analyses.
Additionally, some of the results appear to contradict each other. For example, they show that there is no impact of habitat change in larger-bodied species, but also that larger-bodied species saw a decrease in Ne during the LGP. In another example, they state that when a species saw an increase in habitat during the LGP, they also had an increase in Ne. However, they also state that this was not the case for non-passerines.
Ecosystems are highly complex; there may also be other variables influencing past demographic change other than those explored here. Results should be interpreted with caution.
Reviewer #3 (Public review):
Summary:
This work presents a novel neural network-based framework for parameterizing individual differences in human behavior. Using two distinct decision-making experiments, the authors demonstrate the approach's potential and claims it can predict individual behavior (1) within the same task, (2) across different tasks, and (3) across individuals. While the goal of capturing individual variability is compelling and the potential applications are promising, the claims are weakly supported, and I find that the underlying problem is conceptually ill-defined.
Strengths:
The idea of using neural networks for parameterizing individual differences in human behavior is novel, and the potential applications can be impactful.
Weaknesses:
(1) To demonstrate the effectiveness of the approach, the authors compare a Q-learning cognitive model (for the MDP task) and RTNet (for the MNIST task) against the proposed framework. However, as I understand it, neither the cognitive model nor RTNet is designed to fit or account for individual variability. If that is the case, it is unclear why these models serve as appropriate baselines. Isn't it expected that a model explicitly fitted to individual data would outperform models that do not? If so, does the observed superiority of the proposed framework simply reflect the unsurprising benefit of fitting individual variability? I think the authors should either clarify why these models constitute fair control or validate the proposed approach against stronger and more appropriate baselines.
(2) It's not very clear in the results section what it means by having a shorter within-individual distance than between-individual distances. Related to the comment above, is there any control analysis performed for this? Also, this analysis appears to have nothing to do with predicting individual behavior. Is this evidence toward successfully parameterizing individual differences? Could this be task-dependent, especially since the transfer is evaluated on exceedingly similar tasks in both experiments? I think a bit more discussion of the motivation and implications of these results will help the reader in making sense of this analysis.
(3) The authors have to better define what exactly he meant by transferring across different "tasks" and testing the framework in "more distinctive tasks". All presented evidence, taken at face value, demonstrated transferring across different "conditions" of the same task within the same experiment. It is unclear to me how generalizable the framework will be when applied to different tasks.
(4) Conceptually, it is also unclear to me how plausible it is that the framework could generalize across tasks spanning multiple cognitive domains (if that's what is meant by more distinctive). For instance, how can an individual's task performance on a Posner task predict task performance on the Cambridge face memory test? Which part of the framework could have enabled such a cross-domain prediction of task performance? I think these have to be at least discussed to some extent, since without it the future direction is meaningless.
(5) How is the negative log-likelihood, which seems to be the main metric for comparison, computed? Is this based on trial-by-trial response prediction or probability of responses, as what usually performed in cognitive modelling?
(6) None of the presented evidence is cross-validated. The authors should consider performing K-fold cross-validation on the train, test, and evaluation split of subjects to ensure robustness of the findings.
(7) The authors excluded 25 subjects (20% of the data) for different reasons. This is a substantial proportion, especially by the standards of what is typically observed in behavioral experiments. The authors should provide a clear justification for these exclusion criteria and, if possible, cite relevant studies that support the use of such stringent thresholds.
(8) The authors should do a better job of creating the figures and writing the figure captions. It is unclear which specific claim the authors are addressing with the figure. For example, what is the key message of Figure 2C regarding transfer within and across participants? Why are the stats presentation different between the Cognitive model and the EIDT framework plots? In Figure 3, it's unclear what these dots and clusters represent and how they support the authors' claim that the same individual forms clusters. And isn't this experiment have 98 subjects after exclusion, this plot has way less than 98 dots as far as I can tell. Furthermore, I find Figure 5 particularly confusing, as the underlying claim it is meant to illustrate is unclear. Clearer figures and more informative captions are needed to guide the reader effectively.
(9) I also find the writing somewhat difficult to follow. The subheadings are confusing, and it's often unclear which specific claim the authors are addressing. The presentation of results feels disorganized, making it hard to trace the evidence supporting each claim. Also, the excessive use of acronyms (e.g., SX, SY, CG, EA, ES, DA, DS) makes the text harder to parse. I recommend restructuring the results section to be clearer and significantly reducing the use of unnecessary acronyms.
for - article - substack - Annick De Witt - Toxic Polarization is killing us. A new worldview can save us - from - article - LinkedIn - Bayo Akomolafe - I am against "worldview"\ - https://hyp.is/oqgW2ivdEfCmu9M8EYHozw/www.linkedin.com/posts/bayoakomolafe_i-am-against-worldview-the-term-seems-activity-7319799984663535616-fpVW/ - to - book - Embracing Paradox, Evolving Language - https://hyp.is/go?url=https%3A%2F%2Fipfs.indy0.net%2Fipfs%2Fbafybeihk6dcr7dfruu65z5e5ze2rkeiydkmgbbpadhyulckm4afnqbtdgy&group=world - from - Substack article - Can and should expect a spiritual Revolution any time soon? - Michel Bauwens - https://hyp.is/JDDTADInEfCKmLNKpwhsng/4thgenerationcivilization.substack.com/p/can-and-should-we-expect-a-spiritual
summary - Annick de Witt takes the reader on a journey of discovery of that looks at the nuances of the complex set of entangled crisis we face today, by referring to the idea of worldviews - She shows how the quagmires now emerging are the result of interplay between three major worldviews, traditional, modern and post-modern and how each represents a partial truth that denies the partial truths held by the others - The article takes the example of Trumpism and the MAGA movement to illustrate, but the same analysis could be extended to the many different cultural worldviews found in different peoples around the globe - In particular, with Trump's recent decision to bomb Iran's nuclear facilities, tensions between the traditional Islamic culture and the West's traditional, modern and post-modern segments of society are again on the rise - The insightful analysis culminates in the proposal for an integral worldview that includes all three but transcends each one - It may be useful to introduce Annick to Greg Henrique's Unified Theory of Knowledge (UToK), - https://www.unifiedtheoryofknowledge.org/ - Gregg works with John Vervaeke that Annick has cited - Regarding Bayo Akomolafe's short LinkedIn note on the word "worldview", I respect both Annick's detailed analysis as well as Bayo's interpretation and look forward to a comparative analylsis of these two perspectives around the word "worldview" - I am also in the middle of annotating Lisa E. Maroski's book, Embracing Paradox, Evolving Language, which is salient here as well
Indyweb dev - new Plexmark - analog affirmation slider - while reading the passage I was annotating, I realised that I was in agreement with a lot of what the author was articulating. However, I have no way to indicate this match because it would be too much - this gave rise to a new Plexmark: <br /> - Have an analog control slider for each sentence that indicates - agreement on one side and - disagreement on the other side as well as a - 'don't know' button. - This gives a running indication of resonance with your own salience landscape - This can then be used in conjunction with the Indranet - If there is an indication of strong agreement, then the reader may have strong motivation to investigate that author's mindplex, - especially if there is a strong salience mismatch between the author and the reader, indicating a possible learning event
Retrospective reflections - (See below) adjacency - sacred - relationship with - free - open source - what is your relationship with the sacred? - this is the same as asking - how do you feel in your time of solitude and aloneness? - do you feel deep connection and a sense of not being lonely while you are alone? - to be alienated if not to feel disconnected with others - as it is to be disconnected with the ceaseless sacred that continuously surrounds you, from birth to death
it means integration of both traditional and modern
two central ideas of Deep Humanity praxis fit into these three worldview
death awareness is a major focus on traditional knowledge systems but
June 27, 2025 - modernism - objective reality - validation - example - personal experience - beeping electricity meter
But if the author stops short of ultimate revelation and explicit description, the imagination of the reader is allowed to make up the deficit with his or her own idea of what constitutes the ‘unspeakably’ terrible.
the oppsosite of the despriiosn of dorians portraits suggesting a sense of humanity that is ugly and unavoidable and realistic is moral precaution iOS not taken. he is instead ba real person not an actual monster and the onlyy supernatural nature of the text is reflected in that dorian is 'eternally' young.
. P
Rappelle peut être que le gouvernement doit présenter un plan de 40 Mds d'économies d'ici la mi-juillet.
Reconduire en 2026 les montants de l’ensemble des prestations sociales (minima sociaux, pensions de retraites, allocations chômage) versées à leur valeur de 2025.
Il y a plusieurs versions de l'année blanche. Là c'est la version la plus basique. Mais l'année blanche globale est censée porter sur un gel de l'ensemble de la dépense publique en valeur et pas uniquement sur les prestas indexées. C'est pour ça que le chiffre de 24 Mds d’économies est évoqué avec l'année blanche
Reviewer #1 (Public review):
In this study, Nishi et al. claim that the ratio of long-term hematopoietic stem cell (LT-HSC) versus short-term HSC (ST-HSC) determines the lineage output of HSCs and reduced ratio of ST-HSC in aged mice causes myeloid-biased hematopoiesis. Authors used Hoxb5 reporter mice to isolated LT-HSC and ST-HSC and performed molecular analyses and transplantation assays to support their arguments. How hematopoietic system becomes myeloid-biased upon aging is an important question with many implications in disease context as well. However, this study needs more definitive data.
(1) Authors' experimental designs have some caveats to definitely support their claims. Authors claimed that aged LT-HSCs have no myeloid-biased clone expansion using transplantation assays. In these experiments, authors used 10 HSCs and young mice as recipients. Given the huge expansion of old HSC by number and known heterogeneity in immunophenotypically defined HSC populations, it is questionable how 10 out of so many old HSCs (an average of 300,000 up to 500,000 cells per mouse; Mitchell et al., Nature Cell Biology, 2023) can faithfully represent old HSC population. The Hoxb5+ old HSC primary and secondary recipient mice data (Fig. 2C and D) support this concern. In addition, they only used young recipients. Considering the importance of inflammatory aged niche in the myeloid-biased lineage output, transplanting young vs old LT-HSCs into aged mice will complete the whole picture.
In response to the above comments, the authors calculated the required sample size as approximately 384 cells to represent 500,000 HSCs per old mouse. Based on the total 1260 cells used throughout the whole manuscript (Figures 2, 3, 5, 6, S3, and S6), the authors claimed that the data is reflecting old HSC behavior. However, 384 cells represent HSCs from one old mouse. Following the authors' logic, they did only 3.2 mice (1260/384) experiment for the whole manuscript to make their argument. N of 3 is not enough, especially for old mice experiments considering the heterogeneity of aged mice. Also, they did not address the comment regarding inflammatory aged niche effects.
(2) Authors' molecular data analyses need more rigor with unbiased approaches. They claimed that neither aged LT-HSCs nor aged ST-HSCs exhibited myeloid or lymphoid gene set enrichment but aged bulk HSCs, which are just a sum of LT-HSCs and ST-HSCs by their gating scheme (Fig. 4A), showed the "tendency" of enrichment of myeloid-related genes based on the selected gene set (Fig. 4D). Although the proportion of ST-HSCs is reduced in bulk HSCs upon aging, since ST-HSCs do not exhibit lymphoid gene set enrichment based on their data, it is hard to understand how aged bulk HSCs have more myeloid gene set enrichment compared to young bulk HSCs. This bulk HSC data rather suggest that there could be a trend toward certain lineage bias (although not significant) in aged LT-HSCs or ST-HSCs. Authors need to verify the molecular lineage priming of LT-HSCs and ST-HSCs using another comprehensive dataset.
(3) Although authors could not find any molecular evidence for myeloid-biased hematopoiesis from old HSCs (either LT or ST), they argued that the ratio between LT-HSC and ST-HSC causes myeloid-biased hematopoiesis upon aging based on young HSC experiments (Fig. 6). However, old ST-HSC functional data showed that they barely contribute to blood production unlike young Hoxb5- HSCs (ST-HSC) in the transplantation setting (Fig. 2). Is there any evidence that in unperturbed native old hematopoiesis, old Hoxb5- HSCs (ST-HSC) still contribute to blood production? To answer this question, authors performed additional experiments with increased cell number (Fig. S6). Although Fig. S6.D data has a statistical significance, it is questionable how biologically meaningful it is. More fundamental question is back to the representability. Can this cell number used in this experiment represent old HSC (either LT or ST) behavior?
Reviewer #2 (Public review):
In the present manuscript, Li et al. use biochemical fractionation of "RNA granules" from P5 wildtype and FMR1 knock-out mouse brains to analyze their protein/RNA content, determine a single particle cryo-EM structure of contained ribosomes, and perform ribo-seq analysis of ribosome-protected RNA fragments (RPFs). The authors conclude from these that neither the composition of the ribosome granules, nor the state of their contained ribosomes, nor the mRNA positions with high ribosome occupancy change significantly. Besides minor changes in mRNA occupancy, the one change the authors identified is a decrease in puromycylated punctae in distal neurites of cultured primary neurons of the same mice, and their enhanced resistance to different pharmacological treatments. These results directly build on their earlier work (Anadolu et al., 2023) using analogous preparations of rat brains; the authors now perform a very similar study using WT and FMR1-KO mouse brains. This is an important topic, aiming to identify the molecular underpinnings of the FMRP protein, which is the basis of a major neurological disease. Unfortunately, several limitations of this study prevent it from being more convincing in its present form.
In order to improve this study, our main suggestions are as follows:
(1) The authors equate their biochemically purified "RG" fraction with their imaging-based detection of puromycin-positive punctae. They claim essentially no differences in RGs, but detect differences in the latter (mostly their abundance and sensitivity to DHPG/HHT/Aniso). In the discussion the authors acknowledge the inconsistency between these two modalities: "An inconsistency in our findings is the loss of distal RPM puncta coupled with an increase in the immunoreactivity for S6 in the RG." and "Thus, it may be that the RG is not simply made up of ribosomes from the large liquid-liquid phase RNA granules."
How can the authors be sure that they are analysing the same entities in both modalities? A more parsimonious explanation of their results would be that, while there might be some overlap, two different entities are analyzed. Much of the main message rests on this equivalence, and I believe the authors should show its validity.
(2) The authors show that increased nuclease digestion (and magnesium concentration) led to a reduction of their RPF sizes down to levels also seen by other researchers. Analyzing these now properly digested RPFs, the authors state that the CDS coverage and periodicity drastically improved, and that spurious enrichments of secretory mRNAs, which made up one of the major fractions in their previous work, are now reduced. In my opinion, this would be more appropriately communicated as a correction to their previous work, not as a main Figure in another manuscript.
(3) The fold changes reported in Figure 7 (ranging between log2(-0.2) and log2(+0.25)) are all extremely small and in my opinion should not be used to derive claims such as "The loss of FMRP significantly affected the abundance and occupancy of FMRP-Clipped mRNAs in WT and FMR1-KO RG (Fig 7A, 7B), but not their enrichment between RG and RCs".
(4) Figure 8 / S8-1 - The authors show that ~2/3 of their reads stem from PCR duplicates, but that even after removing those, the majority of peaks remain unaltered. At the same time, Figure S8-1 shows the total number of peaks to be 615 compared with 1392 before duplicate removal. Can the authors comment on this discrepancy? In addition, the dataset with properly removed artefacts should be used for their main display item instead of the current Figure 8.
(5) Figure 9 / S9-1, the density of punctae in both WT and FMR1-KO actually increases after treatment of HHT or Anisomycin (Figure S9-1 B-C). Even if a large fraction would now be "resistant to run-off", there should not be an increase. While this effect is deemed not significant, a much smaller effect in Figure 9C is deemed significant. Can the authors explain this? Given how vastly different the sample sizes are (ranging from 23 neurites in Figures S9-1 to 5,171 neurites in Figure 9), the authors should (randomly) sample to the same size and repeat their statistical analysis again, to improve their credibility.
Author response:
Reviewer #1 (Public review):
Summary:
The authors have investigated the role of FMRP in the formation and function of RNA granules in mouse brain/cultured hippocampal neurons. Most of their results indicate that FMRP does not have a role in the formation or function of RNA granules with specific mRNAs, but may have some role in distal RNA granules in neurons and their response to synaptic stimulation. This is an important work (though the results are mostly negative) in understanding the composition and function of neuronal RNA granules. The last part of the work in cultured neurons is disjointed from the rest of the manuscript, and the results are neither convincing nor provide any mechanistic insight.
Strengths:
(1) The study is quite thorough, the methods and analysis used are robust, and the conclusion and interpretation are diligent.
(2) The comparative study of Rat and Mouse RNA granules is very helpful for future studies.
(3) The conclusion that the absence of FMRP does not affect the RNA granule composition and many of its properties in the system the authors have chosen to study is well supported by the results.
(4) The difference in the response to DHPG stimulation concerning RNA granules described here is very interesting and could provide a basis for further studies, though it has some serious technical issues.
Weaknesses:
(1) The system used for the study (P5 mouse brain or DIV 8-10 cultured neuron) is surprising, as the majority of defects in the absence of FMRP are reported in later stages (P30+ brain and DIV 14+ neurons). It is important to test if the conclusions drawn here hold good at different developmental stages.
(2) The term 'distal granules' is very vague. Since there is no structural or biochemical characterization of these granules, it is difficult to understand how they are different from the proximal granules and why FMRP has an effect only on these granules.
(3) Since the manuscript does not find any effect of FMRP on neuronal RNA granules, it does not provide any new molecular insight with respect to the function of FMRP
Thank you for your comments and for pointing out the strengths of the manuscript. Unfortunately, we will not be able to respond to point #1. The protocol for purification of the ribosomes from RNA granules does not work in older brains (See Khandjian et al, 2004 PNAS 101:13357), presumably due to the presence of large concentrations of myelin. While it would be possible to repeat our results later in culture, we have no expectation that it would be different since we do observe DHPG induction of elongation dependent, initiation independent mGLUR-LTD in later cultures (Graber et al, 2017 J. Neuroscience 37:9116)..We will strengthen this caveat in the discussion that our results are only at a snapshot of development and that it is certainly possible that different results may be seen at different times. We agree with point 2 that ‘distal granules’ is a vague term. We will remove the term and clarify that we only quantified granules larger than 50 microns from the cell soma. We do not know if these granules are distinct. We would respectfully disagree with point #3 that the study does not provide molecular insight into the function of FMRP, as disproving that FMRP is important for stalling and determining the position of stalling removes a major hypothesis about the function of FMRP, and showing that something is not true, is at least to me, providing insight.
Reviewer #2 (Public review):
In the present manuscript, Li et al. use biochemical fractionation of "RNA granules" from P5 wildtype and FMR1 knock-out mouse brains to analyze their protein/RNA content, determine a single particle cryo-EM structure of contained ribosomes, and perform ribo-seq analysis of ribosome-protected RNA fragments (RPFs). The authors conclude from these that neither the composition of the ribosome granules, nor the state of their contained ribosomes, nor the mRNA positions with high ribosome occupancy change significantly. Besides minor changes in mRNA occupancy, the one change the authors identified is a decrease in puromycylated punctae in distal neurites of cultured primary neurons of the same mice, and their enhanced resistance to different pharmacological treatments. These results directly build on their earlier work (Anadolu et al., 2023) using analogous preparations of rat brains; the authors now perform a very similar study using WT and FMR1-KO mouse brains. This is an important topic, aiming to identify the molecular underpinnings of the FMRP protein, which is the basis of a major neurological disease. Unfortunately, several limitations of this study prevent it from being more convincing in its present form.
In order to improve this study, our main suggestions are as follows:
(1) The authors equate their biochemically purified "RG" fraction with their imaging-based detection of puromycin-positive punctae. They claim essentially no differences in RGs, but detect differences in the latter (mostly their abundance and sensitivity to DHPG/HHT/Aniso). In the discussion the authors acknowledge the inconsistency between these two modalities: "An inconsistency in our findings is the loss of distal RPM puncta coupled with an increase in the immunoreactivity for S6 in the RG." and "Thus, it may be that the RG is not simply made up of ribosomes from the large liquid-liquid phase RNA granules."
How can the authors be sure that they are analysing the same entities in both modalities? A more parsimonious explanation of their results would be that, while there might be some overlap, two different entities are analyzed. Much of the main message rests on this equivalence, and I believe the authors should show its validity.
(2) The authors show that increased nuclease digestion (and magnesium concentration) led to a reduction of their RPF sizes down to levels also seen by other researchers. Analyzing these now properly digested RPFs, the authors state that the CDS coverage and periodicity drastically improved, and that spurious enrichments of secretory mRNAs, which made up one of the major fractions in their previous work, are now reduced. In my opinion, this would be more appropriately communicated as a correction to their previous work, not as a main Figure in another manuscript.
(3) The fold changes reported in Figure 7 (ranging between log2(-0.2) and log2(+0.25)) are all extremely small and in my opinion should not be used to derive claims such as "The loss of FMRP significantly affected the abundance and occupancy of FMRP-Clipped mRNAs in WT and FMR1-KO RG (Fig 7A, 7B), but not their enrichment between RG and RCs".
(4) Figure 8 / S8-1 - The authors show that ~2/3 of their reads stem from PCR duplicates, but that even after removing those, the majority of peaks remain unaltered. At the same time, Figure S8-1 shows the total number of peaks to be 615 compared with 1392 before duplicate removal. Can the authors comment on this discrepancy? In addition, the dataset with properly removed artefacts should be used for their main display item instead of the current Figure 8.
(5) Figure 9 / S9-1, the density of punctae in both WT and FMR1-KO actually increases after treatment of HHT or Anisomycin (Figure S9-1 B-C). Even if a large fraction would now be "resistant to run-off", there should not be an increase. While this effect is deemed not significant, a much smaller effect in Figure 9C is deemed significant. Can the authors explain this? Given how vastly different the sample sizes are (ranging from 23 neurites in Figures S9-1 to 5,171 neurites in Figure 9), the authors should (randomly) sample to the same size and repeat their statistical analysis again, to improve their credibility.
Thank you for your comments. We agree with the issue in point #1 that the equivalence of RPM puncta with the RG fraction is an issue and while we believe that we show in a number of ways that the two are related (anisomycin-resistant puromycylation, puromyclation only at high concentrations consistent with the hybrid state, etc), we would respectfully disagree that our main message results from the equivalence of the RPM-labeled RNA granules in neurites and the ribosomes isolated by sedimentation. We will make this point clearer in our revision. For point #2, we agree that the changes with increased nuclease is somewhat out of place in a narrative sense, but it is clearly relevant to this work. Whether or not one sees this as a ‘correction’ or an interesting point will depend on a better characterization of the structures of the stalled polysomes. My personal view is that the nuclease resistance of cleavage near the RNA entrance site is quite interesting. Since we reproduce our results with a similar nuclease treatment in mice, as reported in our previous publication, I believe the comparison could be of interest in the future and would like to retain it. We agree with point #3 and will temper these claims in our revised version. For point #4, we will determine more carefully why the number of peaks differs and switch the main and supplemental figures. We apologize for the typo in the figure legend in Figure 9, 171, not 5171. The box plot line shows the median not the average and the data is clearly skewed such that the median and average are different (i.e. there is a two-fold decrease in the average density of distal puncta between WT and FMRP, but the average density is actually slightly decreased with HHT and A, although the median increases slightly. We will now report the results in distinct modalities to clarify this, and we will reexamine the statistics to better address the skewed distribution of values in the revised version.
Summary:
Li et al describe a set of experiments to probe the role of FMRP in ribosome stalling and RNA granule composition. The authors are able to recapitulate findings from a previous study performed in rats (this one is in mice).
Strengths:
(1) The work addresses an important and challenging issue, investigating mechanisms that regulate stalled ribosomes, focusing on the role of FMRP. This is a complicated problem, given the heterogeneity of the granules and the challenges related to their purification. This work is a solid attempt at addressing this issue, which is widely understudied.
(2) The interpretation of the results could be interesting, if supported by solid data. The idea that FMRP could control the formation and release of RNA granules, rather than the elongation by stalled ribosomes is of high importance to the field, offering a fresh perspective into translational regulation by FMRP.
(3) The authors focused on recapitulating previous findings, published elsewhere (Anadolu et al., 2023) by the same group, but using rat tissue, rather than mouse tissue. Overall, they succeeded in doing so, demonstrating, among other findings, that stalled ribosomes are enriched in consensus mRNA motifs that are linked to FMRP. These interesting findings reinforce the role of FMRP in formation and stabilization of RNA granules. It would be nice to see extensive characterization of the mouse granules as performed in Figure 1 of Anadolu and colleagues, 2023.
(4) Some of the techniques incorporated aid in creating novel hypotheses, such as the ribopuromycilation assay and the cryo-EM of granule ribosomes.
Weaknesses:
(1) The RNA granule characterization needs to be more rigorous. Coomassie is not proper for this type of characterization, simply because protein weight says little about its nature. The enrichment of key proteins is not robust and seems to not reach significance in multiple instances, including S6 and UPF1. Furthermore, S6 is the only proxy used for ribosome quantification. Could the authors include at least 3 other ribosomal proteins (2 from small, 2 from large subunit)?
(2) Page 12-13 - The Gene Ontology analysis is performed incorrectly. First, one should not rank genes by their RPKM levels. It is well known that housekeeping genes such as those related to actin dynamics, molecular transport and translation are highly enriched in sequencing datasets. It is usually more informative when significantly different genes are ranked by p adjust or log2 Fold Change, then compared against a background to verify enrichment of specific processes. However, the authors found no DEGs. I would suggest the removal of this analysis, incorporation of a gene set enrichment analyses (ranked by p adjust). I further suggest that the authors incorporate a dimensionality reduction analysis to demonstrate that the lack of significance stems from biology and not experimental artifacts, such as poor reproducibility across biological replicates.
Thank you for your comments on the strengths of the manuscript. We agree with point #1 that the mouse RNA granule characterization needs to be more rigorous and we plan to accomplish this in our revised version. Similarly, we will incorporate the additional statistical analysis suggested by the reviewer in a revised version.
Document de Synthèse : Regards Croisés sur le Temps Long de l'Action Publique
Les interventions de * Math Anton (Secrétaire d'État, Ministère des Affaires Étrangères de Finlande), * Sophie Péder (Cheffe du bureau parisien du magazine The Economist) et * Pierre Rosanvallon (Professeur Émérite au Collège de France) ont éclairé cette problématique sous des angles historiques, comparatifs et pratiques.
I. Le Poids du Court Terme et ses Racines Historiques
Pierre Rosanvallon a d'emblée mis en lumière le phénomène du "présentisme", caractéristique du monde moderne, et les raisons profondes de la préférence pour le court terme en démocratie.
La politique pré-démocratique était "nécessairement inscrite dans une durée et dans une forme de perpétuité même". * La rupture démocratique : La modernité démocratique introduit une "double rupture dans les cadres politiques et disons dans les cadres mentaux qui permettaient d'inscrire le long terme dans le court terme". Le problème n'est pas simplement de penser le long terme, mais de savoir "comment on intègre le long terme dans le court terme". * Manifestations de la préférence pour le court terme : * Dans le champ politique : Volonté d'éliminer le temps long, avec des mandats très courts pendant la Révolution française (mandats parlementaires d'un an, président de l'Assemblée changé toutes les semaines).
La modernité est perçue comme une "invention permanente" de la société. * En termes sociologiques : Détachement de l'individu des corps (corporations, couple) qui le rattachent au passé et l'enchaînent à l'avenir (ex: instauration du divorce). * Dans le rapport à la nature : Rupture avec l'idée de perpétuité de la nature (ex: abrogation de l'édit de Colbert sur les forêts de 1669 par la Constituante, affirmant le droit de propriété comme droit d'agir immédiatement au court terme).
Tentatives d'organisation des compromis :
II. L'Intéressement à l'Avenir : Un Enjeu Crucial
Pierre Rosanvallon a souligné un défi majeur : "ce qui fait le plus défaut (...) c'est le fait que les individus démocratiques n'intègrent pas eux-mêmes le long terme".
La connaissance des problèmes (ex: climat) est souvent suffisante, mais l'inaction persiste.
Critique des modèles classiques :
III. Expériences Nationales et Leçons Comparatives
Les interventions ont offert un éclairage sur les approches de la Finlande et du Royaume-Uni, contrastant souvent avec les pratiques françaises.
A. Le Modèle Finlandais : Survivre pour Réussir
L'ambassadeur Math Anton a présenté le cas de la Finlande, souvent citée en exemple pour sa capacité à penser le temps long, illustrant le mot d'ordre : "survivre pour réussir".
Institutions et pratiques favorisant le temps long :
Responsabilisation des acteurs :
Institutions dédiées au temps long :
B. Le Cas du Royaume-Uni : Le "Muddling Through" et les Défis du Brexit
Sophie Péder a décrit une culture britannique du "muddling through" (faire avec les moyens du bord) plutôt que de la vision à long terme, avec des conséquences notables pour l'action publique.
IV. Défis et Perspectives pour l'Action Publique
Plusieurs défis majeurs ont été soulevés, invitant à une réflexion approfondie sur les réponses politiques à apporter.
En conclusion, la réflexion sur le temps long dans l'action publique est une question complexe, dont les réponses résident moins dans la création de nouvelles institutions que dans la promotion de fonctionnalités, la responsabilisation de tous les acteurs (étatiques, sociaux, locaux, individuels), une meilleure éducation et un engagement scientifique accessible, afin d'organiser une "familiarité avec les temporalités" nécessaire pour relever les défis futurs.
Note de synthèse : Temps Long et Outils de l'Action Publique
Introduction
Thèmes Majeurs et Idées Clés
Conclusion
La conférence a mis en lumière un consensus sur les conditions nécessaires à une action publique efficace à long terme : un diagnostic partagé et basé sur des faits, la fixation d'un cap clair et sa continuité, et l'embarquement de l'ensemble des acteurs et de la population.
Les défis majeurs identifiés incluent la "dictature de l'immédiateté" médiatique et politique, la bureaucratisation et le cloisonnement de l'administration, et la difficulté à assurer la continuité des financements et des équipes politiques.
Un appel a été lancé pour repenser le fonctionnement même de la démocratie, en renforçant la confiance envers les acteurs locaux et la société civile, et en encourageant des compromis qui engagent sur la durée.
Document de synthèse : Temps long et urgences climatiques – Perspectives croisées
Ce document de synthèse vise à analyser les thèmes principaux et les idées ou faits les plus importants issus de la conférence "Troisième conférence : temps long et urgences climatiques", organisée dans le cadre du rapport annuel du Conseil d'État sur l'État stratège et le temps long dans les politiques publiques.
Les interventions de
ont offert des perspectives complémentaires sur l'urgence climatique, la planification écologique, la participation citoyenne et le rôle du secteur de l'assurance.
Thèmes principaux et idées clés :
1. L'Urgence Climatique et la Rupture Anthropique (Perspective Scientifique)
Valérie Masson-Delmotte, climatologue, souligne la rupture fondamentale que représente l'influence humaine sur le climat, visible à travers l'analyse des carottages glaciaires remontant à 800 000 ans.
Elle met en évidence une "augmentation brutale et rapide par rapport aux variations naturelles de la teneur des principaux gaz à effet de serre dans l'atmosphère, notamment le dioxyde de carbone émis par la combustion d'énergie fossile".
2. La Planification Écologique et ses Défis (Perspective de l'État)
Antoine Pellion, Secrétaire général à la planification écologique, détaille la démarche française pour coordonner l'action publique face aux enjeux écologiques, en lien avec le Premier ministre.
3. Le Rôle Crucial de la Démocratie Environnementale (Perspective de la Société Civile)
Antoine Gaté, représentant de France Nature Environnement, insiste sur le rôle des associations dans la protection de l'environnement et met en garde contre un "backlash écologique".
4. Le Secteur de l'Assurance face aux Risques Climatiques (Perspective Économique)
Florence Lustman, Présidente de France Assureurs, partage la vision des assureurs, à la fois gestionnaires de risques à long terme et acteurs de la réparation immédiate.
En conclusion, cette conférence a mis en évidence l'ampleur et la complexité des défis posés par l'urgence climatique. Si des outils et des stratégies sont en place, leur efficacité dépendra de la capacité à surmonter les tensions (entre temps long et court, entre objectifs sectoriels et systémiques, entre choix politiques et délibération citoyenne) et à mobiliser l'ensemble de la société autour d'une vision partagée et d'actions concrètes, en s'appuyant sur la science et en renforçant la démocratie environnementale.
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Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
Summary:
The authors report a study on how stimulation of receptive-field surround of V1 and LGN neurons affects their firing rates. Specifically, they examine stimuli in which a grey patch covers the classical RF of the cell and a stimulus appears in the surround. Using a number of different stimulus paradigms they find a long latency response in V1 (but not the LGN) which does not depend strongly on the characteristics of the surround grating (drifting vs static, continuous vs discontinuous, predictable grating vs unpredictable pink noise). They find that population responses to simple achromatic stimuli have a different structure that does not distinguish so clearly between the grey patch and other conditions and the latency of the response was similar regardless of whether the center or surround was stimulated by the achromatic surface. Taken together they propose that the surround-response is related to the representation of the grey surface itself. They relate their findings to previous studies that have put forward the concept of an ’inverse RF’ based on strong responses to small grey patches on a full-screen grating. They also discuss their results in the context of studies that suggest that surround responses are related to predictions of the RF content or figure-ground segregation. Strengths:
I find the study to be an interesting extension of the work on surround stimulation and the addition of the LGN data is useful showing that the surround-induced responses are not present in the feedforward path. The conclusions appear solid, being based on large numbers of neurons obtained through Neuropixels recordings. The use of many different stimulus combinations provides a rich view of the nature of the surround-induced responses.
Weaknesses:
The statistics are pooled across animals, which is less appropriate for hierarchical data. There is no histological confirmation of placement of the electrode in the LGN and there is no analysis of eye or face movements which may have contributed to the surround-induced responses. There are also some missing statistics and methods details which make interpretation more difficult.
We thank the reviewer for their positive and constructive comments, and have addressed these specific issues in response to the minor comments. For the statistics across animals, we refer to “Reviewer 1 recommendations” point 1. For the histological analysis, we refer to “Reviewer 1 recommendations point 2”. For the eye and facial movements, we refer to “Reviewer 1 recommendations point 5”. Concerning missing statistics and methods details, we refer to various responses to “Reviewer 1 recommendations”. We thoroughly reviewed the manuscript and included all missing statistical and methodological details.
Reviewer #2 (Public review):
Cuevas et al. investigate the stimulus selectivity of surround-induced responses in the mouse primary visual cortex (V1). While classical experiments in non-human primates and cats have generally demonstrated that stimuli in the surround receptive field (RF) of V1 neurons only modulate activity to stimuli presented in the center RF, without eliciting responses when presented in isolation, recent studies in mouse V1 have indicated the presence of purely surround-induced responses. These have been linked to prediction error signals. In this study, the authors build on these previous findings by systematically examining the stimulus selectivity of surround-induced responses.
Using neuropixels recordings in V1 and the dorsal lateral geniculate nucleus (dLGN) of head-fixed, awake mice, the authors presented various stimulus types (gratings, noise, surfaces) to the center and surround, as well as to the surround only, while also varying the size of the stimuli. Their results confirm the existence of surround-induced responses in mouse V1 neurons, demonstrating that these responses do not require spatial or temporal coherence across the surround, as would be expected if they were linked to prediction error signals. Instead, they suggest that surround-induced responses primarily reflect the representation of the achromatic surface itself.
The literature on center-surround effects in V1 is extensive and sometimes confusing, likely due to the use of different species, stimulus configurations, contrast levels, and stimulus sizes across different studies. It is plausible that surround modulation serves multiple functions depending on these parameters. Within this context, the study by Cuevas et al. makes a significant contribution by exploring the relationship between surround-induced responses in mouse V1 and stimulus statistics. The research is meticulously conducted and incorporates a wide range of experimental stimulus conditions, providing valuable new insights regarding center-surround interactions.
However, the current manuscript presents challenges in readability for both non-experts and experts. Some conclusions are difficult to follow or not clearly justified.
I recommend the following improvements to enhance clarity and comprehension:
(1) Clearly state the hypotheses being tested at the beginning of the manuscript.
(2) Always specify the species used in referenced studies to avoid confusion (esp. Introduction and Discussion).
(3) Briefly summarize the main findings at the beginning of each section to provide context.
(4) Clearly define important terms such as “surface stimulus” and “early vs. late stimulus period” to ensure understanding.
(5) Provide a rationale for each result section, explaining the significance of the findings.
(6) Offer a detailed explanation of why the results do not support the prediction error signal hypothesis but instead suggest an encoding of the achromatic surface.
These adjustments will help make the manuscript more accessible and its conclusions more compelling.
We thank the reviewer for their constructive feedback and for highlighting the need for improved clarity regarding the hypotheses and their relation to the experimental findings.
• We have strongly improved the Introduction and Discussion section, explaining the different hypotheses and their relation to the performed experiments.
• In the Introduction, we have clearly outlined each hypothesis and its predictions, providing a structured framework for understanding the rationale behind our experimental design. • In the Discussion, we have been more explicit in explaining how the experimental findings inform these hypotheses.
• We explicitly mentioned the species used in the referenced studies.
• We provided a clearer rationale for each experiment in the Results section.
We have also always clearly stated the species that previous studies used, both in the Introduction and Discussion section.
Reviewer #3 (Public review):
Summary:
This paper explores the phenomenon whereby some V1 neurons can respond to stimuli presented far outside their receptive field. It introduces three possible explanations for this phenomenon and it presents experiments that it argues favor the third explanation, based on figure/ground segregation.
Strengths:
I found it useful to see that there are three possible interpretations of this finding (prediction error, interpolation, and figure/ground). I also found it useful to see a comparison with LGN responses and to see that the effect there is not only absent but actually the opposite: stimuli presented far outside the receptive field suppress rather than drive the neurons. Other experiments presented here may also be of interest to the field.
Weaknesses:
The paper is not particularly clear. I came out of it rather confused as to which hypotheses were still standing and which hypotheses were ruled out. There are numerous ways to make it clearer.
We thank the reviewer for their constructive feedback and for highlighting the need for improved clarity regarding the hypotheses and their relation to the experimental findings.
• We have strongly improved the Introduction and Discussion section, explaining the different hypotheses and their relation to the performed experiments.
• In the Introduction, we have clearly outlined each hypothesis and its predictions, providing a structured framework for understanding the rationale behind our experimental design. • In the Discussion, we have been more explicit in explaining how the experimental findings inform these hypotheses.
** Recommendations for the Authors:**
Reviewer #1 (Recommendations for the Authors):
(1) Given the data is hierarchical with neurons clustered within 6 mice (how many recording sessions per animal?) I would recommend the use of Linear Mixed Effects models. Simply pooling all neurons increases the risk of false alarms.
To clarify: We used the standard method for analyzing single-unit recordings, by comparing the responses of a population of single neurons between two different conditions. This means that the responses of each single neuron were measured in the different conditions, and the statistics were therefore based on the pairwise differences computed for each neuron separately. This is a common and standard procedure in systems neuroscience, and was also used in the previous studies on this topic (Keller et al., 2020; Kirchberger et al., 2023). We were not concerned with comparing two groups of animals, for which hierarchical analyses are recommended. To address the reviewer’s concern, we did examine whether differences between baseline and the gray/drift condition, as well as the gray/drift compared to the grating condition, were consistent across sessions, which was indeed the case. These findings are presented in Supplementary Figure 6.
(2) Line 432: “The study utilized three to eight-month-old mice of both genders”. This is confusing, I assume they mean six mice in total, please restate. What about the LGN recordings, were these done in the same mice? Can the authors please clarify how many animals, how many total units, how many included units, how many recording sessions per animal, and whether the same units were recorded in all experiments?
We have now clarified the information regarding the animals used in the Methods section.
• We state that “We included female and male mice (C57BL/6), a total of six animals for V1 recordings between three and eight months old. In two of those animals, we recorded simultaneously from LGN and V1.”
• We state that“For each animal, we recorded around 2-3 sessions from each hemisphere, and we recorded from both hemispheres.”
• We noted that the number of neurons was not mentioned for each figure caption. We apologize for this omission. We have now added the number for all of the figures and protocols to the revised manuscript. We note that the same neurons were recorded for the different conditions within each protocol, however because a few sessions were short we recorded more units for the grating protocol. Note that we did not make statistical comparisons between protocols.
(3) I see no histology for confirmation of placement of the electrode in the LGN, how can they be sure they were recording from the LGN? There is also little description of the LGN experiments in the methods.
For better clarity, we have included a reconstruction of the electrode track from histological sections of one animal post-experiment (Figure S4). The LGN was targeted via stereotactical surgery, and the visual responses in this area are highly distinct. In addition, we used a flash protocol to identify the early-latency responses typical for the LGN, which is described in the Methods section: “A flash stimulus was employed to confirm the locations of LGN at the beginning of the recording sessions, similar to our previous work in which we recorded from LGN and V1 simultaneously (Schneider et al., 2023). This stimulus consisted of a 100 ms white screen and a 2 s gray screen as the inter-stimulus interval, designed to identify visually responsive areas. The responses of multi-unit activity (MUA) to the flash stimulus were extracted and a CSD analysis was then performed on the MUA, sampling every two channels. The resulting CSD profiles were plotted to identify channels corresponding to the LGN. During LGN recordings, simultaneous recordings were made from V1, revealing visually responsive areas interspersed with non-responsive channels.”
(4) Many statements are not backed up by statistics, for example, each time the authors report that the response at 90degree sign is higher than baseline (Line 121 amongst other places) there is no test to support this. Also Line 140 (negative correlation), Line 145, Line 180.
For comparison purposes, we only presented statistical analyses across conditions. However, we have now added information to the figure captions stating that all conditions show values higher than the baseline.
(5) As far as I can see there is no analysis of eye movements or facial movements. This could be an issue, for example, if the onset of the far surround stimuli induces movements this may lead to spurious activations in V1 that would be interpreted as surround-induced responses.
To address this point, we have included a supplementary figure analyzing facial movements across different sessions and comparing them between conditions (Supplementary Figure 5). A detailed explanation of this analysis has been added to the Methods section. Overall, we observed no significant differences in face movements between trials with gratings, trials with the gray patch, and trials with the gray screen presented during baseline. Animals exhibited similar face movements across all three conditions, supporting the conclusion that the observed neural firing rate increases for the gray-patch condition are not related to face movements.
(6) The experiments with the rectangular patch (Figure 3) seem to give a slightly different result as the responses for large sizes (75, 90) don’t appear to be above baseline. This condition is also perceptually the least consistent with a grey surface in the RF, the grey patch doesn’t appear to occlude the surface in this condition. I think this is largely consistent with their conclusions and it could merit some discussion in the results/discussion section.
While the effect is maybe a bit weaker, the total surround stimulated also covers a smaller area because of the large rectangular gray patch. Furthermore, the early responses are clearly elevated above baseline, and the responses up to 70 degrees are still higher than baseline. Hence we think this data point for 90 degrees does not warrant a strong interpretation.
Minor points:
(1) Figure 1h: What is the statistical test reported in the panel (I guess a signed rank based on later figures)? Figure 4d doesn’t appear to be significantly different but is reported as so. Perhaps the median can be indicated on the distribution?
We explained that we used a signed rank test for Figure 1h and now included the median of the distributions in Figure 4d.
(2) What was the reason for having the gratings only extend to half the x-axis of the screen, rather than being full-screen? This creates a percept (in humans at least) that is more consistent with the grey patch being a hole in the grating as the grey patch has the same luminance as the background outside the grating.
We explained in the Methods section that “We presented only half of the x-axis due to the large size of our monitor, in order to avoid over-stimulation of the animals with very large grating stimuli.”. Perceptually speaking, the gray patch appears as something occluding the grating, not as a “hole”.
(3) Line 103: “and, importantly, had less than 10degree sign (absolute) distance to the grating stimulus’ RF center.” Re-phrase, a stimulus doesn’t have an RF center.
We corrected this to “We included only single units into the analysis that met several criteria in terms of visual responses (see Methods) and, importantly, the RF center had less than 10(absolute) distance to the grating stimulus’ center. ”.
(4) Line 143: “We recorded single neurons LGN” - should be “single LGN neurons”.
We corrected this to “we recorded single LGN neurons”.
(5) Line 200: They could spell out here that the latency is consistent with the latency observed for the grey patch conditions in the previous experiments. (6) Line 465: This is very brief. What criteria did they use for single-unit assignation? Were all units well-isolated or were multi-units included?
We clarified in the Methods section that “We isolated single units with Kilosort 2.5 (Steinmetz et al., 2021) and manually curated them with Phy2 (Rossant et al., 2021). We included only single units with a maximum contamination of 10 percent.”
(7) Line 469: “The experiment was run on a Windows 10”. Typo.
We corrected this to “The experiment was run on Windows 10”.
(9) Line 481: “We averaged the response over all trials and positions of the screen”. What do they mean by ’positions of the screen’?
We changed this to “We computed the response for each position separately right, by averaging the response across all the trials where a square was presented at a given position.”
(9) Line 483: “We fitted an ellipse in the center of the response”. How?
We additionally explain how we preferred the detection of the RF using an ellipse fitting: “A heatmap of the response was computed. This heatmap was then smoothed, and we calculated the location of the peak response. From the heatmap we calculated the centroid of the response using the function regionprops.m that finds unique objects, we then selected the biggest area detected. Using the centroids provided as output. We then fitted an ellipse centered on this peak response location to the smoothed heatmap using the MATLAB function ellipse.m.“
(10) Line 485 “...and positioned the stimulus at the response peak previously found”. Unclear wording, do you mean the center of the ellipse fit to the MUA response averaged across channels or something else? (11) Line 487: “We performed a permutation test of the responses inside the RF detected vs a circle from the same area where the screen was gray for the same trials.”. The wording is a bit unclear here, can they clarify what they mean by the ’same trials’, what is being compared to what here?
We used a permutation test to compare the neuron’s responses to black and white squares inside the RF to the condition where there was no square in the RF (i.e. the RF was covered by the gray background).
(12) Was the pink noise background regenerated on each trial or as the same noise pattern shown on each trial?
We explain that “We randomly presented one of two different pink noise images”
(13) Line 552: “...used a time window of the Gaussian smoothing kernel from-.05 to .05”. Missing units.
We explained that “we used a time window of the Gaussian smoothing kernel from -.05 s to .05 s, with a standard deviation of 0.0125 s.”
(14) Line 565: “Additionally, for the occluded stimulus, we included patch sizes of 70 degree sign and larger.”. Not sure what they’re referring to here.
We changed this to: “For the population analyses, we analyzed the conditions in which the gray patch sizes were 70 degrees and 90 degrees”.
(15) Line 569: What is perplexity, and how does changing it affect the t-SNE embeddings?
Note that t-SNE is only used for visualization purposes. In the revised manuscript, we have expanded our explanation regarding the use of t-SNE and the choice of perplexity values. Specifically, we have clarified that we used a perplexity value of 20 for the Gratings with circular and rectangular occluders and 100 for the black-and-white condition. These values were empirically selected to ensure that the groups in the data were clearly separable while maintaining the balance between local and global relationships in the projected space. This choice allowed us to visually distinguish the different groups while preserving the meaningful structure encoded in the dissimilarity matrices. In particular, varying the perplexity values would not alter the conclusions drawn from the visualization, as t-SNE does not affect the underlying analytical steps of our study.
(16) Line 572: “We trained a C-Support Vector Classifier based on dissimilarity matrices”. This is overly brief, please describe the construction of the dissimilarity matrices and how the training was implemented. Was this binary, multi-class? What conditions were compared exactly?
In the revised manuscript, we have expanded our explanation regarding the construction of the dissimilarity matrices and the implementation of the C-Support Vector Classification (C-SVC) model (See Methods section).
The dissimilarity matrices were calculated using the Euclidean distance between firing rate vectors for all pairs of trials (as shown in Figure 6a-b). These matrices were used directly as input for the classifier. It is important to note that t-SNE was not used for classification but only for visualization purposes. The classifier was binary, distinguishing between two classes (e.g., Dr vs St). We trained the model using 60% of the data for training and used 40% for testing. The C-SVC was implemented using sklearn, and the classification score corresponds to the average accuracy across 20 repetitions.
Reviewer #2 (Recommendations for the Authors):
The relationship between the current paper and Keller et al. is challenging to understand. It seems like the study is critiquing the previous study but rather implicitly and not directly. I would suggest either directly stating the criticism or presenting the current study as a follow-up investigation that further explores the observed effect or provides an alternative function. Additionally, defining the inverse RF versus surround-induced responses earlier than in the discussion would be beneficial. Some suggestions:
(1) The introduction is well-written, but it would be helpful to clearly define the hypotheses regarding the function of surround-induced responses and revisit these hypotheses one by one in the results section.
Indeed, we have generally improved the Introduction of the manuscript, and stated the hypotheses and their relationships to the Experiments more clearly.
(2) Explicitly mention how you compare classic grating stimuli of varying sizes with gray patch stimuli. Do the patch stimuli all come with a full-field grating? For the full-field grating, you have one size parameter, while for the patch stimuli, you have two (size of the patch and size of the grating).
We now clearly describe how we compare grating stimuli of varying sizes with gray patch stimuli.
(3) The third paragraph in the introduction reads more like a discussion and might be better placed there.
We have moved content from the third paragraph of the Introduction to the Discussion, where it fits more naturally.
(4) Include 1-2 sentences explaining how you center RFs and detail the resolution of your method.
We have added an explanation to the Methods: “To center the visual stimuli during the recording session, we averaged the multiunit activity across the responsive channels and positioned the stimulus at the center of the ellipse fit to the MUA response averaged across channels.”.
(5) Motivate the use of achromatic stimuli. This section is generally quite hard to understand, so try to simplify it.
We explained better in the Introduction why we performed this particular experiment.
(6) The decoding analysis is great, but it is somewhat difficult to understand the most important results. Consider summarizing the key findings at the beginning of this section.
We now provide a clearer motivation at the start of the Decoding section.
Reviewer #3 (Recommendations for the Authors):
I have a few suggestions to improve the clarity of the presentation.
Abstract: it lists a series of observations and it ends with a conclusion (“based on these findings...”). However, it provides little explanation for how this conclusion would arise from the observations. It would be more helpful to introduce the reasoning at the top and show what is consistent with it.
We have improved the abstract of the paper incorporating this feedback.
To some extent, this applies to Results too. Sometimes we are shown the results of some experiment just because others have done a similar experiment. Would it be better to tell us which hypotheses it tests and whether the results are consistent with all 3 hypotheses or might rule one or more out? I came out of the paper rather confused as to which hypotheses were still standing and which hypotheses were ruled out.
We have strongly improved our explanation of the hypotheses and the relationships to the experiments in the Introduction.
It would be best if the Results section focused on the results of the study, without much emphasis on what previous studies did or did not measure. Here, instead, in the middle of Results we are told multiple times what Keller et al. (2020) did or did not measure, and what they did or did not find. Please focus on the questions and on the results. Where they agree or disagree with previous papers, tell us briefly that this is the case.
We have revised the Results section in the revised manuscript, and ensured that there is much less focus on what previous studies did in the Results. Differences to previous work are now discussed in the Discussion section.
The notation is extremely awkward. For instance “Gc” stands for two words (Gray center) but “Gr” stands for a single word (Grating). The double meaning of G is one of many sources of confusion.
This notation needs to be revised. Here is one way to make it simpler: choose one word for each type of stimulus (e.g. Gray, White, Black, Drift, Stat, Noise) and use it without abbreviations. To indicate the configuration, combine two of those words (e.g. Gray/Drift for Gray in the center and Drift in the surround).
We have corrected the notation in the figures and text to enhance readability and improve the reader’s understanding.
Figure 1e and many subsequent ones: it is not clear why the firing rate is shown in a logarithmic scale. Why not show it in a linear scale? Anyway, if the logarithmic scale is preferred for some reason, then please give us ticks at numbers that we can interpret, like 0.1,1,10,100... or 0.5,1,2,4... Also, please use the same y-scale across figures so we can compare.
To clarify: it is necessary to normalize the firing rates relative to baseline, in order to pool across neurons. However such a divisive normalization would be by itself problematic, as e.g. a change from 1 to 2 is the same as a change from 1 to 0.5, on a linear scale. Furthermore such division is highly outlier sensitive. For this reason taking the logarithm (base 10) of the ratio is an appropriate transformation. We changed the tick labels to 1, 2, 4 like the reviewer suggested.
Figure 3: it is not clear what “size” refers to in the stimuli where there is no gray center. Is it the horizontal size of the overall stimulus? Some cartoons might help. Or just some words to explain.
Figure 3: if my understanding of “size” above is correct, the results are remarkable: there is no effect whatsoever of replacing the center stimulus with a gray rectangle. Shouldn’t this be remarked upon?
We have added a paragraph under figure 3 and in the Methods section explaining that the sizes represent the varying horizontal dimensions of the rectangular patch. In this protocol, the classical condition (i.e. without gray patch) was shown only as full-field gratings, which is depicted in the plot as size 0, indicating no rectangular patch was present.
DETAILS The word “achromatic” appears many times in the paper and is essentially uninformative (all stimuli in this study are achromatic, including the gratings). It could be removed in most places except a few, where it is actually used to mean “uniform”. In those cases, it should be replaced by “uniform”.
Ditto for the word “luminous”, which appears twice and has no apparent meaning. Please replace it with “uniform”.
We have replaced the words achromatic and luminous with “uniform” stimuli to improve the clarity when we refer to only black or white stimuli.
Page 3, line 70: “We raise some important factors to consider when describing responses to only surround stimulation.” This sentence might belong in the Discussion but not in the middle of a paragraph of Results.
We removed this sentence.
Neuropixel - Neuropixels (plural)
“area LGN” - LGN
We corrected for misspellings.
References
Keller, A.J., Roth, M.M., Scanziani, M., 2020. Feedback generates a second receptive field in neurons of the visual cortex. Nature 582, 545–549. doi:10.1038/s41586-020-2319-4.
Kirchberger, L., Mukherjee, S., Self, M.W., Roelfsema, P.R., 2023. Contextual drive of neuronal responses in mouse V1 in the absence of feedforward input. Science Advances 9, eadd2498. doi:10. 1126/sciadv.add2498.
Rossant, C., et al., 2021. phy: Interactive analysis of large-scale electrophysiological data. https://github.com/cortex-lab/phy.
Schneider, M., Tzanou, A., Uran, C., Vinck, M., 2023. Cell-type-specific propagation of visual flicker. Cell Reports 42.
Steinmetz, N.A., Aydin, C., Lebedeva, A., Okun, M., Pachitariu, M., Bauza, M., Beau, M., Bhagat, J., B¨ohm, C., Broux, M., Chen, S., Colonell, J., Gardner, R.J., Karsh, B., Kloosterman, F., Kostadinov, D., Mora-Lopez, C., O’Callaghan, J., Park, J., Putzeys, J., Sauerbrei, B., van Daal,R.J.J., Vollan, A.Z., Wang, S., Welkenhuysen, M., Ye, Z., Dudman, J.T., Dutta, B., Hantman, A.W., Harris, K.D., Lee, A.K., Moser, E.I., O’Keefe, J., Renart, A., Svoboda, K., H¨ausser, M., Haesler, S., Carandini, M., Harris, T.D., 2021. Neuropixels 2.0: A miniaturized high-density probe for stable, long-term brain recordings. Science 372, eabf4588. doi:10.1126/science.abf4588.
Reviewer #3 (Public review):
Smit and Robbins' manuscript investigates the dynamics of aggression among female groupmates across five gorilla groups. The authors utilize longitudinal data to examine how reproductive state, group size, presence of males, and resource availability influence patterns of aggression and overall dominance rankings as measured by Elo scores. The findings underscore the important role of group composition and reproductive status, particularly pregnancy, in shaping dominance relationships in wild gorillas. While the study addresses a compelling and understudied topic, I have several comments and suggestions that may enhance clarity and improve the reader's experience.
(1) Clarification of longitudinal data - The manuscript states that 25 years of behavioral data were used, but this number appears unclear. Based on my calculations, the maximum duration of behavioral observation for any one group appears to be 18 years. Specifically: - ATA: 6 years - BIT: 8 years - KYA: 18 years - MUK: 6 years - ORU: 8 years I recommend that the authors clarify how the 25-year duration was derived.
(2) Consideration of group size - The authors mention that group size was excluded from analyses to avoid conflating the opposing effects of female and male group members. While this is understandable, it may still be beneficial to explore group size effects in supplementary analyses. I suggest reporting statistics related to group size and potentially including a supplementary figure. Additionally, given that the study includes both mountain and wild gorillas, it would be helpful to examine whether any interspecies differences are apparent.
(3) Behavioral measures clarification - Lines 112-116 describe the types of aggressive behaviors observed. It would be helpful to clarify how these behaviors differ from those used to calculate Elo scores, or whether they overlap. A brief explanation would improve transparency regarding the methodology.
(4) Aggression rates versus Elo scores - The manuscript uses aggression rates rather than dominance rank (as measured by Elo scores) as the main outcome variable, but there is no explanation on why. How would the results differ if aggression rates were replaced or supplemented with Elo scores? The current justification for prioritizing aggression rates over dominance rank needs to be more clearly supported.
Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
This work aims to improve our understanding of the factors that influence female-on-female aggressive interactions in gorilla social hierarchies, using 25 years of behavioural data from five wild groups of two gorilla species. Researchers analysed aggressive interactions between 31 adult females, using behavioural observations and dominance hierarchies inferred through Elo-rating methods. Aggression intensity (mild, moderate, severe) and direction (measured as the rank difference between aggressor and recipient) were used as key variables. A linear mixed-effects model was applied to evaluate how aggression direction varied with reproductive state (cycling, trimester-specific pregnancy, or lactation) and sex composition of the group. This study highlights the direction of aggressive interactions between females, with most interactions being directed from higher- to lower-ranking adult females close in social rank. However, the results show that 42% of these interactions are directed from lower- to higher-ranking females. Particularly, lactating and pregnant females targeted higher-ranking individuals, which the authors suggest might be due to higher energetic needs, which increase risk-taking in lactating and pregnant females. Sex composition within the group also influenced which individuals were targeted. The authors suggest that male presence buffers female-on-female aggression, allowing females to target higher-ranking females than themselves. In contrast, females targeted lower-ranking females than themselves in groups with a larger ratio of females, which supposes a lower risk for the females since the pool of competitors is larger. The findings provide an important insight into aggression heuristics in primate social systems and the social and individual factors that influence these interactions, providing a deeper understanding of the evolutionary pressures that shape risk-taking, dominance maintenance, and the flexibility of social strategies in group-living species.
The authors achieved their aim by demonstrating that aggression direction in female gorillas is influenced by factors such as reproductive condition and social context, and their results support the broader claim that aggression heuristics are flexible. However, some specific interpretations require further support. Despite this, the study makes a valuable contribution to the field of behavioural ecology by reframing how we think about intra-sexual competition and social rank maintenance in primates.
Strengths:
One of the study's major strengths is the use of an extensive dataset that compiles 25 years of behavioural data and 6871 aggressive interactions between 31 adult females in five social groups, which allows for a robust statistical analysis. This study uses a novel approach to the study of aggression in social groups by including factors such as the direction and intensity of aggressive interactions, which offers a comprehensive understanding of these complex social dynamics. In addition, this study incorporates ecological and physiological factors such as the reproductive state of the females and the sex composition of the group, which allows an integrative perspective on aggression within the broader context of body condition and social environment. The authors successfully integrate their results into broader evolutionary and ecological frameworks, enriching discussions around social hierarchies and risk sensitivity in primates and other animals.
Thank you for the positive assessment of our work and the nice summary of the manuscript!
Weaknesses:
Although the paper has a novel approach by studying the effect of reproductive state and social environment on female-female aggression, the use of observational data without experimental manipulation limits the ability to establish causation. The authors suggest that the difference observed in female aggression direction between groups with different sex composition might be indicative of male presence buffering aggression, which seems speculative, as no direct evidence of male intervention or support was reported. Similarly, the use of reproductive state as a proxy for energetic need is an indirect measure and does not account for actual energy expenditure or caloric intake, which weakens the authors' claims that female energetic need induces risk-taking. Overall, this paper would benefit from stronger justification and empirical support to strengthen the conclusions of the study about the mechanisms driving female aggression in gorillas.
We agree that experimental manipulation would allow us to extend our work. Unfortunately, this is not possible with wild, endangered gorillas.
We have now added more references (Watts 1994; Watts 1997) and enriched our arguments regarding male presence buffering aggression. Previous research suggests that male gorillas may support lower-ranking females and they may intervene in female-female conflicts (Sicotte 2002). Unfortunately, our dataset did not allow us to test for male protection. We conduct proximity scans every 10 minutes and these scans are not associated to each interaction, meaning that we cannot reliably test if proximity to a male influence the likelihood to receive aggression.
We have now clearly stated that reproductive state is an indirect proxy for energetic needs. We agree with your point about energy intake and expenditure, but unfortunately, we do not have data on energy expenditure or caloric intake to allow us to delve into more fine-grained analyses.
Overall, we have tried to enrich the justification and empirical support to strengthen our conclusions by clarifying the text and adding more examples and references.
Reviewer #2 (Public review):
Summary:
The authors' aim in this study is to assess the factors that can shift competitive incentives against higher- or lower-ranking groupmates in two gorilla species.
Strengths:
This is a relevant topic, where important insights could be gained. The authors brought together a substantial dataset: a long-term behavioral dataset representing two gorilla species from five social groups.
Weaknesses:
The authors have not fully shown the data used in the model and explored the potential of the model. Therefore, I remain cautious about the current results and conclusions.
Some specific suggestions that require attention are
(1) The authors described how group size can affect aggression patterns in some species (line 54), using a whole paragraph, but did not include it as an explanation variable in their model, despite that they stated the overall group size can "conflate opposing effects of females and males" (line 85). I suggest underlining the effects of numbers of males or/and females here and de-emphasizing the effect of group size in the Introduction.
We did not use group size as a main predictor, as has been commonly done in other species, because of potentially conflating opposing effects of males and females. To further stress this point, we have specifically added in the introduction: “group size, the overall number of individuals in the group, might not be a good predictor of aggression heuristics, as it can conflate the effects of different kinds of individuals on aggression (see Smit & Robbins 2024 for an example of opposing effects of the number of females and number of males on female gorilla aggression).”
We also “ran our analysis testing for group size (number of weaned individuals in the group), instead of the numbers of females and males, [and] its influence on interaction score was not significant (estimate=-0.001, p-value=0.682).”
(2) There should be more details given about how the authors calculated individual Elo-ratings (line 98). It seems that authors pooled all avoidance/displacement behaviors throughout the study period. But how often was the Elo-rating they included in the model calculated? By the day or by the month? I guess it was by the day, as they "estimate female reproductive state daily" (line 123). If so, it should be made clear in the text.
We rephrased accordingly: “We used all avoidance and displacement interactions throughout the study period and we used the function elo.seq from R package EloRating to infer daily individual female Elo-scores”. We also clarified that “This method takes into account the temporal sequence of interactions and updates an individual’s Elo-scores each day the individual interacted with another...”
In addition, all groups were long-term studied, and the group composition seems fluctuant based on the Table 1 in Reference 11. When an individual enters/leaves the group with a stable hierarchy, it takes time before the hierarchy turns stable again. If the avoidance/displacement behaviors used for the rank relationship were not common, it would take a few days or maybe longer. Also, were the aggressive behaviors more common during rank fluctuations? In other words, if avoidance/displacement behaviors and aggressive behaviors occur simultaneously during rank fluctuations, how did the authors deal with it and take it into consideration in the analysis?
We have shown in Reference 25 (Smit & Robbins 2025) after Reference 11 (Smit & Robbins 2024) that females form highly stable hierarchies, and that dyadic dominance relationships are not influenced by dispersal or death of third individuals. Notably, new immigrant females usually start at and remain low ranking, without large fluctuations in rank. Therefore, the presence of any fluctuation periods have limited influence in the aggressive interactions in our study system.
The authors emphasized several times in the text that gorillas "form highly stable hierarchical relationships". Also, in Reference 25, they found very high stabilities of each group's hierarchy. However, the number of females involved in that analysis was different from that used here. They need to provide more basic info on each group's dominance hierarchy and verify their statement. I strongly suggest that the authors display Elo-rating trajectories and necessary relevant statistics for each group throughout the study period as part of the supplementary materials.
In fact, the females involved in the present analysis and the analysis of Smit & Robbins 2025 are the same. Our present analysis is based on the hierarchies of Smit & Robbins 2025. Note that female gorillas disperse and occasionally immigrate to another study group. This is why some females may appear in the hierarchies of more than one group, giving the impression that there are more females involved in the analysis of Smit & Robbins 2025 (e.g. by counting the lines in the Elo-rating plots). We now specifically state that “We present these interactions and hierarchies in detail in Smit & Robbins 2025”, to clarify that the hierarchies are the same.
(3) The authors stated why they differentiated the different stages based on female reproductive status. They also referred to the differences in energetic needs between stages of pregnancy and lactation (lines 127-128). However, in the mixed model, they only compared the interaction score between the female cycling stage and other stages. The model was not well explained, and the results could be expanded. I suggest conducting more pairwise comparisons in the model and presenting the statistics in the text, if there are significant results. If all three pregnancy stages differed significantly from cycling and lactating stages but not from each other, they may be merged as one pregnancy stage. More in-depth analysis would help provide better answers to the research questions.
Thank you for pointing this out. First, when we considered one pregnancy stage, pregnant females showed indeed a significantly greater interaction score than females in other reproductive stages. We have now included that in the manuscript. However, we still find relevant to test for the different stages of pregnancy, given the difference of energetic needs in these stages. We have now included the pairwise comparisons in a new table (Table 2).
Reviewer #3 (Public review):
Smit and Robbins' manuscript investigates the dynamics of aggression among female groupmates across five gorilla groups. The authors utilize longitudinal data to examine how reproductive state, group size, presence of males, and resource availability influence patterns of aggression and overall dominance rankings as measured by Elo scores. The findings underscore the important role of group composition and reproductive status, particularly pregnancy, in shaping dominance relationships in wild gorillas. While the study addresses a compelling and understudied topic, I have several comments and suggestions that may enhance clarity and improve the reader's experience.
(1) Clarification of longitudinal data - The manuscript states that 25 years of behavioral data were used, but this number appears unclear. Based on my calculations, the maximum duration of behavioral observation for any one group appears to be 18 years. Specifically:
ATA: 6 years
BIT: 8 years
KYA: 18 years
MUK: 6 years
ORU: 8 years
I recommend that the authors clarify how the 25-year duration was derived.
Indeed none of the five study “groups” has been studied for 25 years in a row. However, MUK emerged from a fission of group KYA in early 2016. So, from the start of group KYA in October 1998 to the end of group MUK in December 2023, there are 25 years and 2 months. We have now rephrased to “...starting in 1998 in one of the mountain gorilla groups” in the introduction, and to “We use a long-term behavioural dataset on five wild groups of the two gorilla species, starting in 1998” in the abstract.
(2) Consideration of group size - The authors mention that group size was excluded from analyses to avoid conflating the opposing effects of female and male group members. While this is understandable, it may still be beneficial to explore group size effects in supplementary analyses. I suggest reporting statistics related to group size and potentially including a supplementary figure. Additionally, given that the study includes both mountain and wild gorillas, it would be helpful to examine whether any interspecies differences are apparent.
We have now added the suggested extra test: “When we ran our analysis testing for group size (number of weaned individuals in the group), instead of the numbers of females and males, its influence on interaction score was not significant (estimate=-0.001, p-value=0.682).”
Regarding species differences: In our analysis, we test for species (mountain vs western) and we find no significant differences between the two. This is stated in the results.
(3) Behavioral measures clarification - Lines 112-116 describe the types of aggressive behaviors observed. It would be helpful to clarify how these behaviors differ from those used to calculate Elo scores, or whether they overlap. A brief explanation would improve transparency regarding the methodology.
We now added short explanations into brackets for behaviours that are not obvious. We also added a sentence in the text to clarify the difference with the behaviours used to calculate Elo scores: “These two behaviours [avoidance and displacement] are ritualized, occurring in absence of aggression, they are considered a more reliable proxy of power relationships over aggression, and they are typically used to infer gorilla hierarchical relationships”.
(4) Aggression rates versus Elo scores - The manuscript uses aggression rates rather than dominance rank (as measured by Elo scores) as the main outcome variable, but there is no explanation on why. How would the results differ if aggression rates were replaced or supplemented with Elo scores? The current justification for prioritizing aggression rates over dominance rank needs to be more clearly supported.
The sentence we added above (“These two behaviours [avoidance and displacement] are ritualized, occurring in absence of aggression, they are considered a more reliable proxy of power relationships over aggression, and they are typically used to infer gorilla hierarchical relationships”) and the first paragraph of the results hopefully clarify that ritualized agonistic interactions are generally directionally consistent and more reliably capture the highly stable dominance relationships of female gorillas. This approach has been used to calculate dominance rank in gorillas in all studies that have considered it, dating back to the 1970s (namely in studies by Harcourt and Watts). On the other hand, aggression can be context dependent (we now clearly note that in the beginning of the Methods paragraph on aggressive interactions). Therefore, we use Eloscores inferred from ritualized interactions as base and a reliable proxy of power relationships; then we test if the direction of aggression within these relationships is driven also by energetic needs or the social environment.
Compte Rendu Détaillé : Le Handicap et l'École Inclusive en France
Ce document est un compte rendu détaillé des thèmes principaux et des faits marquants abordés lors d'un débat radiophonique sur France Culture, intitulé "Handicap : l'école est-elle la clé de l'inclusion ?".
Les intervenantes,
explorent les défis et les perspectives de l'inclusion des élèves en situation de handicap dans le système éducatif français.
1. La Loi de 2005 et la Réalité de l'Inclusion
La discussion s'ouvre sur le constat que, malgré la loi de 2005 affirmant le droit de chaque enfant à une scolarisation en milieu ordinaire, la réalité est loin d'être satisfaisante.
Une étude récente de la Fédération UNAPEI révèle que "3/4 des enfants en situation de handicap n'ont pas accès à une scolarité normale, 23 % d'entre eux n'ayant même aucune heure de scolarisation par semaine."
Ce chiffre contraste avec l'annonce présidentielle de 430 000 élèves handicapés scolarisés à la dernière rentrée, soulevant la question de la "qualité de l'accueil de ces élèves."
2. Définition et Ambivalence de l'École Inclusive
Les intervenantes s'accordent sur l'idée que l'expression "école inclusive" devrait être redondante, car l'école, par essence et mission, se doit d'être inclusive.
Isabelle Keval : "Déjà, l'école inclusive, c'est une expression qui devrait être redondante parce que l'école dans ses textes, dans son histoire et dans ses missions, elle est inclusive.
Or, dans la réalité des faits, comme on vient de l'entendre et comme le rappelle de nombreuses enquêtes chaque année à la rentrée, elle ne l'est pas. Voilà. Donc, l'école inclusive, c'est une école qui accueille tout le monde."
Sonia Ainou : L'école inclusive est "celle qui permet d'accueillir tous les élèves quel qu'ils soient, qu'ils s'appuient sur les leviers sur lesquels ils peuvent progresser, gagner en autonomie et c'est celle qui va mettre en œuvre tous les moyens et les ressources nécessaires pour que l'élève puisse rentrer dans les apprentissages, grandir comme tous les autres élèves parmi tout le monde."
Servane Hug met l'accent sur la "l'accessibilité pédagogique des pratiques des enseignants" comme levier essentiel pour une réelle inclusion.
Une difficulté soulignée par Isabelle Keval est l'ambivalence inhérente à la notion d'inclusion : "la difficulté de l'inclusion, c'est qu'au fond, dans la notion, il y a cette ambivalence que pour pouvoir inclure, il faut pouvoir identifier et que dès lors qu'on identifie, on risque de stigmatiser."
3. La "Jungle de Sigles" et la Nécessité de Dispositifs Adaptés
Le vocabulaire complexe (dispositifs d'autorégulation, professeurs ressources, unités d'enseignement, Ulis, AESH, MDPH) est évoqué.
Sonia Ainou clarifie que ces termes désignent les diverses formes d'accompagnement nécessaires aux élèves ayant des besoins spécifiques : "c'est que cet élève-là a besoin qu'on intervienne de façon plus ou moins intensive auprès de lui parce qu'il a des des compétences, mais on a besoin d'accompagner ses compétences, de les renforcer.
Ça peut être des aides humaines comme les AESH, mais ça peut aussi être des aides techniques. Ça peut être aussi un aménagement pédagogique par l'enseignant. Ça peut être la formation des AESH."
4. Le Rôle Crucial de la Formation des Enseignants et la Coopération Médico-Sociale
Les trois intervenantes s'accordent sur le fait que l'augmentation du nombre d'accompagnants (AESH) ne suffit pas.
Le point central de l'avancement vers une école réellement inclusive réside dans la formation des enseignants et une coopération renforcée entre le monde médico-social et l'Éducation Nationale.
Servane Hug insiste sur la nécessité pour les enseignants de "se rendre compte aujourd'hui que accueillir un enfant en situation de handicap dans sa classe, ce n'est pas essayer de faire disparaître la différence mais au contraire c'est de se de de de rendre accessible ces pratiques."
Elle ajoute : "je crois que c'est c'est sur ça qu'il faut appuyer, c'est cette coopération entre les enseignants et les professionnels du médico-social, les éducateurs, ce qui permettra réellement, je pense, une avancée." Isabelle Keval corrobore en pointant un "déficit de formation" dans le cursus des enseignants, soulignant l'augmentation des demandes de formation continue sur ces questions à l'INSEI.
Cette appréhension des enseignants est liée à la "variété de handicap" et aux "résistances des parents des autres enfants".
5. Les Chiffres et les Avancées Gouvernementales
Elle souligne le déploiement des "unités d'enseignement externalisé" et les futurs "dispositifs intégrés médico-éducatifs (DIY)" d'ici 2027, visant à prendre en charge les enfants polyhandicapés ou "lourdement handicapés" qui ne peuvent actuellement pas intégrer l'école ordinaire.
Cependant, Sonia Ainou tempère en affirmant qu'il ne s'agit pas d'une "amélioration" mais plutôt d'"engagements" et d'"annonces", et que la plateforme marentree.org continue de recueillir de nombreux témoignages d'enfants sans solution de scolarisation.
Le manque de places en établissements spécialisés reste criant, avec "12000 enfants qui n'ont pas de place".
6. L'Accessibilité au-delà du Technique : Sociale et Intellectuelle
Isabelle Keval élargit la notion d'accessibilité au-delà de la simple installation de rampes ou ascenseurs.
Pour elle, l'accessibilité doit aussi être "développé au niveau social, sociétal, intellectuel dans le regard qu'on porte sur les enfants en situation de handicap".
Il s'agit de les considérer comme des "sujets, des personnes qui ont la possibilité de choisir quelque chose, de décider", plutôt que de les stigmatiser par un "fléchage" technique.
7. L'Inclusion Hors Scolaire et la Communauté d'Expérience
Le débat explore également l'importance de l'inclusion dans les activités extrascolaires. Servane Hug ne croit pas que l'école soit "l'outil ultime de l'inclusion" en raison de l'attente de performances cognitives.
Elle suggère d'investir les centres de loisirs, les colonies, et les activités sportives et artistiques, où les enfants n'ont pas à s'évaluer les uns les autres.
Sonia Ainou renchérit en affirmant qu'il faut "saisir tous les leviers dans tous les lieux de vie et d'apprentissage des enfants", car l'école est la "porte d'entrée de l'avenir", mais l'endossement du statut d'élève se fait aussi "dans les temps hors scolaires."
Isabelle Keval insiste sur la "similarité d'expérience" comme levier d'inclusion, en utilisant des contextes comme l'eau ou l'air où les différences s'estompent au profit d'un point commun : "ce que nous partageons c'est l'eau. Voilà.
Et nous avons là un point commun. Nous sommes deux humains dans ce milieu aquatique."
L'objectif est de viser l'"universel" et non de "continuer à distinguer ceux qui sont dehors et ceux qui sont dedans."
8. Le Cas Belge : Un Modèle de Coopération
Servane Hug, dans le cadre de sa mission d'information parlementaire, a étudié le modèle belge.
Elle note que "1500 enfants sont aujourd'hui accueillis en Belgique" faute de solutions en France. La réussite belge s'explique par un "renforcement entre la coopération éducation nationale en Belgique et professionnel du médico-social."
En Belgique, les éducateurs sortent des IME et il y a l'équivalent d'une unité d'enseignement externalisée par groupe scolaire.
Sonia Ainou questionne pourquoi la France finance ces départs plutôt que d'investir dans une transformation du système national.
9. Les Freins et la "Plasticité" du Système
Les freins à l'inclusion sont multiples : les résistances des parents d'autres élèves, l'appréhension des professionnels, et la difficulté de modifier les habitudes. Isabelle Keval introduit le concept de "plasticité" : l'école inclusive et la société inclusive ne sont pas des touts déjà constitués dans lesquels on essaie de faire rentrer un élément.
Il s'agit plutôt d'"adapter ce système, le faire changer pour que finalement il y ait plus de cette frontière".
Sonia Ainou utilise le terme d'"agilité" pour décrire la capacité du système à répondre à toute forme de vulnérabilité.
Elle insiste sur la nécessité de faire travailler ensemble tous les acteurs (collectivités, financeurs, professionnels de l'éducation nationale, AESH, cuisiniers, professionnels médico-sociaux et libéraux), avec une attention particulière aux personnes concernées et aux familles, qui doivent être des "acteurs principaux".
10. Conclusion : Un Long Chemin à Parcourir Le débat se termine sur la reconnaissance du "long chemin à parcourir" pour atteindre une inclusion véritable.
La "plasticité" et l'"agilité" du système, la formation continue des enseignants, et une coopération intersectorielle sont identifiées comme les clés de cette transformation.
Servane Hug, en tant que députée, s'engage à œuvrer pour un "changement de paradigme" lors du prochain projet de loi de financement.
L'objectif ultime est de donner à tous les enfants en situation de handicap une "véritable chance [...] de rentrer dans les apprentissages et dans la vie tout simplement."
Dossier de briefing : Faire face à l'inceste et au silence
Source : Extraits de "France Inter - Faire face à l'inceste 16173-13.10.2021-ITEMA_22805012-2021F26104S0286-22.mp3"
Thèmes Principaux et Idées Essentielles
Ce briefing aborde la problématique de l'inceste en France, en soulignant l'ampleur du phénomène, les mécanismes du silence qui l'entourent, les conséquences dévastatrices pour les victimes et les voies de prévention et de réparation.
1. L'ampleur de l'inceste et la conspiration du silence
Cependant, Bruno Clavier, psychanalyste et psychologue clinicien, estime que ce chiffre est largement sous-estimé en raison du déni et de l'amnésie des victimes. Il suggère que le chiffre réel pourrait être de trois à quatre personnes sur dix. * Le silence comme arme : Le silence est décrit comme une "arme de destruction massive" érigée par les agresseurs, entraînant de graves dégâts collatéraux. Ce silence est imposé par les abuseurs et les violeurs, qui sont dans l'écrasante majorité des hommes. * Complicité sociétale : La société est interrogée sur sa complicité dans cette "conspiration du silence".
Charlotte Pudlovski, cofondatrice de Louis Media, souligne que la société a longtemps ignoré ou minimisé le problème, comme en témoignent les réticences des médias à aborder le sujet avant des événements médiatisés.
L'amnésie est particulièrement fréquente chez les victimes, et peut durer des décennies, le cerveau cherchant à se protéger de la violence. Certains patients disent même : "Si je me souviens, je meurs."
2. Le traumatisme et ses conséquences
Grégoire de la Cour témoigne de douleurs physiques inexpliquées (mal au ventre pendant 55 ans) dues à ce traumatisme.
Le corps devient un "pire ennemi", un "traître", dont on a honte. Bruno Clavier compare les violences sexuelles à une "déflagration", un "incendie" qui brûle les circuits nerveux et laisse des "traces multiples" invisibles aux médecins.
Beaucoup de victimes leur en veulent d'abord pour ne pas les avoir protégées. Cependant, les experts soulignent que ces mères ont souvent elles-mêmes été victimes d'abus, reproduisant un "formatage" familial du silence. "Elles donnent la soupe qu'elles ont mangé."
3. La libération de la parole et les défis persistants
4. Prévention et chemins de réparation
Bien sûr, voici une synthèse détaillée des thèmes principaux et des idées essentielles abordées dans l'extrait audio "L'inceste, la loi du silence" de France Culture :
Synthèse détaillée : L'Inceste, la Loi du Silence
Cet extrait du podcast France Culture, "L'inceste, la loi du silence", présente une exploration poignante et multifacette de l'inceste, s'appuyant sur des témoignages de victimes, des analyses d'experts (anthropologue, historienne, philosophe), et une réflexion sur l'évolution de la perception sociale et juridique de ce crime.
Le document met en lumière la fréquence "effarante" de l'inceste et les mécanismes complexes de silence et de domination qui l'entourent.
Thèmes Principaux et Idées Essentielles :
Le Silence : Une Règle Fondamentale et Ses Mécanismes :
En conclusion, cet extrait de France Culture offre une analyse profonde de l'inceste, soulignant non seulement sa nature dévastatrice pour les victimes, mais aussi sa place structurelle dans l'ordre social, alimentée par des siècles de silence, de déni et de justification de la domination.
Il met en lumière l'espoir d'un changement radical grâce à la libération de la parole et à une remise en question fondamentale des structures patriarcales.
Compte rendu détaillé : "Y a-t-il une culture de l'inceste en France ?" (France Culture, 12.10.2022)
L'émission, en se basant sur le rapport de la CIVISE (Commission Indépendante sur l'Inceste et les Violences Sexuelles), questionne l'existence d'une "culture de l'inceste" en France, remettant en cause le mythe du tabou anthropologique et soulignant la réalité omniprésente de ces violences.
1. L'ampleur et la sous-estimation de l'inceste en France
Le débat s'ouvre sur un chiffre choc issu du rapport de la CIVISE : "160 000 enfants subissent des violences sexuelles chaque année en France."
Ce chiffre, longtemps "sous-estimé voire complètement négligé", contredit l'idée reçue d'un tabou anthropologique sur ce crime.
Au contraire, les enquêtes récentes montrent que "ce crime touche un français ou une française sur 10 et est présent dans toutes les classes sociales."
Cette affirmation résume la dissonance entre la perception publique de l'inceste comme un interdit absolu et sa réalité fréquente et dissimulée.
Édouard Duran, juge des enfants et co-président de la CIVISE, confirme que "16 500 personnes sont venues [à la CIVISE], nous ont fait confiance [...] toutes nous disent cela, que les violences commencent quelques jours après la naissance ou qu'elles durent jusqu'à la majorité ou au-delà de la majorité." Il insiste sur le fait que "la maison est pour beaucoup le lieu du danger, de la confrontation à la terreur et à la mort même."
2. Le mythe du tabou anthropologique et la réalité de la "culture de l'inceste"
Ils affirment que cette idée, véhiculée par des "anthropologues depuis leur position située, c'est-à-dire d'hommes blancs qui sont arrivés sans vouloir parler de violence sexuelle mais simplement en voulant étudier les règles du mariage", a conforté le silence autour de l'inceste.
Pour eux, le concept de tabou du mariage "n'a rien à voir avec les pratiques d'inceste. Marier ou pas marier avec certaines personnes, ça n'empêche pas d'incester ces certaines personnes."
Le terme de "culture de l'inceste" est utilisé dans plusieurs sens par Juliette Drouard :
Elles peuvent "romantiser l'inceste comme dans Game of Thrones avec le frère, la sœur" ou, comme dans le porno, où le "stepmom" est un hashtag très recherché.
D'autres œuvres "n'adoptent pas le point de vue de la victime" ou reprennent des mythes comme celui de Lolita, où "ce serait la personne victime qui vient séduire l'agresseur."
3. L'évolution historique et juridique de la perception de l'inceste
Julie Doyon, historienne, apporte un éclairage diachronique sur la question. Elle souligne que l'inceste, dans l'Ancien Régime, était "beaucoup dit, montré, écrit" dans la littérature et était un crime considéré comme tel dans la doctrine pénale.
Cependant, il n'était "pas du tout la même signification qu'aujourd'hui.
C'est-à-dire qu'il n'est pas indexé à une forme de violence ni spécifiquement à la catégorie de l'enfance." L'inceste était alors un "crime sans victime.
Un crime avec deux coupables", considéré comme un crime de mœurs et de péché entre personnes apparentées.
Le "point de bascule" se situe entre le 18e et le 19e siècle, où l'inceste passe d'une conception de "couple incestueux" à celle d'"acte d'agression sexuelle commis par un adulte sur un enfant dans le cadre familial."
La Révolution française, en voulant séculariser le droit pénal, a supprimé le crime d'inceste, le considérant comme relevant de la sphère religieuse et de la "vie privée".
Aujourd'hui, Édouard Duran déplore cette persistance de l'idée que "la maison est éminemment essentiellement le lieu du privé."
Il insiste sur la nécessité que "ce qui doit régner dans la maison, c'est la loi commune et pas la loi d'un seul, pas la loi du dominant."
4. La spécificité de la violence incestueuse et la vulnérabilité des enfants
Édouard Duran insiste sur la vulnérabilité des enfants : "les agresseurs recherchent toujours une proie en raison de sa vulnérabilité.
Et l'enfant parmi les êtres vulnérables dans la société est le plus vulnérable et parmi les enfants vulnérables, il y a les enfants handicapés, plus vulnérables et plus invisibilisés encore."
Il récuse l'argument souvent avancé par les agresseurs : "Je n'ai jamais entendu en audience, en cours d'assise, au tribunal correctionnel ou au tribunal pour enfants un agresseur dire autre chose que c'est l'enfant qui m'a séduit." Édouard Duran refuse de "chercher à comprendre" dans le sens de "chercher dans la psychologie de l'agresseur ce qui pourrait l'excuser." Pour lui, l'impératif moral est de "mettre en sécurité les enfants victimes d'inceste et les personnes victimes de violence."
Juliette Drouard souligne l'importance de parler de "pédocriminalité de manière générale", car "les adultes qui commettent des agressions sur des enfants, les commettent aussi bien sur leurs enfants que sur les enfants des autres."
Elle met en évidence une "communauté de traumatisme" et de destruction pour toutes les victimes, avec seulement une "différence de degré dans l'échelle de la trahison éthologique" selon Sortnaf.
Édouard Duran, citant Christine Ang, décrit l'inceste comme un "crime absolument spécifique, un crime contre l'humanité du sujet, un crime généalogique."
Il explique que "en venant à elle sexuellement, il se refuse à elle comme père.
C'est une humiliation sociale avant tout par laquelle l'enfant n'a plus de place dans l'histoire des humains."
Il n'y a "pas d'amour dans l'inceste," comme le souligne Juliette Drouard : "L'excitant ça n'est pas l'amour mais le pouvoir et les fractions."
5. Le silence, la prescription et la difficile écoute de la parole des victimes
Le silence est présenté comme un facteur mortifère : "Ce qui tue c'est le silence. C'est de ne pas parler. C'est de ne pas dire, de ne pas pouvoir dire."
L'extrait du documentaire "Inceste, le dire et l'entendre" illustre le ressenti des victimes : "On t'a juste dit que l'agression sexuelle c'est dehors que ça se passe.
C'est des étrangers qui peuvent t'attaquer. C'est des étrangers. C'est jamais dedans la famille. Et que tu pressens, tu ressens que quand il t'arrive un truc à l'intérieur de la famille, il faut fermer sa gueule."
La question de la prescription est abordée. Le ministre de la Justice, Éric Dupond-Moretti, évoque l'allongement du délai de 20 à 30 ans à compter de la majorité depuis 2018.
Édouard Duran souligne l'importance de cet allongement, car les traumatismes générés par ces violences "ne sont pas cachés dans un passé lointain.
C'est un présent perpétuel qui s'immisce dans toutes les sphères de l'existence, des plus sociales au plus intimes." Il insiste sur "l'aspiration profonde à ce que justice soit rendue."
L'expression d'Iris Bray, "Mon corps est une archive vivante de mon inceste," résonne avec cette idée de persistance du traumatisme.
Malgré une apparente "libération de la parole" dans l'espace public, Juliette Drouard et Édouard Duran soulignent que le tabou reste "absolu" là où l'inceste a lieu.
Seulement "1000 condamnations" pour "160 000 enfants victimes de violence sexuelle chaque année" révèlent un "système d'impunité des agresseurs."
Les enfants n'ont pas les outils pour décrire ce qui leur arrive et sont souvent "tués ou resilenciés" lorsqu'ils parlent.
Édouard Duran révèle que "dans 9 cas sur 10, le confident de l'enfant ne fait rien." Le processus de "silenciation" est au cœur de la stratégie de l'agresseur, qui vise à "imposer le silence à l'enfant victime" et à "contaminer le groupe."
Julie Doyon nuance l'idée d'un silence absolu en soulignant l'existence de moments passés où l'inceste a été discuté publiquement, comme la fin des années 1980 avec les "dossiers de l'écran."
Elle insiste sur le fait que le vrai problème n'est peut-être "pas tant de le parler que de l'entendre."
Elle met en lumière les dynamiques complexes au sein des familles, où le "silence familial n'est pas un bloc monolithique" et où les rôles et statuts des individus influencent la manière dont la parole circule ou est étouffée.
Conclusion
Le débat met en lumière une réalité complexe et souvent douloureuse de l'inceste en France.
Loin d'être un tabou universellement respecté, il est une violence omniprésente, souvent dissimulée par des mécanismes de silence, d'impunité et une certaine "culture" qui minimise ou romantise la souffrance des victimes.
Les intervenants appellent à une meilleure compréhension historique, juridique et sociétale de l'inceste, une protection accrue des enfants victimes, et une capacité collective à écouter et croire la parole de ceux qui osent briser le silence.
Numéro de téléphone Inceste : 0805 802 804 (anonyme et gratuit)
Compte Rendu Détaillé : "La culture de l'inceste" sur France Inter
1. L'Inceste : Non une Déviance Individuelle, mais un Phénomène Culturel et Systémique
Le thème central de l'émission et de l'ouvrage est la remise en question de la vision traditionnelle de l'inceste comme une "déviance, d'exception pathologiques, de monstres à la marge".
Au contraire, les invitées soutiennent que l'inceste est un phénomène "massif" et "systématique" ancré au "cœur même de notre organisation sociale".
C'est notre société, c'est nous, c'est nos amis, c'est nos pères. C'est ça qu'on doit regarder." La responsabilité est ainsi déplacée de l'individu "monstrueux" vers le collectif et le système social. * Continuité avec la Culture du Viol : Juliette Drouar explique que le terme "culture de l'inceste" est décalqué de l'expression "culture du viol", visant à souligner un aspect "culturel" et non une "exception, une pathologie, une monstruosité". * L'Inceste comme Outil de Domination Patriarcale : L'ouvrage postule que l'inceste est "une expression, c'est une reconduction d'un fonctionnement social qui s'appuie sur l'idée de domination". Iris Brey affirme que c'est un "système qui est mis en place pour que le corps des enfants et que le corps des femmes continue à être dominé par le patriarcat et par les hommes". Les agresseurs, à 76% des hommes, se sentent "autorisé[s] partout et depuis toujours" à agresser le corps "le plus faible".
2. Le Tabou de l'Inceste : Ne pas Parler, Plutôt que Ne pas Exister
Les auteures déconstruisent l'idée reçue selon laquelle l'inceste serait un interdit social fondamental.
Elles affirment que l'inceste n'est pas un tabou dans sa pratique, mais plutôt un tabou dans sa discussion et sa reconnaissance.
Les Enfants Parlent, les Parents n'Entendent pas : Le véritable tabou n'est pas le silence des enfants victimes – "les enfants en parlent" – mais plutôt l'incapacité des adultes à les entendre : "C'est que les parents ne veulent pas entendre ou ne peuvent pas entendre."
L'ouvrage met en lumière la vulnérabilité intrinsèque des enfants dans le système social et familial, où leur dépendance est naturalisée et leurs droits sont "déprivés".
4. Représentations Médiatiques et Culturelles de l'Inceste : Banalisation et Distorsion
Une part importante de la discussion est consacrée à la manière dont l'inceste est représenté ou non représenté dans la culture populaire, contribuant à sa banalisation et à la culpabilisation des victimes.
Elle rappelle que la Lolita de Nabokov était "une enfant violée par son beau-père". * Distorsion des Représentations de l'Inceste :Inceste père-fille : Souvent présenté avec la culpabilisation de la jeune fille. * Inceste mère-fils : Souvent "montré comme une démarche d'émancipation, comme une relecture du d'Œdipe". * Inceste frère-sœur : Bien que les plus rares dans la réalité, ils sont "montrés beaucoup dans les séries et notamment dans Game of Thrones comme quelque chose d'érotisé et de normal". * L'Inceste et le Pornographie Grand Public : Ovidi (co-auteure) et Juliette Drouar abordent l'infiltration de l'inceste dans le porno grand public, notamment via le mythe de la "MILF" (Mother I'd Like to F***) qui a évolué vers la "Stepmom" (belle-mère) comme hashtag principal.
Ce phénomène, initialement américain, s'est "très largement diffusé", banalisant une "représentation érotisée de l'inceste" où les violences sont déniées au profit d'une sexualisation "sexy" et "fun".
Les auteures déplorent que ces représentations "ne représente[nt] jamais l'inceste comme un acte de violence et de domination".
5. Une Lutte Collective pour une Pensée Collective
L'écriture de ce livre est présentée comme une "lutte, un combat", rendue possible uniquement par un effort collectif.
Le suicide de Tal Piter Bro Merx pendant l'écriture du livre témoigne de l'épreuve que représente l'engagement sur ce sujet, même dans une approche théorique.
En conclusion, "La culture de l'inceste" est un ouvrage politique et théorique qui vise à déconstruire les mythes entourant l'inceste, le présentant non pas comme un fait divers isolé, mais comme un symptôme d'un système de domination patriarcale et d'une invisibilisation de la vulnérabilité et des droits des enfants.
L'émission met en lumière la nécessité d'une prise de conscience collective et d'une relecture critique des représentations culturelles pour démanteler ce système.
Note de Synthèse : Réussite à l'École, Réussite de l'École
Cette note de synthèse analyse les thèmes principaux et les idées ou faits les plus importants extraits des interventions du Conseil économique, social et environnemental (CESE) sur le sujet de la réussite à l'école et la réussite de l'école.
Elle met en lumière les constats, les défis et les préconisations du CESE et des différents groupes de la société civile.
1. Le CESE : Rôle, Composition et Missions
Le CESE est présenté comme la troisième assemblée citée dans la Constitution française, après l'Assemblée Nationale et le Sénat.
Il est composé de 175 conseillers et conseillères, désignés pour 5 ans par des organisations représentatives de la société civile (entreprises, syndicats, associations, ONG, organisations étudiantes, mouvements de jeunesse, etc.).
C'est un "mini France réunie dans un hémicycle où tous les points de vue se rencontrent", un des rares endroits où "des chasseurs peuvent discuter et débattre avec des défenseurs des oiseaux".
Ses quatre missions principales sont :
2. Le Contexte Politique Actuel et l'Urgence de Faire Entendre la Voix de la Société Civile
Plusieurs interventions soulignent le contexte politique troublé en France, marqué par la dissolution de l'Assemblée nationale et la montée de l'extrême droite.
La société civile organisée exprime une vive inquiétude face à cette situation, considérant l'arrivée de l'extrême droite au pouvoir comme un "véritable danger pour notre démocratie et pour le monde du travail".
Les craintes exprimées incluent :
Le bureau du CESE insiste sur la nécessité de mieux associer la société civile à l'action publique, estimant que "la mécanique de nos institutions s'est rigidifiée et a fini par faire perdre la culture du dialogue et du débat".
Le CESE se positionne comme un "lieu de résistance par le respect des divergences et des différences" et un "lieu de recueil de toutes les voix des espoirs et des préoccupations".
3. L'École Française : Constats d'Inégalités et Défis
Le CESE aborde en profondeur le sujet de l'école, reconnaissant son rôle fondamental dans la République.
Malgré un budget important et un taux d'alphabétisation et d'éducation sans précédent, l'école française "peine à faire réussir tous les jeunes". Plusieurs constats alarmants sont mis en évidence :
La corrélation entre les inégalités scolaires et sociales est forte : "le facteur essentiel de la difficulté scolaire et de l'échec scolaire provient des inégalités sociales".
L'enquête PISA montre que "l'école française réussit aux élèves les plus favorisés". 30% des élèves issus de milieux populaires sont en difficulté.
4. Philosophie de la Réussite : De l'Égalité des Chances à la Réussite de Tous
Le CESE prône un changement de paradigme, passant du concept d'«égalité des chances» à celui de la «réussite de toutes et tous».
5. Axes de Préconisations du CESE
Le projet d'avis du CESE s'articule autour de quatre axes majeurs :
A. Redéfinir les finalités de l'école et mieux articuler l'ensemble des politiques publiques
Préconisation 1 : Organiser un grand débat démocratique pour clarifier et redéfinir collectivement les finalités de l'école. L'école a été historiquement construite pour former une élite, non pour faire réussir tous les élèves. Préconisation 15 : Améliorer la concertation et l'articulation des politiques publiques complémentaires (logement, transport, santé, culture, sport) qui concourent à l'éducation, car "l'école seule n'arrivera pas à faire de l'égalité".
B. Favoriser l'égalité et la mixité dans les territoires
C. Conforter les acteurs de la communauté éducative et en priorité les élèves
D. Transformer l'école et conforter le rôle des personnels
6. Perspectives et Appel à l'Action
Le CESE insiste sur l'urgence d'un message positif et d'espoir pour les jeunes. L'école est vue comme un "moyen pour chacun et chacune de se construire, de s'émanciper, de surmonter ses difficultés, de grandir, d'apprendre, d'imaginer le monde de demain pour faire société ensemble".
Malgré des réserves de certains groupes (entreprises, agriculture) sur des points spécifiques (financement, applicabilité de certaines mesures, pertinence de redéfinir les finalités pour l'agriculture), le projet d'avis "Réussite à l'école, réussite de l'école" a été adopté avec 97 voix pour, 1 contre et 23 abstentions.
Le CESE s'engage à porter et prolonger cet avis, réaffirmant son rôle d'assemblée de combat et de solution face aux défis sociaux et éducatifs.
DOCUMENT DE BRIEFING
Sujet : Le rôle et les missions du Conseil Économique, Social et Environnemental (CESE) et l'importance de l'éducation à la vie affective, relationnelle et sexuelle (EVARS) en France.
Date : Septembre 2024
1. Le Conseil Économique, Social et Environnemental (CESE) : Un Acteur Clé de la Démocratie Participative en France
Missions Principales :
Composition et Représentativité :
Mécanismes de Participation Citoyenne :
Importance dans le Contexte Actuel :
Défis et Critiques :
2. L'Éducation à la Vie Affective, Relationnelle et Sexuelle (EVARS) : Un Impératif Social et Démocratique
Un avis du CESE sur l'EVARS met en lumière l'urgence de cette éducation pour faire face à des enjeux sociétaux majeurs.
Cadre et Enjeux de l'EVARS :
Un Constat Alarmant :
Trois associations (Planning Familial, S.O.S. Homophobie, S.O.S. Homophobie) ont même saisi le tribunal administratif pour cette non-application. * Augmentation des Violences et Maladies :"Les taux de MST augmentent" ("48% pour les cas de gonorrhée, 34% pour la syphilis, 16% pour les clamidias"). * "160 000 enfants sont chaque année victimes de violence sexuelle" en France. La Commission Indépendante sur l'Inceste et les Violences Sexuelles faites aux Enfants (CIVISE) a souligné que "77% des violences sexuelles sur les enfants se déroulent au sein de la famille" et le risque est "presque triplé pour les enfants porteurs d'un handicap". * "Un quart des jeunes hommes de 18 à 24 ans adhère à l'idée que quand une femme dit non cela veut dire oui". * "Deux tiers des enfants de moins de 15 ans et un tiers des enfants de moins de 12 ans ont déjà eu accès à des images pornographiques non souhaitées". La pornographie "éduque malgré eux" les enfants à des "images violentes". * La "montée préoccupante des représentations sexistes, du machisme et de la misogynie, notamment chez les plus jeunes" est observée. * Manque d'Information et de Ressources : Les jeunes "estiment ne pas avoir accès à une information de qualité sur la vie affective et sexuelle". Les professionnels manquent de formation et de moyens. * Résistance et Désinformation : De "nombreux mouvements réactionnaires s'opposent à toute éducation affective ou sexuelle au nom d'un risque de perversion de l'enfant". La consultation en ligne du CESE a été "attaquée tout d'abord juridiquement puis sur la plateforme elle-même afin de fausser les résultats". * Recommandations Clés du CESE (24 préconisations au total) : * Garantir l'Application de la Loi : Nécessité d'un "courage politique" pour appliquer la loi de 2001, avec une "politique coordonnée, pilotée et dotée de moyens suffisants". * Formation des Professionnels : Mise en place d'un "plan de formation national accessible gratuitement à tout intervenant éducatif", incluant les spécificités des publics fragiles (traumatisme, violence sexuelle, interculturalité). * Soutien aux Familles : Les familles sont centrales mais n'ont pas toujours les ressources. Le CESE propose de "compléter et élargir les dispositifs actuels de soutien à la parentalité" et de leur donner "accès à une information claire et fiable sur les besoins et les droits des enfants". * Campagne Nationale d'Information : Lancer une "campagne nationale d'information sur les contenus réels de l'EVARS afin de déconstruire les discours qui alimentent les peurs et les fantasmes". * Rôle de l'École : Proposer la mise en place d'une "éducation aux compétences psychosociales et à l'égalité dotée d'au moins 30 heures d'enseignement dès le collège", assurée par des enseignants dédiés avec l'appui d'interventions extérieures. * Prise en Compte dans Tous les Espaces : Inclure l'EVARS dans les projets d'établissement, les contrats de délégation des fédérations sportives, et soutenir les "centaines de milliers de professionnels et bénévoles" agissant dans les lieux de loisirs, sport, aide sociale à l'enfance, etc. * Lutte contre l'Infantisme : Combattre ce "système qui autorise tout adulte à déconsidérer la parole des enfants et leur place dans la société du seul fait qu'il et elle soient des enfants". * Exploiter le Numérique : Utiliser les "supports numériques pour nous rendre actrices et acteurs de l'éveil à la vie affective, relationnelle et sexuelle" et renforcer la régulation des réseaux sociaux pour un "cadre vertueux" et lutter contre les contenus violents.
Perspective :
L'EVARS est "une politique publique globale de tous les instants qui aujourd'hui pêche par le manque d'ambition et de moyens".
C'est une "composante essentielle de nos vies, de nos rapports aux autres", qui participe à une "transformation en profondeur de la société" pour plus "d'inclusivité, plus de respect et moins de violence entre les individus".
Les membres du CESE saluent la qualité du rapport et soulignent que les jeunes sont "en demande" d'informations sur la sexualité.
Note de synthèse détaillée : Vivre dans les Territoires Ruraux
Date : 19 juin 2024 Sujet : Synthèse des thèmes majeurs et faits marquants de l'événement "Vivre dans les territoires ruraux" organisé par la Commission Territoire, Agriculture et Alimentation.
Introduction
Loin d'être une "diagonale du vide uniforme", ces territoires regorgent d'initiatives et sont essentiels pour relever les défis sociétaux actuels et futurs. Cette réunion a permis de confronter des analyses sociologiques historiques et contemporaines aux témoignages concrets d'acteurs de terrain, offrant une vision nuancée des défis et des opportunités.
1. La Ruralité : Une Entité Complexe et en Évolution
Historiquement perçue comme un résidu voué à l'uniformisation face à l'urbanisation, la ruralité a connu une "disparition des écrans radars des politiques publiques nationales" avant de revenir au cœur du débat public, notamment avec la crise des gilets jaunes et la crise sanitaire.
Évolution Sociologique et Démographique :
Enjeux et Attributs des Territoires Ruraux :
2. Freins et Défis Majeurs
Malgré leur dynamisme, les territoires ruraux font face à des contraintes importantes qui entravent leur plein développement :
3. Initiatives et Modèles de Réussite
Les témoignages des acteurs de terrain ont mis en évidence la capacité des territoires ruraux à innover et à se développer grâce à des initiatives locales fortes et à la coopération :
Société d'Économie Mixte (SEM) en Lozère (Roger Cruis) :
4. Recommandations et Perspectives
Plusieurs pistes de réflexion et d'action émergent de ces échanges :
Conclusion
La ruralité française n'est ni figée ni uniforme, mais un "cœur battant de notre économie et de notre souveraineté alimentaire", un "atout puissant pour permettre la transition écologique".
Les initiatives citoyennes et la coopération entre tous les acteurs locaux sont les moteurs essentiels de son dynamisme. Il appartient aux politiques publiques de les soutenir efficacement en adaptant les cadres existants et en facilitant l'émergence de nouveaux modèles, pour que la ruralité puisse pleinement jouer son rôle dans la société de demain. "Le pays a besoin de sa ruralité."
Ce document est un compte rendu détaillé de la session plénière du Conseil Économique, Social et Environnemental (CESE) consacrée à un projet d'avis intitulé "La protection de l’enfance est en danger : les préconisations du CESE".
Il met en lumière les discussions, les constats alarmants, et les propositions concrètes visant à améliorer la protection de l'enfance en France.
Thèmes Principaux et Idées Clés :
Le Rôle du CESE et la Participation Citoyenne :
La Crise de la Protection de l'Enfance :
Désorganisation Institutionnelle et Disparités Territoriales : L'État est "totalement désengagé de la politique de la protection de l'enfance". Il manque de données statistiques consolidées au niveau national, ce qui "organise la cécité de l'État et organise l'invisibilité des bénéficiaires".
Problèmes de Santé et Scolarité : Les enfants victimes de violence ou de négligence grave ont une espérance de vie réduite de 20 ans. Moins de 30% d'entre eux ont un bilan de santé global à l'entrée dans le dispositif de protection, et moins de 10% ont un suivi effectif. La déscolarisation est fréquente chez les enfants placés.
Gouvernance et Coordination :
Amélioration de la Protection :
Respect des Droits de l'Enfant :
Soutien aux Professionnels :
Citations Clés :
En conclusion, ce briefing met en évidence l'urgence d'une refonte profonde de la politique de protection de l'enfance en France.
Il souligne la nécessité d'une action concertée de l'État, des départements et de la société civile pour garantir le respect des droits fondamentaux de chaque enfant, en s'appuyant sur des professionnels mieux formés, mieux rémunérés et mieux accompagnés, et en assurant une gouvernance transparente et efficace.
Note de synthèse : Sortir de la Crise Démocratique - Rapport Annuel sur l'État de la France 2024
Introduction
Il s'appuie également sur une grande enquête menée avec l'Institut Ipsos et les discussions tenues lors de sa présentation.
Le rapport et les débats mettent en lumière les préoccupations majeures des Français et proposent un diagnostic des défis démocratiques du pays, soulignant le rôle crucial du CESE en tant que "troisième assemblée" et lieu de "participation citoyenne".
1. La Crise Démocratique et l'Impact des Inégalités
Le rapport dresse un tableau alarmant de la "crise démocratique" en France, qu'il juge "forte et multifactorielle".
Cette crise est intrinsèquement liée à la prolifération et à l'accentuation des inégalités dans la société française.
Multiplicité des Inégalités : Les sources soulignent la présence d'inégalités à plusieurs niveaux :
Elle ajoute que "la concentration de la richesse et des revenus... a conduit à un sentiment d'injustice sociale très fort qui érode la confiance dans les institutions démocratiques et réduit la participation politique." Le sondage Ipsos révèle que "plus vous avez accès à des services, plus vous faites confiance en la démocratie... inversement, un moindre accès à ces services-là délite la démocratie." Mael Nisan, présidente de la FAGE, souligne que "les jeunes n'auront pas confiance envers le système" si l'État ne leur fait pas confiance. * Déconnexion et Défiance : Le sentiment que les responsables politiques sont "déconnectés des réalités des citoyens" est partagé par 76% des Français. Eric Chenu, président de la Mutualité Française, évoque une société "démantelée petit à petit parce qu'on... a rendu technique un certain nombre de questions qui sont éminemment politiques." Patricia Drevon, de Force Ouvrière, rappelle l'importance de la démocratie sociale et de la négociation collective. * Pessimisme et Optimisme Relatif : Si les Français sont majoritairement optimistes quant à leur avenir personnel (82% de satisfaction moyenne sur leur bien-être), ils sont nettement plus pessimistes concernant l'avenir de la France et de la planète.
Patrick Martin, président du MEDEF, appelle à ne pas auto-alimenter un "pessimisme Crass" et rappelle les initiatives positives en France, comme la création d'entreprises. Cependant, Maël Nisan et Claire Touri rappellent qu'il ne faut pas "confondre pessimisme et réalisme".
2. Le Rôle du CESE et la Démocratie Participative
Le CESE se positionne comme un acteur essentiel pour "sortir de la crise démocratique" en renforçant la démocratie participative et en assurant le lien entre les pouvoirs publics et la société civile.
Il est le "lieu où s'organise le débat avec les représentants de la société civile organisée et avec les citoyens."
Marie-Lise Léon, secrétaire générale de la CFDT, insiste sur l'importance de "rendre visible ces millions de personnes" qui ne rentrent pas dans les statistiques macro. François Asselin, président de la Confédération des petites et moyennes entreprises, défend le CESE en affirmant que "si on le fout en l'air, on se dira, c'était quand même mieux avant." * Missions et Contribution : Le CESE a quatre missions principales : conseiller le gouvernement et le Parlement, favoriser le dialogue social, évaluer les politiques publiques et renforcer la démocratie participative. Des exemples concrets de propositions du CESE ayant eu un impact sont cités, comme la "garantie jeune" et les dispositifs d'alerte pour les violences faites aux femmes. * Pétitions Citoyennes : Les citoyens peuvent soumettre une pétition au CESE dès l'âge de 16 ans. Si elle recueille plus de 150 000 signatures, le Conseil "doit obligatoirement étudier le sujet".
Même sans atteindre ce seuil, une pétition peut retenir l'attention du Conseil. * Méthodes Participatives : Le CESE utilise des méthodes participatives telles que le tirage au sort de citoyens, l'organisation de conventions citoyennes ou les consultations en ligne.
Claire Touri, rapporteure du rapport, souligne que "participation ne veut pas dire consultation, c'est bien de coconstruction dont il s'agit." Elle ajoute que "les citoyens n'ont jamais été aussi éduqués, informés, connectés ; ils veulent participer davantage."
3. Les Solutions Proposées et les Défis à Relever
Le rapport et les débats identifient des pistes pour "sortir de l'urgence" et "retrouver du pouvoir d'agir", en mettant l'accent sur la proximité, la co-construction et une vision à long terme.
Il est crucial d'apporter des "réponses ciblées" aux situations diverses, car "construire des réponses trop macro empêche d'appréhender finement le vécu, les aspérités et donc d'apporter des solutions adaptées aux besoins des individus."
Dominique Charger, président de la coopération agricole, insiste sur l'importance de l'ancrage et des "réalités vécues". * Reconstruire la Confiance et le Sens Commun : La demande d'écoute et de considération est "extrêmement forte" chez les Français.
Il est essentiel de "retisser le lien" entre l'action publique et les citoyens.
Laurent Escure, secrétaire général de l'UNSA, met en avant le besoin d'une "fabrique de commun et de commodités" pour réduire les fractures sociales et territoriales. Noël Léandri, président du collectif Alerte, souligne le "besoin de dignité pour nourrir notre cohésion sociale." * Dette Publique et Investissement Stratégique : La dette publique est une "préoccupation majeure" pour les Français. Le rapport suggère de l'utiliser pour des "politiques structurelles" et pour "investir" plutôt que pour couvrir des dépenses de fonctionnement. Jacques Landriot, président de la Confédération des Scops et des Scop, cite une étude sur la transition écologique montrant que "ne pas investir dans la transition écologique pourrait coûter plus cher que de le faire." Jean-Charles Deschamp, du groupe des associations, appelle à aborder la "réforme fiscale" sans tabou. * Importance de l'Éducation et du Travail : L'éducation est perçue comme un "outil de lutte contre les inégalités" (27% des sondés). Le travail et l'emploi sont également "toujours extrêmement valorisé par les Français", comme des "leviers d'insertion" et des ressources économiques déterminantes. * Interdépendance et Coopération : Marie-Lise Léon met en avant l'importance de l'"interdépendance entre acteurs de la société civile" et avec les acteurs politiques et économiques. Patrick Livet, président de France Tiers-Lieu, souligne que "personne n'a la réponse tout seul" et que les crises sont "communes" et "se rejoignent". Marc Clamel, présidente de la CoFaC, insiste sur le fait que "l'intérêt général ne se décrète pas, qu'il se construit pas à pas avec les intérêts individuels, les intérêts collectifs pour aboutir à l'intérêt général." * Politisiser la Société : Claire Touri appelle à passer d'une société engagée à une société "beaucoup plus politisée", non pas au sens partisan, mais en développant "une approche plus systémique" et en créant des "espaces où s'expriment les désaccords", car "ce n'est pas grave de ne pas être d'accord".
Sylvain Boucherin, de l'association Humanité Biodiversité, évoque une "inquiétude" sur le fait que la transition écologique aura un coût, mais aussi un "point positif" sur la capacité de la biodiversité à repartir.
Conclusion
Le rapport et les discussions soulignent l'urgence d'une refonte du contrat social français, axée sur la lutte contre les inégalités, le renforcement de la démocratie participative et une approche holistique des défis.
Le CESE, en tant qu'instance unique de dialogue entre la société civile et les pouvoirs publics, se positionne comme un acteur central pour initier ces transformations, en encourageant l'écoute, le respect et la co-construction des solutions adaptées aux réalités des citoyens.
Ce document est un compte rendu détaillé des discussions et des propositions issues de la session du Conseil Économique, Social et Environnemental (CESE) consacrée aux violences faites aux femmes, en particulier dans les Outre-mer. Il met en lumière les rôles du CESE, l'ampleur du problème des violences, les obstacles à leur éradication, les initiatives de la société civile, et les axes d'amélioration des politiques publiques.
Rôle et Missions du CESE Le CESE est présenté comme la "troisième assemblée citée dans la Constitution" après l'Assemblée Nationale et le Sénat. Sa mission principale est de "conseiller le gouvernement et le Parlement dans l'élaboration des lois et des politiques publiques". Plus récemment, il est également devenu la "chambre de la participation citoyenne", agissant comme un "trait d'union entre les pouvoirs publics et la société civile".
Les quatre missions principales du CESE sont :
Conseiller et éclairer le gouvernement et le Parlement. Favoriser le dialogue social et la recherche de consensus. Contribuer à évaluer l'efficacité des politiques publiques. Prendre le pouls des régions en lien constant avec les CESER. Le CESE a également pour mission de "renforcer la démocratie participative en permettant aux citoyens de faire entendre leur voix". Les citoyens peuvent saisir le CESE via pétition, et si elle recueille plus de 150 000 signatures, le Conseil est "obligatoirement" tenu d'étudier le sujet.
Ampleur et Spécificités des Violences dans les Outre-mer Les statistiques et témoignages révèlent une situation alarmante :
En 2023, 96 féminicides sur 119 décès au sein des couples et 451 tentatives d'homicide en France, chiffres en "hausse constante depuis 2019". Un viol ou une tentative de viol "toutes les 2 minutes et demi" en France. Les violences faites aux femmes sont "toujours aussi prégnantes dans le monde et en France", et les Outre-mer ne font "pas exception, bien au contraire". Entre 2017 et 2023, le nombre de féminicides a augmenté dans les Outre-mer, tout comme les violences sexuelles et psychologiques, y compris dans la sphère professionnelle. "11% des féminicides interviennent dans les territoires ultramarins qui ne concentrent que 4% de la population". Les taux de morts violentes au sein du couple placent la Guyane, la Nouvelle-Calédonie, et la Polynésie française comme "les territoires ayant les niveaux de violence les plus élevés en France". Forte augmentation des violences psychologiques, pour lesquelles les campagnes de prévention sont "quasi inexistantes". Un enfant est tué "tous les 6 jours au sein de sa famille" en France, et "160 000 sont agressés sexuellement chaque année". Plusieurs facteurs aggravants et spécificités sont soulignés pour les Outre-mer :
Contexte socio-économique : "creusement des inégalités familiales, sociales et économiques", "taux des familles monoparentales", "revenus très bas". "Un enfant sur deux à la Réunion vit dans un foyer pauvre, 37% de la population vit en dessous du seuil de pauvreté." Culture patriarcale : "culture patriarcale de domination masculine toujours très enracinée", "tabou" autour de l'éducation à la vie affective, relationnelle et sexuelle. Spécificités géographiques et sociales : "interconnaissances plus fortes" sur les îles rendant la dissimulation des violences plus facile, difficultés de fuite dues à l'éloignement ("océan tout autour"). Poids des coutumes : En Nouvelle-Calédonie, le "statut civil coutumier" rend la situation des femmes particulièrement difficile. Une femme mariée coutumièrement "est mariée à un clan", et si elle part, c'est "sans ses enfants parce qu'en fait ses enfants appartiennent au clan". Le divorce est également complexe, nécessitant l'accord des deux clans. Méconnaissance des formes de violence : Beaucoup de femmes ne réalisent pas qu'elles sont victimes, surtout pour les violences psychologiques et économiques. Obstacles et Défis à la Lutte Plusieurs obstacles majeurs sont identifiés :
Manque de financement : Les associations sont le "premier acteur de terrain", mais manquent cruellement de moyens financiers et de "visibilité à moyen terme". Des exemples concrets sont donnés sur l'annulation de formations faute d'inscrits, ou l'absence de versement des subventions annuelles. Manque de cohérence et de coordination : Les actions sont souvent "ponctuelles", il manque une "grosse dose de cohérence, coordination, coopération". Données lacunaires : Les données sur les violences sont "trop éparses et incomplètes", ce qui "montre en creux l'absence de vision globale et de coordination des politiques publiques". Accès insuffisant aux droits et à la protection : Les victimes ont un "accès insuffisant au droit". Le manque de places d'hébergement est criant, forçant des femmes à "dormir dans leur voiture". Le 115 est "saturé". Méconnaissance des outils existants : Les "lois sont méconnues", y compris des acteurs de terrain. L'ordonnance de protection est "très peu utilisée". Implication inégale des élus locaux : "La prise de conscience des élus [...] est très inégale et elle est souvent insuffisante pour faire bouger les choses". Formation insuffisante des professionnels : Malgré des formations dispensées, elles restent "très inégales d'un territoire à l'autre". Les encadrants d'enfants, par exemple, ne sont pas toujours formés, ce qui "dilue" l'impact des actions de sensibilisation. Adaptation des outils nationaux : Des dispositifs comme le 3919 sont "totalement inadaptés à la diversité des populations ultra-marines" (langue, horaires, interconnaissance). Initiatives et Solutions Proposées Plusieurs pistes et actions sont suggérées :
Soutien aux associations : Reconnaître leur rôle "primordial" et leur accorder des "moyens pérennes" et des "financements à hauteur des besoins", connus "tôt dans l'année". Prévention et éducation : Développer la "prévention dès le plus jeune âge", notamment via l'éducation à la vie affective, relationnelle et sexuelle (EVARS), obligatoire mais non appliquée partout. Il faut "rompre avec les stéréotypes sexistes" et "adapter les messages aux populations locales". Amélioration des données et observatoires : Étendre les enquêtes "Virage" à l'ensemble des territoires et alimenter un "observatoire national dédié aux violences faites aux femmes dans les Outre-mer" pour disposer de "chiffres fiables et homogènes". Renforcement de la coordination : Création de "référents dans un peu toutes les administrations et aussi des associations qui montent en compétence sur l'écoute des femmes victimes de violence". Importance des "délégations régionales aux droits des femmes" si elles sont dotées de moyens suffisants. Formation des professionnels : Renforcer la "formation initiale et continue des professionnels" (gendarmes, intervenants sociaux, magistrats, personnel de santé, élus). Décloisonnement de la justice : Les "pôles violences intrafamiliales" visent à spécialiser les magistrats et à favoriser les échanges entre les différents acteurs judiciaires pour une prise en charge globale. Lois et politiques publiques ambitieuses : Nécessité d'une "loi cadre" similaire à la loi intégrale espagnole pour une approche globale et coordonnée. Les plans pluriannuels d'investissement ("PPI") et les "contrats de convergence et de transformation" devraient inclure des "financements pluriannuels fléchés" pour la lutte contre les violences. Mobilisation de la société civile : Impliquer davantage les élus locaux, les organisations patronales, syndicales, et les associations. Le CESE lui-même peut servir de lieu de "débat avec les représentants de la société civile organisée et avec les citoyens". Innovation technologique et outils de protection : Utilisation d'outils comme "Mémo de vie" (coffre-fort numérique), le "bouton Monchérif" (alerte géolocalisée), le "sifflet anti-relou" (interpellation de l'entourage). Sensibilisation et médiatisation : L'importance de montrer la "cruauté" des violences pour "faire comprendre l'ampleur" et inciter à la dénonciation. Conclusion Le CESE, sept ans après son premier avis en 2017, réitère son appel aux pouvoirs publics. La résolution actuelle, fruit d'un travail collaboratif et de l'écoute des acteurs de terrain, souligne l'urgence d'une "volonté politique" forte et de "moyens dédiés" pour enrayer ce fléau. Le consensus général est que les violences faites aux femmes sont "un coût humain individuel et collectif aussi dramatique qu'inacceptable", et qu'il est "l'affaire de toutes et de tous" de les combattre. La résolution, adoptée à l'unanimité (111 votants, 111 voix pour), est un appel clair à l'action.
Synthèse : La Restauration de la Nature – Face à l'Urgence, Donnons l'Envie d'Agir * Ce briefing se base sur les discussions et le projet d'avis du Conseil Économique, Social et Environnemental (CESE) intitulé "La restauration de la nature : face à l'urgence, donnons l'envie d'agir". Il met en lumière le rôle du CESE, les enjeux cruciaux de la restauration de la nature, les défis et les propositions concrètes pour une action efficace, ainsi que les divers points de vue exprimés par les groupes d'intérêts.
1. Le CESE : Un Trait d'Union et un Moteur de Démocratie Participative
Ses missions principales sont :
Les citoyens peuvent saisir le CESE via une pétition de plus de 150 000 signatures. * Des exemples concrets de l'impact du CESE sont cités, tels que la "garantie jeune" ou les "dispositifs d'alerte pour lutter contre les violences faites aux femmes".
2. L'Urgence de la Restauration de la Nature : Constats et Enjeux
Constats alarmants :
Plus de "80 % des habitats européens sont dégradés". "85 % des zones humides ont disparu" dans le monde.
La France a perdu "70 % de ses haies depuis 1950 [et] la moitié de ces zones humides entre 1960 et 1990". * Dépendance économique : Près de "50 % du PIB mondial repose sur la nature", et "80 % des emplois en France dépendent directement ou indirectement de la nature". * Santé humaine : Selon l'OMS, "23 % des décès et 25 % des pathologies chroniques dans le monde peuvent être attribués à des facteurs environnementaux et comportementaux."
Objectifs et Définition de la Restauration :
3. Les Défis et Freins à l'Action
Plusieurs obstacles entravent la mise en œuvre de la restauration :
Faible appropriation et fragmentation : Le plan national se heurte à une "faible appropriation [des enjeux], à une fragmentation des responsabilités et à un manque de moyens".
4. Préconisations du CESE : Une Approche Pragmaticque et Collaborative
Axe 1 : Donner le cap et accompagner
Axe 2 : Embarquer les acteurs en faveur de la restauration de la nature
Axe 3 : Mobiliser outils et moyens
5. Messages Clés et Perspectives
Optimisme et action concrète : Malgré l'ampleur des défis, l'avis se veut optimiste : "La restauration de la nature ça fonctionne, ça se voit et ça peut embarquer les gens à cette objectif à cet engagement collectif."
Rôle du CESE : Le CESE prouve sa capacité à "co-construire avec les acteurs, par les acteurs, à partir du terrain", et à faire discuter des points de vue divergents.
Compte-rendu détaillé de la matinée : L'IA, la voie citoyenne Date : [Non précisé, mais fait référence à des événements de 2023 et 2024] Lieu : Palais d'Iéna, siège du Conseil Économique Social et Environnemental (CESE) Organisateurs : CESE, Conseil National du Numérique (CNNum), en partenariat avec Make.org, The Future Society, Sciences Po, ENS.
Points clés :
Document de Synthèse : Les Politiques Publiques en Faveur de la Jeunesse – Constats, Défis et Recommandations
Source Principale : Extraits de la "Présentation du rapport annuel de la Cour des comptes - En direct", par Pierre Moscovici, Premier Président de la Cour des comptes, et compléments par Jacques Cressel, Président de la Commission Économie et Finances du CE.
Ce rapport, fruit d'un travail collectif des chambres de la Cour et des Chambres Régionales et Territoriales des Comptes (CRTC), est enrichi par l'éclairage d'un groupe d'experts universitaires et chercheurs spécialistes de la jeunesse.
Il s'inscrit dans une nouvelle approche de la Cour, caractérisée par une publication thématique annuelle et une transparence accrue, avec 180 rapports publiés par an.
I. La Jeunesse en France : Portrait et Enjeux Démographiques
II. Coût et Défis des Politiques Publiques en Faveur de la Jeunesse
III. Principaux Enseignements et Recommandations par Domaine
Le rapport se structure autour de 16 enquêtes réparties en quatre parties :
IV. Messages Transversaux et Orientations Stratégiques de la Cour des Comptes
V. La Situation des Finances Publiques (Constat Général de la Cour)
VI. Compléments du CE et Perspectives
Conclusion : Le rapport de la Cour des comptes, tout en dressant un état des lieux lucide des défis posés par les inégalités et la fragmentation des politiques, insuffle une note d'optimisme.
Il ne s'agit pas de déplorer un manque d'investissement, mais bien d'améliorer la "qualité de la dépense" et son "ciblage".
La jeunesse française est perçue comme un "atout", une "ressource inestimable" et "porteuse d'un dynamisme", justifiant ainsi un "témoignage de confiance".
Les recommandations visent à structurer une véritable "politique de et pour la jeunesse", intégrant planification, coordination et évaluation rigoureuse pour garantir une meilleure égalité des chances.
Compte-rendu détaillé : La prévention en santé, passons aux actes !
Il vise à identifier les thèmes principaux, les idées clés et les faits marquants soulevés par les différents intervenants, en incluant des citations pertinentes.
1. La Prévention : Un Enjeu Sociétal Majeur et Sous-Estimé
1.1 Prévenir Plutôt que Guérir : Une Évidence non Appliquée
Le constat est unanime : "Prévenir plutôt que guérir, voilà qui semble évident et pourtant la prévention est encore trop souvent le parent pauvre des politiques publiques." (Déclaration introductive).
Il est souligné que la santé ne se limite pas aux hôpitaux, médecins et médicaments, mais est une affaire de société.
1.2 Un Investissement, non un Coût
De plus, elle redonne aux citoyens un "pouvoir sur leur propre santé", les plaçant comme "acteur de tout" plutôt que comme patient.
1.3 Historique et Concepts : Prévention vs Promotion de la Santé
1.4 Efficacité et Retour sur Investissement
Le retour sur investissement est "une évidence" pour les études scientifiques, l'exemple de la prévention du tabagisme montrant "1900 % de retour sur investissement".
Malgré cela, le financement reste difficile, nécessitant des "dispositifs de financement incitatifs et pérennes" et pluriannuels.
2. Les Déterminants de la Santé et les Inégalités
La discussion met en évidence la multiplicité des déterminants qui influencent la santé, soulignant leur rôle dans la création et l'aggravation des inégalités.
2.1 Déterminants Sociaux et Économiques
2.2 Déterminants Environnementaux et Risques Émergents
2.3 Déterminants Commerciaux et Influence de l'Industrie
2.4 L'Approche Genrée en Santé
Emmanuel Cambois et Lormier soulignent que la santé des femmes et les défis auxquels elles sont confrontées (troubles musculosquelettiques, troubles anxiodépressifs, carrières hachées) sont souvent sous-estimés ou mal compris.
Il est crucial d'adopter des "approches différenciées entre les hommes et les femmes" dans la prévention et la personnalisation des soins, car les symptômes et les parcours de vie peuvent varier considérablement.
3. Innovations et Défis dans la Prévention
La discussion explore les nouvelles méthodes et outils, notamment le numérique, tout en identifiant les freins persistants à une prévention efficace.
3.1 Le Numérique : Opportunité et Défi
Les données de santé massives et l'intelligence artificielle permettent une "détection précoce" (ex: radiologie), un "soutien personnalisé" (applications mobiles, chatbots) et une "télésurveillance" des paramètres vitaux.
Cependant, des "freins" persistent : un "décalage culturel et organisationnel" du système de santé axé sur le curatif, la nécessité de "former" les professionnels de santé, et les "déterminants numériques de la santé" (accès, connectivité, confiance). L'objectif est de passer "d'une médecine épisodique à un suivi continu".
3.2 Financement et Volonté Politique
Le succès de la lutte contre le tabagisme, principalement par l'augmentation des taxes, en est la preuve. Il souligne que "ça ne demande pas de financement public, au contraire, c'est des taxes, ça apporte des financements publics".
Il critique le recours au "bon sens" plutôt qu'aux "données probantes" pour certaines initiatives de prévention coûteuses (ex: bilans de santé périodiques).
Il insiste sur la nécessité d'investir dans les réseaux de prévention de base (médecine scolaire, PMI, médecine du travail), qui sont "en difficulté".
3.3 Gouvernance et Coordination
Le Professeur Ruche et Emmanuel Cambois insistent sur la nécessité d'une "intersectorialité et interministérialité" au niveau national, et d'une "déclinaison territoriale au plus près des territoires et des populations".
La promotion de la santé plaide pour "introduire la santé dans toutes les politiques publiques".
La CNS recommande une "stratégie nationale de santé" sur 10 ans et des "feuilles de route prévention promotion de la santé" au niveau territorial avec un "rendu de compte".
4. La Santé au Travail : Un Pilier de la Prévention
La deuxième partie de la séance est spécifiquement dédiée à la santé au travail, soulignant ses défis et les pistes d'amélioration.
4.1 Des Chiffres Alarmants
Cela montre que "malgré un accord interprofessionnel national sur la prévention au travail une loi en décembre 2020 une loi en août 2021 sur la prévention au travail, on est à un niveau qui stagne en terme de prise en charge de prévention primaire au travail".
4.2 Des Bouleversements qui Pèsent
Le monde du travail est confronté à des "bouleversements" majeurs :
Les troubles musculosquelettiques, première cause de maladie professionnelle, touchent "trois femmes sur 5 et un homme sur deux". La "répartition genrée du travail domestique" impacte aussi la santé mentale des femmes. * Santé mentale : Les principaux facteurs de risque sont le "stress chronique" (80%, surcharge mentale, burnout, troubles du sommeil, suicide) et les "violences internes ou externes" (20%, incivilités, harcèlement, discrimination).
La "fatigue liée aux outils et à l'utilisation des outils numériques" est un nouveau défi. * Pratiques managériales : Elles sont "déterminantes" mais apparaissent "trop verticales et trop hiérarchiques" en France, avec un manque de "confiance au salariés" et un "besoin de maîtrise et de contrôle encore très important" (Dr. Florence Bénichou).
4.3 Les Nouveaux Visages du Travail
L'étude met en lumière la situation des travailleurs indépendants et des plateformes. Les livreurs et VTC subissent des "risques forts" (accidents, TMS, problèmes de santé mentale dus à la "pression" des algorithmes et à l'angoisse de la perte de revenus). L'accès aux assurances est "très peu connu" et utilisé.
4.4 Pistes d'Amélioration : Vers une Prévention Primaire Renforcée
Les rapporteurs proposent trois axes pour améliorer la prévention au travail :
4.5 Des Exemples de Succès et une Volonté Politique
Cette approche, qui combine "volonté politique, des moyens, une mobilisation des différents acteurs publics comme privés", montre qu'il n'y a "pas de fatalité en matière d'accidentologie".
Madame Astrid Panosian Bouvet, Ministre chargée du travail et de l'emploi, salue le rapport et confirme l'importance du sujet. Elle rappelle que la santé au travail n'est pas "assez haut sur l'agenda public".
Elle insiste sur la "lutte contre les accidents du travail grave et mortel", un phénomène qui n'est "pas une fatalité" et dont beaucoup sont "évitables".
Elle confirme la volonté de "capitaliser sur ce succès" des JO et de "dupliquer la méthode" notamment via le "dialogue social au sein des branches" et une "meilleure coopération interministérielle".
Elle souligne que la prévention doit être au "cœur des préoccupations" et non un "codicille au contrat de travail".
5. Conclusion Générale
Cela implique une approche globale et transversale, intégrant les déterminants sociaux, environnementaux et commerciaux.
Le numérique offre des outils prometteurs, mais leur déploiement doit être inclusif et accompagné.
Le financement n'est pas le seul obstacle ; la capacité à remettre en question des intérêts privés et la volonté politique sont primordiales.
La santé au travail, avec ses défis liés aux changements climatiques, aux inégalités de genre et aux nouvelles formes de travail, est un exemple criant de la nécessité d'une prévention primaire renforcée, basée sur le dialogue social et l'écoute des travailleurs.
Synthèse du Briefing : L'Accès à une Alimentation de Qualité et la Précarité Alimentaire en France
Cet avis met en lumière une situation alarmante en France où l'accès à une alimentation saine et équilibrée, en quantité suffisante, qui est un droit fondamental, n'est pas pleinement effectif.
La précarité alimentaire s'aggrave, touchant près de 16% de la population, soit 8 millions de personnes ayant eu recours à l'aide alimentaire en 2023, un chiffre ayant triplé en 10 ans.
De plus, les conséquences sanitaires sont préoccupantes, avec un tiers des adultes et plus d'un enfant sur dix en surpoids, et une augmentation des maladies chroniques liées à une alimentation déséquilibrée, représentant un coût de 11 milliards d'euros par an pour l'assurance maladie.
L'avis propose une approche systémique, interministérielle, et territoriale pour adresser ces défis majeurs.
Thèmes Principaux et Idées Clés :
Constat Alarmant de la Précarité Alimentaire et de ses Conséquences Sanitaires et Économiques :
Le Professeur Daniel Nizeri, président du comité de suivi du Plan National Nutrition Santé (PNNS), souligne que l'alimentation déséquilibrée est "directement impliquée dans le développement des principales pathologies chroniques", coûtant "55 milliards d'euros" et entraînant "plus de 50000 décès par an".
Rôle des Banques Alimentaires et Aggravation de la Précarité :
Leurs objectifs sont de "lutter simultanément contre la précarité alimentaire et le gaspillage alimentaire, utiliser l'aide alimentaire comme créatrice de lien social, participer à l'amélioration de l'alimentation distribuée, s'adapter constamment à l'évolution des besoins." * Impact de l'inflation : La crise inflationniste est la "première cause du recours à l'aide alimentaire." * Profil des personnes aidées : 42% touchent un revenu mensuel inférieur ou égal à 1000 €, une personne sur deux vit en logement social, et un tiers des personnes en emploi vit en milieu rural, signe de l'aggravation de la précarité en milieu rural et de l'émergence des "travailleurs pauvres". * Problèmes de santé et renoncement aux soins : "78 % déclarent au moins un problème de santé" et "déclare avoir renoncé à ses soins pour des raisons financières." * Amélioration de la qualité de l'aide alimentaire : Les banques alimentaires ont progressé de 8 points en 10 ans pour les fruits et légumes et intègrent l'accompagnement social via des ateliers (anti-gaspi, activité physique, nutrition santé, etc.) pour favoriser l'autonomie et la participation.
Propositions et Préconisations du CESE (16 au total) :
Points de Débat et de Divergence au sein du CESE :
Cette initiative, saluée comme "une très belle entreprise" et "une œuvre une action qui s'inscrit directement dans la volonté de se placer ailleurs et même de combattre ces remugles insupportables", met en lumière la capacité de l'entrepreneuriat social à concilier qualité alimentaire et insertion professionnelle.
Elle est présentée comme un exemple des valeurs civiques fortes et de la démocratie sociale.
Conclusion du Vote :
Le projet d'avis "Permettre à tous de bénéficier d’une alimentation de qualité en quantité suffisante" a été adopté avec 94 votes pour, 17 contre et 11 abstentions.
Ce vote témoigne d'un consensus large sur l'urgence d'agir face à la précarité alimentaire et aux enjeux de santé publique, malgré des divergences notables sur les moyens et l'étendue des mesures à prendre.
Le CESE réaffirme ainsi son rôle dans l'éclairage de la décision publique sur des sujets de société fondamentaux.
Note de synthèse : Évaluation des Politiques Publiques Environnementales
Source : Extraits de "Quelles évaluations des politiques publiques environnementales ? - En direct" (enregistrement d'une session plénière du Conseil Économique, Social et Environnemental - CESE)
Date : Session plénière du 13 juin [année non précisée, mais post-2008 et avec des références à des événements de 2023-2025].
Objet : Examen d'un projet d'avis intitulé "L'évaluation des politiques publiques environnementales : un pilier démocratique à consolider".
Cette session aborde également des sujets d'actualité variés tels que la publicité des paris sportifs, la jurisprudence Depardieu, les coupes budgétaires dans la branche famille de la sécurité sociale, le gel de MaPrimeRénov', les reculs écologiques, et la situation à Gaza.
Résumé
L'avis présenté vise à renforcer la faisabilité, la légitimité démocratique et l'utilité de ces évaluations.
Il met en lumière les lacunes actuelles, notamment le manque d'objectifs clairs, d'indicateurs et de suivi, et propose 14 préconisations concrètes pour y remédier.
Les discussions insistent sur l'importance d'anticiper l'évaluation dès la conception des politiques, d'associer toutes les parties prenantes (y compris les citoyens, les jeunes et les acteurs territoriaux), et d'utiliser les conclusions pour éclairer les décisions futures et le débat public.
Un thème récurrent est la nécessité de changer la culture de l'évaluation, la percevant non comme une sanction, mais comme un outil d'amélioration continue et de redevabilité.
Thèmes Principaux
Le rôle et les missions du CESE :
Citations Clés
Les principales recommandations incluent :
Institutionnaliser l'évaluation : Intégrer l'évaluation comme une étape fondamentale et obligatoire dès la conception de toute politique environnementale, avec des objectifs et des indicateurs clairs.
Renforcer la participation citoyenne et territoriale : Associer activement un large éventail de parties prenantes, y compris les citoyens, les jeunes, et les conseils territoriaux (Césars et Codèves), pour garantir la légitimité et l'acceptabilité des politiques.
Transparence et redevabilité : Assurer un suivi public et structuré des évaluations, avec la création d'une plateforme nationale, pour éclairer le débat public et permettre aux citoyens de comprendre et d'agir.
Changer la culture de l'évaluation : Éduquer les décideurs et le public à percevoir l'évaluation non comme une menace ou une sanction, mais comme un outil d'apprentissage, d'amélioration continue et d'efficacité de l'action publique.
Prendre en compte le coût de l'inaction : Développer des outils pour chiffrer les conséquences économiques, sociales et environnementales du non-agir, afin de justifier des politiques ambitieuses.
Adopter une pensée systémique et la robustesse : Aller au-delà de la performance et des chiffres isolés pour considérer les interactions complexes des écosystèmes et privilégier des solutions qui augmentent la résilience des systèmes face aux fluctuations.
L'accent devrait être mis sur la biodiversité comme levier systémique.
Briefing Document : Désenclaver pour un espace de vie apaisé - Quartiers urbains populaires #2 : En direct
Date : 14 juin 2024 (référence aux dates mentionnées dans les sources)
L'événement fait suite aux violences de 2023 et s'inscrit dans une démarche d'écoute active et d'immersion dans la réalité de ces quartiers. L'objectif principal est de "désenclaver les quartiers pour y faire advenir des espaces de vie apaisés".
Thèmes Majeurs et Idées Clés :
1. Revalorisation et Reconnaissance des Quartiers Populaires :
2. Le Désenclavement : Au-delà de l'Infrastructure, un Enjeu Humain et Social :
3. Le Rôle Central de la Culture et de l'Éducation :
4. Les Défis et la Nécessité d'une Action Collective et Durable :
Conclusion : Cette conférence met en lumière la richesse et la vitalité des quartiers populaires, tout en soulignant les défis structurels auxquels ils sont confrontés, notamment le manque de moyens et la stigmatisation.
Les solutions présentées reposent sur une approche participative, une valorisation des initiatives locales, un réinvestissement dans l'accès à la culture et à l'éducation, et une volonté de "désenclaver" au sens large : briser les barrières physiques et mentales, recréer du lien social et donner le pouvoir d'agir aux habitants pour qu'ils soient les acteurs de leur propre développement et du "vivre ensemble".
Le message final est clair : "il n'y a pas de fatalité" et l'action collective est la clé pour un avenir apaisé.
Note de Synthèse : Réflexion sur les Politiques de l'Enfance en France
Date : 27 Octobre 2023 Sujet : Bilan et perspectives des politiques publiques de l'enfance en France, rôle du Haut Commissariat à l'Enfance, défis et pistes d'action.
Source : Extraits de "CCTE - Session#1 : Échanges avec Sarah EL HAÏRY - En direct"
1. Création et Mission du Haut Commissariat à l'Enfance (HCE)
Le Haut Commissariat à l'Enfance est une institution nouvellement créée, ayant moins de trois mois d'existence au moment de l'intervention.
Sa création répond à un besoin jugé "criant" de repenser la place de l'enfant dans la société française de manière globale.
L'objectif est de créer un environnement qui pense l'ensemble des temps et spécificités de l'enfant.
Sarah El Haïry se décrit comme celle qui va "pousser la place de l'enfant ou la voie de l'enfant quel que soit le ministère et donc quelle que soit la politique publique pensée".
Son rôle n'est pas opérationnel mais garantit la prise en compte des besoins et des droits de l'enfant dans toutes les instances, même celles éloignées de l'univers de l'enfance (ex: secteur du tourisme avec la question du "No Kids").
Elle travaille en étroite collaboration avec Matignon pour assurer l'interministérialité et peut mobiliser toutes les administrations.
Elle peut demander des expertises mais surtout comprendre "Qu'est-ce qui a bloqué ? Pourquoi lui tout seul il l'a pas fait ?".
2. Défis Majeurs des Politiques de l'Enfance
Plusieurs défis sont soulevés, illustrant la complexité et les lacunes actuelles du système :
Sarah El Haïry confirme ce point : "rien ça a été fait par silo". La prise en charge des enfants en situation de handicap, par exemple, implique de multiples acteurs (département pour le transport, État pour l'éducation, ministère de la santé pour les soins) sans coordination globale, menant à des "inégalités territoriales" et une situation de "petit bonheur la chance".
Place de l'enfant dans la société : La question des "No Kids" (établissements interdisant l'accès aux enfants) est soulevée comme une illustration du manque de prise en compte de la place de l'enfant, qui est pourtant un "premier des impératifs".
Protection et bien-être matériel : La situation des "enfants à la rue" est un "engagement" du Président de la République mais reste une réalité criante.
La Haute Commissaire insiste sur la "faiblesse ou la fragilité parfois de notre système" où la responsabilité est renvoyée entre l'État (pour les familles) et les départements (pour les enfants seuls), menant à "3000" enfants à la rue.
Elle souligne que "quelle que soit sa nationalité quelle que soit son identité c'est pas aujourd'hui un élément qui doit être différenciant".
Une citoyenne soulève la question des enfants jugés "dysfonctionnants" à l'école, qui sont trop souvent médicalisés au lieu d'être accompagnés pour transformer leurs particularités en "chance et une force".
La Haute Commissaire mentionne l'absence de "jaune budgétaire" pour l'enfance, rendant difficile une vision d'ensemble des investissements.
3. Soutien à la Parentalité et Rôle des Parents
Le rôle des parents et le besoin de soutien sont des thèmes centraux des échanges :
Sarah El Haïry répond que l'objectif est de "donner à nos enfants les apprentissages essentiels" et de les "accompagner dans cette éducation au consentement", en complémentarité avec les parents.
4. Valorisation des Métiers de l'Enfance
La crise des vocations et le manque de reconnaissance des professionnels travaillant auprès des enfants sont un sujet de préoccupation :
5. Engagement et Perspectives
Sarah El Haïry affirme son engagement personnel et la singularité de sa mission :
Optimisme : Elle exprime un optimisme mesuré, voyant des ouvertures et une volonté collective : "je vois toute la force des équipes de l'Élysée toute la force des équipes de Matignon la force des ministères avec qui je travaille en se disant 'Bah OK mais toi tu vas suivre dans la durée.' Bah oui moi je vais suivre dans la durée".
En conclusion, cette séance d'échanges met en lumière la volonté de la France de dépasser les approches fragmentées des politiques de l'enfance par la création d'un Haut Commissariat dédié.
Les défis sont immenses, allant de la protection des enfants vulnérables et de l'accès à un environnement décent, au soutien d'une parentalité en mutation et à la valorisation des métiers de l'enfance.
L'accent est mis sur la transversalité, la coordination et la nécessité d'une vision globale et durable pour assurer le bien-être de tous les enfants.
Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
Summary:
The authors aimed to elucidate the molecular mechanisms underlying HIV-1 persistence and host immune dysfunction in CD4+ T cells during early infection (<6 months). Using single-cell multi-omics technologies-including scRNA-seq, scATAC-seq, and single-cell multiome analyses-they characterized the transcriptional and epigenomic landscapes of HIV-1-infected CD4+ T cells. They identified key transcription factors (TFs), signaling pathways, and T cell subtypes involved in HIV-1 persistence, particularly highlighting KLF2 and Th17 cells as critical regulators of immune suppression. The study provides new insights into immune dysregulation during early HIV-1 infection and reveals potential epigenetic regulatory mechanisms in HIV-1-infected T cells.
Strengths:
The study excels through its innovative integration of single-cell multi-omics technologies, enabling detailed analysis of gene regulatory networks in HIV-1-infected cells. Focusing on early infection stages, it fills a crucial knowledge gap in understanding initial immune responses and viral reservoir establishment. The identification of KLF2 as a key transcription factor and Th17 cells as major viral reservoirs, supported by comprehensive bioinformatics analyses, provides robust evidence for the study's conclusions. These findings have immediate clinical relevance by identifying potential therapeutic targets for HIV-1 reservoir eradication.
We sincerely appreciate the reviewer’s positive evaluation of our work.
Weaknesses:
Despite its strengths, the study has several limitations. By focusing exclusively on CD4+ T cells, the study overlooks other relevant immune cells such as CD14+ monocytes, NK cells, and B cells. Additionally, while the authors generated their own single-cell datasets, they need to validate their findings using other publicly available single-cell data from HIV-1-infected PBMCs.
Thank you to Reviewer #1 for your feedback on our work. In response to this feedback, we have examined cell-cell interactions between HIV-1-infected CD4+ T cells and other innate immune cells, including monocytes and NK cells. We identified altered interaction signaling patterns (e.g., MIF, ICAM2, CCL5, CLEC2B) that contribute to immune dysfunction and viral persistence (page 9, Supplementary Fig. 5) In addition, we validated the expression of KLF2 and its target genes using a publicly available scRNA-seq dataset from HIV-1-infected PBMCs [1], which includes both healthy donors and individuals with chronic HIV-1 infection. The upregulation of key KLF2 targets in HIV-1-infected CD4+ T cells from this dataset supports the reproducibility of our findings. We have incorporated into the revised Results, Discussion, and Supplementary Materials (page 8, page 12 and Supplementary Fig. 4A).
Reviewer #2 (Public review):
Summary:
The authors observed gene ontologies associated with upregulated KLF2 target genes in HIV-1 RNA+ CD4 T Cells using scRNA-seq and scATAC-seq datasets from the PBMCs of early HIV-1-infected patients, showing immune responses contributing to HIV pathogenesis and novel targets for viral elimination.
Strengths:
The authors carried out detailed transcriptomics profiling with scRNA-seq and scATAC-seq datasets to conclude upregulated KLF2 target genes in HIV-1 RNA+ CD4 T Cells.
We thank the reviewer for highlighting the strengths of our work.
Weaknesses:
This key observation of up-regulation KLF2 associated genes family might be important in the HIV field for early diagnosis and viral clearance. However, with the limited sample size and in-vivo study model, it will be hard to conclude. I highly recommend increasing the sample size of early HIV-1-infected patients.
Thank you to Reviewer #2 for this important comment. We acknowledge the limitations of our modest sample size, which reflects the challenges of recruiting well-characterized individuals in early HIV-1 infection (<6 months) and obtaining high-quality PBMCs for single-cell multi-omic profiling. To strengthen our findings, we validated the upregulation of KLF2 target genes using a publicly available scRNA-seq dataset from HIV-1-infected PBMCs [1], which showed similar expression patterns in HIV-1 RNA+ CD4+ T cells (page 8 and Supplementary Fig. 4A).
Reviewer #3 (Public review):
Summary:
This manuscript studies intracellular changes and immune processes during early HIV-1 infection with an additional focus on the small CD4+ T cell subsets. The authors used single-cell omics to achieve high resolution of transcriptomic and epigenomic data on the infected cells which were verified by viral RNA expression. The results add to understanding of transcriptional regulation which may allow progression or HIV latency later in infected cells. The biosamples were derived from early HIV infection cases, providing particularly valuable data for the HIV research field.
Strengths:
The authors examined the heterogeneity of infected cells within CD4 T cell populations, identified a significant and unexpected difference between naive and effector CD4 T cells, and highlighted the differences in Th2 and Th17 cells. Multiple methods were used to show the role of the increased KLF2 factor in infected cells. This is a valuable finding of a new role for the major transcription factor in further disease progression and/or persistence.
The methods employed by the authors are robust. Single-cell RNA-Seq from PBMC samples was followed by a comprehensive annotation of immune cell subsets, 16 in total. This manuscript presents to the scientific community a valuable multi-omics dataset of good quality, which could be further analyzed in the context of larger studies.
We sincerely thank the reviewer for the insightful and concise summary of our work.
Weaknesses:
Methods and Supplementary materials
Some technical aspects could be described in more detail. For example, it is unclear how the authors filtered out cells that did not pass quality control, such as doublets and cells with low transcript/UMI content. Next, in cell annotation, what is the variability in cell types between donors? This information is important to include in the supplementary materials, especially with such a small sample size. Without this, it is difficult to determine, whether the differences between subsets on transcriptomic level, viral RNA expression level, and chromatin assessment are observed due to cell type variations or individual patient-specific variations. For the DEG analysis, did the authors exclude the most variable genes?
Thank you to Reviewer #3 for these detailed comments and observations. In the revised Methods section (page 16), we have added information on our quality control filtering process. Specifically, we excluded cells with fewer than 200 detected genes, high mitochondrial content (>30%), or low UMI counts. Doublets were identified and removed using DoubletFinder.
To address inter-donor variability, we included a new supplementary figure (Supplementary Fig. 1B) showing the distribution of major immune cell types across individual donors. While we observed some variation in cell-type composition between individuals, this likely reflects natural biological heterogeneity in early HIV-1 infection. Additionally, we applied fastMNN batch correction to mitigate donor-specific technical variation. After correction, the overall patterns of gene expression within each major CD4+ T cell subset were consistent across individuals (Supplementary Fig. 1C).
Regarding the DEG analysis, we used ‘FindMarkers’ function in Seurat (v.3.2.1), which does not exclude highly variable genes. These details have been clarified in the updated Methods section (page 18).
The annotation of 16 cell types from PBMC samples is impressive and of good quality, however, not all cell types get attention for further analysis. It’s natural to focus primarily on the CD4 T cells according to the research objectives. The authors also study potential interactions between CD4 and CD8 T cells by cell communication inference. It would be interesting to ask additional questions for other underexplored immune cell subsets, such as: 1) Could viral RNA be detected in monocytes or macrophages during early infection? 2) What are the inferred interactions between NK cells and infected CD4 T cells, are interactions similar to CD4-CD8 results? 3) What are the inferred interactions between monocytes or macrophages and infected CD4 T cells?
In line with our study objectives, we initially focused on CD4+ T cells as primary HIV-1 targets. However, in response to the reviewer’s comment, we examined the inferred communications between HIV-1-infected CD4+ T cells and other immune cells.
(1) With regard to the presence of viral RNA in monocytes or macrophages, we observed negligible HIV-1 RNA signal in these cell types in our dataset, consistent with their low permissiveness in early-stage infection [2]. However, we acknowledge the limitations of detecting rare infected cells at the single-cell level.
(2) We identified increased MIF and ICAM2 signaling between NK cells and HIV-1-infected CD4+ T cells, which are associated with KLF2-mediated immune modulation. These patterns are consistent with the CD4–CD8 interaction results observed in our dataset. (Supplementary Fig. 5A)
(3) Through the cell-cell interaction analysis with differential expression analysis, we inferred reduced CCL5 and CD55 signaling between monocytes and HIV-1-infected CD4+ T cells (Supplementary Fig. 5B). These reductions may potentially impair immune responses and antiviral defense.
We appreciate the reviewer’s suggestions and believe that the analysis of underexplored immune subsets strengthens the relevance of our findings. These results have been incorporated into the revised Results (page 9).
Discussion
It would be interesting to see more discussion of the observation of how naïve T cells produce more viral RNA compared to effector T cells. It seems counterintuitive according to general levels of transcriptional and translational activity in subsets.
Another discussion block could be added regarding the results and conclusion comparison with Ashokkumar et al. paper published earlier in 2024 (10.1093/gpbjnl/qzae003). This earlier publication used both a cell line-based HIV infection model and primary infected CD4 T cells and identified certain transcription factors correlated with viral RNA expression.
Thank you to Reviewer #3 for the insightful suggestions. We observed that the proportion of HIV-1-infected naïve CD4 T cells is higher compared to effector T cells. Although effector CD4 T cells are generally more active, previous studies have suggested that naïve CD4 T cells are susceptible to HIV-1 infection during early infection that may associate with initial expansion and rapid progression [3, 4]. This may be due to less restriction by antiviral signaling or more accessible chromatin states in resting cells. We have added this context and cited relevant papers to address this observation (page 11)
In addition, we have incorporated a comparative discussion with the recent study [5], which identified FOXP1 and GATA3 as transcriptional regulators associated with HIV-1 RNA expression. While these TFs were not significantly differentially expressed in our dataset, we discuss potential reasons for this discrepancy—including differences in infection model (in vitro vs. ex vivo), infection stage (latency vs. acute), and T cell subset composition—and emphasize that both studies highlight the importance of transcriptional regulation in HIV-1 persistence (page 12 and Supplementary Fig. 4B).
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
The study has several notable limitations.
First, it was restricted to early-stage HIV-1 infection (<6 months) without longitudinal data, preventing the authors from capturing temporal changes in immune cell populations, gene expression profiles, and epigenetic landscapes throughout disease progression.
Thank you to Reviewer #1 for this important limitation. As noted, our study focused exclusively on early-stage HIV-1 infection (<6 months) to capture the initial immune dysregulation and epigenetic alterations. We agree that longitudinal analysis would provide valuable insights into disease progression. However, due to the limited availability of early-infection patient samples suitable for performing multi-omics profiling, we prioritized capturing a detailed snapshot at this early stage. To address this limitation, future studies incorporating longitudinal sampling—including chronic infection and long-term non-progressors—will be essential to fully elucidate the temporal dynamics of HIV-1 pathogenesis.
Second, while the bioinformatic analysis compared "Uninfected" and "HIV-1-infected" cells from patients, the authors could have strengthened their findings by incorporating publicly available single-cell data from healthy donors and chronically infected HIV-1 patients to validate their arguments across all figures.
To support the robustness of our findings, we incorporated a publicly available single-cell RNA-seq dataset [1], which includes both healthy donors and individuals with chronic HIV-1 infection. In this dataset, we validated the upregulation of KLF2 and its target genes in HIV-1-infected CD4+ T cells and observed generally consistent expression patterns with those in our early-infection cohort (page 8; page 12 and Supplementary Fig. S4). While not all gene-level trends were identically reflecting differences in infection stage and immune activation status, this external comparison reinforces the reproducibility of key observations and highlights the unique transcriptional features associated with early HIV-1 infection.
Third, although the study focused on CD4+ T cells as primary HIV-1 targets, it overlooked other important immune cells such as CD8+ T cells, monocytes, and NK cells, which may contribute to viral persistence and immune dysfunction through cell-cell interactions.
In the revised manuscript, we expanded our analysis to include predicted ligand–receptor interactions between HIV-1-infected and uninfected CD4+ T cells with innate and cytotoxic immune cells using CellChat v.2.1.1. Specifically, we evaluated interactions with NK cells and monocytes and identified altered signaling pathways such as MIF, ICAM2, CCL5, and CLEC2B, which are associated with immune modulation (Supplementary Fig. 5A). We have added these results to the revised Results (page 9).
Lastly, comparing these findings with other chronic viral infections (e.g., HBV, HCV) would have positioned this work more effectively within the broader field of viral immunology and enhanced its impact.
We agree that broader comparisons with other chronic viral infections could enhance the impact of our findings. In the current discussion, we noted similarities in interferon signaling disruption with viruses such as HCV and HSV. (page 11). Our observation that HIV-1-infected CD4+ T cells exhibit impaired interferon responses is consistent with immune evasion mechanisms reported in HCV and HSV infections. These results underscore both the shared and specific features of immune modulation and persistence during HIV-1 early infection.
Reviewer #3 (Recommendations for the authors):
Supplementary Table S1 should indicate which technique was used for sequencing. However, the current version of the table marks no protocol applied to the majority of the samples, which is confusing and needs to be corrected.
Thank you to Reviewer #3 for pointing out this important oversight. We have revised Supplementary Table S1 to clearly indicate the sequencing method used for each sample. Separate columns for scRNA-seq, scATAC-seq, and sc-Multiome now specify whether each technique was applied (“Yes” or “No”) to improve clarity and transparency.
(1) Wang, S., et al., An atlas of immune cell exhaustion in HIV-infected individuals revealed by single-cell transcriptomics. Emerg Microbes Infect, 2020. 9(1): p. 2333-2347.
(2) Arfi, V., et al., Characterization of the early steps of infection of primary blood monocytes by human immunodeficiency virus type 1. J Virol, 2008. 82(13): p. 6557-65.
(3) Douek, D.C., et al., HIV preferentially infects HIV-specific CD4+ T cells. Nature, 2002. 417(6884): p. 95-8.
(4) Jiao, Y., et al., Higher HIV DNA in CD4+ naive T-cells during acute HIV-1 infection in rapid progressors. Viral Immunol, 2014. 27(6): p. 316-8.
(5) Ashokkumar, M., et al., Integrated Single-cell Multiomic Analysis of HIV Latency Reversal Reveals Novel Regulators of Viral Reactivation. Genomics Proteomics Bioinformatics, 2024. 22(1).
Author response:
The following is the authors’ response to the original reviews.
Joint Public Review:
Summary:
The authors sought to elucidate the mechanism by which infections increase sleep in Drosophila. Their work is important because it further supports the idea that the blood-brain barrier is involved in brain-body communication, and because it advances the field of sleep research. Using knock-down and knock-out of cytokines and cytokine receptors specifically in the endocrine cells of the gut (cytokines) as well as in the glia forming the blood-brain barrier (BBB) (cytokines receptors), the authors show that cytokines, upd2 and upd3, secreted by entero-endocrine cells in response to infections increase sleep through the Dome receptor in the BBB. They also show that gut-derived Allatostatin (Alst) A promotes wakefulness by inhibiting Alst A signaling that is mediated by Alst receptors expressed in BBB glia. Their results suggest there may be additional mechanisms that promote elevated sleep during gut inflammation.
The authors suggest that upd3 is more critical than upd2, which is not sufficiently addressed or explained. In addition, the study uses the gut's response to reactive oxygen molecules as a proxy for infection, which is not sufficiently justified. Finally, further verification of some fundamental tools used in this paper would further solidify these findings making them more convincing.
Strengths:
(1) The work addresses an important topic and proposes an intriguing mechanism that involves several interconnected tissues. The authors place their research in the appropriate context and reference related work, such as literature about sickness-induced sleep, ROS, the effect of nutritional deprivation on sleep, sleep deprivation and sleep rebound, upregulated receptor expression as a compensatory mechanism in response to low levels of a ligand, and information about Alst A.
(2) The work is, in general, supported by well-performed experiments that use a variety of different tools, including multiple RNAi lines, CRISPR, and mutants, to dissect both signal-sending and receiving sides of the signaling pathway.
(3) The authors provide compelling evidence that shows that endocrine cells from the gut are the source of the upd cytokines that increase daytime sleep, that the glial cells of the BBB are the targets of these upds, and that upd action causes the downregulation of Alst receptors in the BBB via the Jak/Stat pathways.
We are pleased that the reviewers recognized the strength and significance of our findings describing a gut-to-brain cytokine signaling mechanism involving the blood-brain barrier (BBB) and its role in regulating sleep, and we thank them for their comments.
Weaknesses:
(1) There is a limited characterization of cell types in the midgut which are classically associated with upd cytokine production.
We thank the reviewer for raising this point. Although several midgut cell types (including the absorptive enterocytes) may indeed produce Unpaired (Upd) cytokines, our study specifically focused on enteroendocrine cells (EECs), which are well-characterized as secretory endocrine cells capable of exerting systemic effects. As detailed in our response to Results point #2 (please see below), we show that EEC-specific manipulation of Upd signaling is both necessary and sufficient to regulate sleep in response to intestinal oxidative stress. These findings support the role of EECs as a primary source of gut-derived cytokine signaling to the brain. To acknowledge the possible involvement of other source, we have also added a statement to the Discussion in the revised manuscript noting that other, non-endocrine gut cell types may contribute to systemic Unpaired signaling that modulates sleep.
(2) Some of the main tools used in this manuscript to manipulate the gut while not influencing the brain (e.g., Voilà and Voilà + R57C10-GAL80), are not directly shown to not affect gene expression in the brain. This is critical for a manuscript delving into intra-organ communication, as even limited expression in the brain may lead to wrong conclusions.
We agree with the reviewer that this is an important point. To address it, we performed additional validation experiments to assess whether the voilà-GAL4 driver in combination with R57C10-GAL80 (EEC>) influences upd2 or upd3 expression in the brain. Our results show that manipulation using EEC> alters upd2 and upd3 expression in the gut (Fig. 1a,b), with new data showing that this does not affect their expression levels in neuronal tissues (Fig. S1a), supporting the specificity of our approach. These new data are now included in the revised manuscript and described in the Results section. This additional validation strengthens our conclusion that the observed sleep phenotypes result from gut-specific cytokine signaling, rather than from effects on Unpaired cytokines produced in the brain.
(1) >(3) The model of gut inflammation used by the authors is based on the increase in reactive oxygen species (ROS) obtained by feeding flies food containing 1% H2O2. The use of this model is supported by the authors rather weakly in two papers (refs. 26 and 27 ): The paper by Jiang et al. (ref. 26) shows that the infection by Pseudomonas entomophila induces cytokine responses upd2 and 3, which are also induced by the Jnk pathway. In addition, no mention of ROS could be found in Buchon et al. (ref 27); this is a review that refers to results showing that ROS are produced by the NADPH oxidase DUOX as part of the immune response to pathogens in the gut. Thus, there is no strong support for the use of this model.
We thank the reviewer for raising this point. We agree that the references originally cited did not sufficiently justify the use of H<sub>2</sub>O<sub>2</sub> feeding as a model of gut inflammation. To address this, we have revised the Results section to clarify that we use H<sub>2</sub>O<sub>2</sub> feeding as a controlled method to elevate intestinal ROS levels, rather than as a general model of inflammation. This approach allows us to investigate the specific effects of ROS-induced cytokine signaling in the gut. We have also added additional citations to support the physiological relevance of this model. For instance, Tamamouna et al. (2021) demonstrated that H<sub>2</sub>O<sub>2</sub> feeding induces intestinal stem-cell proliferation – a response also observed during bacterial infection – and Jiang et al. (2009) showed that enteric infections increase upd2 and upd3 expression, which we similarly observe following H<sub>2</sub>O<sub>2</sub> feeding (Fig. 3a). These findings support the use of H<sub>2</sub>O<sub>2</sub> as a tool to mimic specific ROS-linked responses in the gut. We believe this targeted and tractable model is a strength of our study, enabling us to dissect how intestinal ROS modulates systemic physiology through cytokine signaling
Additionally, we have included a statement in the Discussion acknowledging that ROS generated during infection may activate signaling mechanisms distinct from those triggered by chemically induced oxidative stress, and that exploring these differences in future studies may yield important insights into gut–brain communication. These revisions provide a stronger justification for our model while more accurately conveying both its relevance and its limitations.
(2) >(4) Likewise, there is no support for the use of ROS in the food instead a direct infection by pathogenic bacteria. Furthermore, it is known that ROS damages the gut epithelium, which in turn induces the expression of the cytokines studied. Thus the effects observed may not reflect the response to infection. In addition, Majcin Dorcikova et al. (2023). Circadian clock disruption promotes the degeneration of dopaminergic neurons in male Drosophila. Nat Commun. 2023 14(1):5908. doi: 10.1038/s41467-02341540-y report that the feeding of adult flies with H2O2 results in neurodegeneration if associated with circadian clock defects. Thus, it would be important to discuss or present controls that show that the feeding of H2O2 does not cause neuronal damage.
We thank the reviewer for this thoughtful follow-up point. We would like to clarify that we do not claim that the effects observed in our study directly reflect the full response to enteric infection. As outlined in our revised response to comment 3, we have updated the manuscript to more precisely describe the H<sub>2</sub>O<sub>2</sub>-feeding paradigm as a model that induces local intestinal ROS responses comparable to, but not equivalent to, those observed during pathogenic challenges. This revised framing highlights both the potential similarities and differences between chemically induced oxidative stress and infection-induced responses. Indeed, in the revised Discussion, we now explicitly acknowledge that ROS generated during infection may engage distinct signaling mechanisms compared to exogenous H<sub>2</sub>O<sub>2</sub> and emphasize the value of future studies in delineating these pathways. We are currently pursuing this direction in an independent ongoing study investigating the effects of enteric infections. However, for the present work, we chose to focus on the effects of ROS-induced responses in isolation, as this provides a clean and well-controlled context to dissect the specific contribution of oxidative stress to cytokine signaling and sleep regulation.
To further address the reviewer’s concern, we have also included new data (a TUNEL stain for apoptotic DNA fragmentation) in the revised manuscript showing that H<sub>2</sub>O<sub>2</sub> feeding does not damage neuronal tissues under our experimental conditions (Fig. S3f,g). This addresses the point raised regarding the potential neurotoxicity of H<sub>2</sub>O<sub>2</sub>, as described by Majcin Dorcikova et al. (2023), and supports the specificity of the sleep phenotypes observed in our study. We believe these revisions and clarifications strengthen the manuscript and make our interpretation more precise.
(3) >(5) The novelty of the work is difficult to evaluate because of the numerous publications on sleep in Drosophila. Thus, it would be very helpful to read from the authors how this work is different and novel from other closely related works such as: Li et al. (2023) Gut AstA mediates sleep deprivation-induced energy wasting in Drosophila. Cell Discov. 23;9(1):49. doi: 10.1038/s41421-023-00541-3.
Our work highlights a distinct role for gut-derived AstA in sleep regulation compared to findings by Lin et al. (Cell Discovery, 2023)[1], who showed that gut AstA mediates energy wasting during sleep deprivation. Their study focused on the metabolic consequences of sleep loss, proposing that sleep deprivation increases ROS in the gut, which then promotes the release of the glucagon-like hormone adipokinetic hormone (AKH) through gut AstA signaling, thereby triggering energy expenditure.
In contrast, our study addresses the inverse question – how ROS in the gut influences sleep. In our model, intestinal ROS promotes sleep, raising the intriguing possibility – cleverly pointed out by the reviewers – that ROS generated during sleep deprivation might promote sleep by inducing Unpaired cytokine signaling in the gut. According to our findings, this suppresses wake-promoting AstA signaling in the BBB, providing a mechanism to promote sleep as a restorative response to gut-derived oxidative stress and potentially limiting further ROS accumulation. Importantly, our findings support a wakepromoting role for EEC-derived AstA, demonstrated by several lines of evidence. First, EEC-specific knockdown of AstA increases sleep. Second, activation of AstA<sup>+</sup> EECs using the heat-sensitive cation channel Transient Receptor Potential A1 (TrpA1) reduces sleep, and this effect is abolished by simultaneous knockdown of AstA, indicating that the sleep-suppressing effect is mediated by AstA and not by other peptides or secreted factors released by these cells. Third, downregulation of AstA receptor expression in BBB glial cells increases sleep, further supporting the existence of a functional gut AstA– glia arousal pathway. We have now included new data in the revised manuscript showing that AstA release from EECs is downregulated during intestinal oxidative stress (Fig. 7k,l,m). This suggests that this wake-promoting signal is suppressed both at its source (the gut endocrine cells), by unknown means, and at its target, the BBB, via Unpaired cytokine signaling that downregulates AstA receptor expression. This coordinated downregulation may serve to efficiently silence this arousal-promoting pathway and facilitate sleep during intestinal stress. These new data, along with an expanded discussion, provide further mechanistic insight into gut-derived AstA signaling and strengthen our proposed model.
This contrasts with the interpretation by Lin et al., who observed increased AstA peptide levels in EECs after antioxidant treatment and interpreted this as peptide retention. However, peptide accumulation may result from either increased production or decreased release, and peptide levels alone are insufficient to distinguish between these possibilities. To resolve this, we examined AstA transcript levels, which can serve as a proxy for production. Following oxidative stress (24 h of 1% H<sub>2</sub>O<sub>2</sub> feeding and the following day), when animals show increased sleep (Fig. 7e), we observed a decrease in AstA transcript levels followed by an increase in peptide levels (Fig. 7k,l,m), suggesting that oxidative stress leads to reduced gut AstA production and release. Furthermore, we recently found that a class of EECs that produce the hormone Tachykinin (Tk) and are distinct from the AstA<sup>+</sup> EECs express the ROSsensitive cation channel TrpA1 (Ahrentløv et al., 2025, Nature Metabolism2). In these Tk<sup>+</sup> EECs, TrpA1 mediates ROS-induced Tk hormone release. In contrast, single-cell RNA-seq data[3] do not support TrpA1 expression in AstA<sup>+</sup> EECs, consistent with our findings that ROS does not promote AstA release – an effect that would be expected if TrpA1 were functionally expressed in AstA<sup>+</sup> EECs. This contradicts the findings of Lin et al., who reported TrpA1 expression in AstA<sup>+</sup> EECs. We have now included relevant single-cell data in the revised manuscript (Fig. S6f) showing that TrpA1 is specifically expressed in Tk<sup>+</sup> EECs, but not in AstA<sup>+</sup> EECs, and we have expanded the discussion to address discrepancies in TrpA1 expression and AstA regulation.
Taken together, our results reveal a dual-site regulatory mechanism in which Unpaired cytokines released from the gut act at the BBB to downregulate AstA receptor expression, while AstA release from EECs is simultaneously suppressed. We thank the reviewers for raising this important point. We have also included a discussion the other point raised by the reviewers – the possibility that ROS generated during sleep deprivation may engage the same signaling pathways described here, providing a mechanistic link between sleep deprivation, intestinal stress, and sleep regulation.
Recommendations for the authors:
A- Material and Methods:
(1) Feeding Assay: The cited publication (doi.org:10.1371/journal.pone.0006063) states: "For the amount of label in the fly to reflect feeding, measurements must therefore be confined to the time period before label egestion commences, about 40 minutes in Drosophila, a time period during which disturbance of the flies affects their feeding behavior. There is thus a requirement for a method of measuring feeding in undisturbed conditions." Was blue fecal matter already present on the tube when flies were homogenized at 1 hour? If so, the assay may reflect gut capacity rather than food passage (as a proxy for food intake). In addition, was the variability of food intake among flies in the same tube tested (to make sure that 1-2 flies are a good proxy for the whole population)?
We agree that this is an important point for feeding experiments. We are aware of the methodological considerations highlighted in the cited study and have extensive experience using a range of feeding assays in Drosophila, including both short- and long-term consumption assays (e.g., dye-based and CAFE assays), as well as automated platforms such as FLIC and FlyPAD (Nature Communications, 2022; Nature Metabolism, 2022; and Nature Metabolism, 2025)[2,4,5].
For the dye-based assay, we carefully selected a 1-hour feeding window based on prior optimization. Since animals were not starved prior to the assay, shorter time points (e.g., 30 minutes) typically result in insufficient ingestion for reliable quantification. A 1-hour period provides a robust readout while remaining within the timeframe before significant label excretion occurs under our experimental conditions. To support the robustness of our findings, we complemented the dye-based assay with data from FLIC, which enables automated, high-resolution monitoring of feeding behavior in undisturbed animals over extended periods. The FLIC results were consistent with the dye-based data, strengthening our confidence in the conclusions. To minimize variability and ensure consistency across experiments, all feeding assays were performed at the same circadian time – Zeitgeber Time 0 (ZT0), corresponding to 10:00 AM when lights are turned on in our incubators. This time point coincides with the animals' natural morning feeding peak, allowing for reproducible comparisons across conditions. Regarding variability among flies within tubes, each biological replicate in the dye assay consisted of 1–2 flies, and results were averaged across multiple replicates. We observed good consistency across samples, suggesting that these small groups reliably reflect group-level feeding behavior under our conditions.
(2) Biological replicates: whereas the number of samples is clearly reported in each figure, the number of biological replicates is not indicated. Please include this information either in Material and methods or in the relevant figure legends. Please also include a description of what was considered a biological replicate.
We have now clarified in the Materials and Methods section under Statistics that all replicates represent independent biological samples, as suggested by the reviewers.
(3) Control Lines: please indicate which control lines were used instead of citing another publication. If preferred, this information could be supplied as a supplementary table.
We now provide a clear description of the control lines used in the Materials and Methods section. Specifically, all GAL4 and GAL80 lines used in this study were backcrossed for several generations into a shared w<sup>1118</sup> background and then crossed to the same w<sup>1118</sup> strain used as the genetic background for the UAS-RNAi, <i.CRISPR, or overexpression lines. This approach ensures, to a strong approximation, that the only difference between control and experimental animals is the presence or absence of the UAS transgene.
(4) Statistical analyses: for some results (e.g., those shown in Figure 3d), it could be useful to test the interaction between genotype and treatment.
We thank the reviewer for this helpful suggestion. In response, we have now performed two-way ANOVA analyses to assess genotype × treatment (diet) interaction effects for the relevant data, including those shown in Figure 3d as well as additional panels where animals were exposed to oxidative stress and sleep phenotypes were measured. We have added the corresponding interaction p-values in the updated figure legends for Figures 3d, 3k, 5a–c, 5f, 5h, 5i, 6c, 6e, and 7e. All of these tests revealed significant interaction effects, supporting the conclusion that the observed differences in sleep phenotypes are specifically dependent on the interaction between genetic manipulation (e.g., cytokine or receptor knockdown) and oxidative stress. These additions reinforce the interpretation that Unpaired cytokine signaling, glial JAK-STAT pathway activity, and AstA receptor regulation functionally interact with intestinal ROS exposure to modulate sleep. We thank the reviewer for suggesting this improvement.
(5) Reporting of p values. Some are reported as specific values whereas others are reported as less than a specific value. Please make this reporting consistent across different figures.
All p-values reported in the manuscript are exact, except in cases where values fall below p < 0.0001. In those instances, we use the inequality because the Prism software package (GraphPad, version 10), which was used for all statistical analyses, does not report more precise values. We believe this reporting approach reflects standard practice in the field.
(6) Please include the color code used in each figure, either in the figure itself or in the legend.
We have now clarified the color coding in all relevant figures. In particular, we acknowledge that the meaning of the half-colored circles used to indicate H<sub>2</sub>O<sub>2</sub> treatment was not previously explained. These have now been clearly labeled in each figure to indicate treatment conditions.
(7) The scheme describing the experimental conditions and the associated chart is confusing. Please improve.
We have improved the schematic by replacing “ROS” with “H<sub>2</sub>O<sub>2</sub>” to more clearly indicate the experimental condition used. Additionally, we have added the corresponding circle annotations so that they now also appear consistently above the relevant charts. This revised layout enhances clarity and helps readers more easily interpret the experimental conditions. We believe these changes address the reviewer’s concern and make the figure significantly more intuitive.
8) Please indicate which line was used for upd-Gal4 and the evidence that it faithfully reflects upd3 expression.
We have now clarified in the Materials and Methods section that the upd3-GAL4 line used in our study is Bloomington stock #98420, which drives GAL4 expression under the control of approximately 2 kb of sequence upstream of the upd3 start codon. This line has previously been used as a transcriptional reporter for upd3 activity. The only use of this line was to illustrate reporter expression in the EECs. To support this aspect of Upd3 expression, we now include new data in the revised manuscript using fluorescent in situ hybridization (FISH) against upd3, which confirms the presence of upd3 transcripts in prospero-positive EECs of the adult midgut (Fig. S1b). Additionally, we show that upd3 transcript levels are significantly reduced in dissected midguts following EEC-specific knockdown using multiple independent RNAi lines driven by voilà-GAL4, both alone and in combination with R57C10-GAL80, consistent with endogenous expression in these cells (Fig. 1a,b).
To further address the reviewer’s concern and provide additional support for the endogenous expression of upd3 in EECs, we performed targeted knockdown experiments focusing on molecularly defined EEC subpopulations. The adult Drosophila midgut contains two major EEC subtypes characterized by their expression of Allatostatin C (AstC) or Tachykinin (Tk), which together encompass the vast majority of EECs. To selectively manipulate these populations, we used AstC-GAL4 and Tk-GAL4 drivers – both knock-in lines in which GAL4 is inserted at the respective endogenous hormone loci. This design enables precise GAL4 expression in AstC- or Tk-expressing EECs based on their native transcriptional profile. To eliminate confounding neuronal expression, we combined these drivers with R57C10GAL80, restricting GAL4 activity to the gut and generating AstC<sup>Gut</sup>> and Tk<sup>Gut</sup>> drivers. Using these tools, we knocked down upd2 and upd3 selectively in the AstC- or Tk-positive EECs. Knockdown of either cytokine in AstC-positive EECs significantly increased sleep under homeostatic conditions, recapitulating the phenotype observed with knockdown in all EECs (Fig. 1m-o). In contrast, knockdown of upd2 or upd3 in Tk-positive EECs had no effect on sleep (Fig. 1p-r). Furthermore, we show in the revised manuscript that selective knockdown of upd2 or upd3 in AstC-positive EECs abolishes the H<sub>2</sub>O<sub>2</sub>-induced increase in sleep (Fig. 3f–h). These findings demonstrate that Unpaired cytokine signaling from AstC-positive EECs is essential for mediating the sleep response to intestinal oxidative stress, highlighting this specific EEC subtype as a key source of cytokine-driven regulation in this context. These new results indicate that AstC-positive EECs are a primary source of the Unpaired cytokines that regulate sleep, while Tk-positive EECs do not appear to contribute to this function. Importantly, upd3 transcript levels were significantly reduced in dissected midguts following AstC<sup>Gut</sup> driven knockdown (Fig. S1r), further confirming that upd3 is endogenously expressed in AstC-positive EECs. Thus we have bolstered our confidence that upd3 is indeed expressed in EECs, as illustrated by the reporter line, through several means.
(9) Please indicate which GFP line was used with upd-Gal4 (CD8, NLS, un-tagged, etc). The Material and Methods section states that it was "UAS-mCD8::GFP (#5137);", however, the stain does not seem to match a cell membrane pattern but rather a nuclear or cytoplasmic pattern. This information would help the interpretation of Figure 1C.
We confirm that the GFP reporter line used with upd3-GAL4 was obtained from Bloomington stock #98420. As noted by the Bloomington Drosophila Stock Center, “the identity of the UAS-GFP transgene is a guess,” and the subcellular localization of the GFP fusion is therefore uncertain. We agree with the reviewer that the signal observed in Figure 1c does not display clear membrane localization and instead appears diffuse, consistent with cytoplasmic or partially nuclear localization. In any case, what we find most salient is the reporter’s labeling of Prospero-positive EECs in the adult midgut, consistent with upd3 expression in these cells. This conclusion is further supported by multiple lines of evidence presented in the revised manuscript, as mentioned above in response to question #8: (1) fluorescent in situ hybridization (FISH) for upd3 confirms expression in EECs (Fig. S1b), (2) EEC-specific RNAi knockdown of upd3 reduces transcript levels in dissected midguts, and (3) publicly available single-cell RNA sequencing datasets[3] also indicate that upd3 is expressed at low levels in a subset of adult midgut EECs under normal conditions. We have also clarified in the revised Materials and Methods section that GFP localization is undefined in the upd3-GAL4 line, to guide interpretation of the reporter signal.
B- Results
(1) Figure 1: According to previous work (10.1016/j.celrep.2015.06.009, http://flygutseq.buchonlab.com/data?gene=upd3%0D%0A), in basal conditions upd3 is expressed as following: ISC (35 RPKM), EB (98 RPKM), EC (57 RPKM), and EEC (8 RPKM). Accordingly, even complete KO in EECs should eliminate only a small fraction of upd3 from whole guts, even less considering the greater abundance of other cell types such as ECs compared to EECs. It would be useful to understand where this discrepancy comes from, in case it is affecting the conclusion of the manuscript. While this point per se does not affect the main conclusions of the manuscript, it makes the interpretation of the results more difficult.
We acknowledge the previously reported low expression of upd3 in EECs. However, the FlyGut-seq site appears to be no longer available, so we could not directly compare other related genes. Nonetheless, our data – based on in situ hybridization, reporter expression, and multiple RNAi knockdowns – consistently support upd3 expression in EECs. These complementary approaches strengthen the conclusion that EECs are an important source of systemic upd3 under the conditions tested.
(2) Figure 1: The upd2-3 mutants show sleep defects very similar to those of EEC>RNAi and >Cas9. It would thus be helpful to try to KO upd3 with other midgut drivers (An EC driver like Myo1A or 5966GS and a progenitor driver like Esg or 5961GS) to validate these results. Such experiments might identify precisely which cells are involved in the gut-brain signaling reported here.
We appreciate the reviewer’s suggestion and agree that exploring other potential sources of Upd3 in the gut is an interesting direction. In this study, we have focused on EECs, which are the primary hormone-secreting cells in the intestine and thus the most likely candidates for mediating systemic effects such as gut-to-brain signaling. While it is possible that other gut cell types – such as enterocytes (e.g., Myo1A<sup>+</sup>) or intestinal progenitors (e.g., Esg<sup>+</sup>) – also contribute to Upd3 production, these cells are not typically endocrine in nature. Demonstrating their involvement in gutto-brain communication would therefore require additional, extensive validation beyond the scope of the current study. Importantly, our data show that manipulating Upd3 specifically in EECs is both necessary and sufficient to modulate sleep in response to intestinal ROS, strongly supporting the conclusion that EEC-derived cytokine signaling underlies the observed phenotype. In contrast, manipulating cytokines in other gut cells could produce indirect effects – such as altered proliferation, epithelial integrity, or immune responses – that complicate the interpretation of behavioral outcomes like sleep. For these reasons, we chose to focus on EECs as the source of endocrine signals mediating gut-to-brain communication. However, to address this point raised by the reviewer, we have now included a statement in the Discussion acknowledging that other non-endocrine gut cell types may also contribute to the systemic Unpaired signaling that modulates sleep in response to intestinal oxidative stress.
(3) Figure 3: "This effect mirrored the upregulation observed with EEC-specific overexpression of upd3, indicating that it reflects physiologically relevant production of upd3 by the gut in response to oxidative stress." Please add (Figure 3a) at the end of this sentence.
We have now added “(Figure 3a)” at the end of the sentence to clearly reference the relevant data.
(4) For Figure 3b, do you have data showing that the increased amount of sleep was due to the addition of H2O2 per se, rather than the procedure of adding it?
We have added new data to address this point. To ensure that the observed sleep increase was specifically due to the presence of H<sub>2</sub>O<sub>2</sub> and not an effect of the food replacement procedure, we performed a control experiment in which animals were fed standard food prepared using the same protocol and replaced daily, but without H<sub>2</sub>O<sub>2</sub>. These animals did not exhibit increased sleep, confirming that the sleep effect is attributable to intestinal ROS rather than the supplementation procedure itself (Fig. S3a). Thanks for the suggestion.
(5) In the text it is stated that "Since 1% H2O2 feeding induced robust responses both in upd3 expression and in sleep behavior, we asked whether gut-derived Unpaired signaling might be essential for the observed ROS-induced sleep modulation. Indeed, EEC-specific RNAi targeting upd2 or upd3 abolished the sleep response to 1% H2O2 feeding." While it is indeed true that there is no additional increase in sleep time due to EEC>upd3 RNAi, it is also true that EEC>upd3 RNAi flies, without any treatment, have already increased their sleep in the first place. It is then possible that rather than unpaired signaling being essential, an upper threshold for maximum sleep allowed by manipulation of these processes was reached. It would be useful to discuss this point.
Several findings argue against a ceiling effect and instead support a requirement for Unpaired signaling in mediating ROS-induced sleep. Animals with EEC-specific upd2 or upd3 knockdown or null mutation not only fail to increase sleep following H<sub>2</sub>O<sub>2</sub> treatment but actually exhibit reduced sleep during oxidative stress (Fig. 3e, k, l; Fig. 5e, f), suggesting that Unpaired signaling is required to sustain sleep under these conditions. Similarly, animals with glial dome knockdown also show reduced sleep under oxidative stress, closely mirroring the phenotype of EEC-specific upd3 RNAi animals (Fig. 5a–c, g–i). These results support the conclusion that gut-to-glia Unpaired cytokine signaling is necessary for maintaining elevated sleep during oxidative stress. In the absence of this signaling, animals exhibit increased wakefulness. We identify AstA as one such wake-promoting signal that is suppressed during intestinal stress. We present new data showing that this pathway is downregulated not only via Unpaired-JAK/STAT signaling in glial cells but also through reduced AstA release from the gut in the revised manuscript. This model, in which Unpaired cytokines promote sleep during intestinal stress by suppressing arousal pathways, is discussed throughout the manuscript to address the reviewer’s point.
(6) In Figure 3k, the dots highlighting the experiment show an empty profile, a full one, and a half one. Please define what the half dots represent.
We have now clarified the color coding in all relevant figures. Specifically, we acknowledge that the meaning of the half-colored circles indicating H<sub>2</sub>O<sub>2</sub> treatment was not previously defined – it indicates washout or recovery time. In the revised version, these symbols are now clearly labeled in each figure to indicate the treatment condition, ensuring consistent and intuitive interpretation across all panels.
(7) The authors used appropriate GAL4 and RNAi lines to the knockdown dome, a upd2/3 JAK-STATlinked receptor, specifically in neurons and glia, respectively, in order to identify the CNS targets of upd2/3 cytokines produced by enteroendocrine cells (EECs). Pan-neuronal dome knockdown did not alter daytime sleep in adult females, yet pan-glial dome knockdown phenocopied effects of upd2/3 knockdown in EECs. They also observed that EEC-specific knockdown of upd2 and upd3 led to a decrease in JAK-STAT reporter activity in repo-positive glial cells. This supports the authors' conclusion that glial cells, not neurons, are the targets by which unpaired cytokines regulate sleep via JAK-STAT signaling. However, they do not show nighttime sleep data of pan-neuronal and pan-glial dome knockdowns. It would strengthen their conclusion if the nighttime sleep of pan-glial dome knockdown phenocopied the upd2/3 knockdowns as well, provided the pan-neuronal dome knockdown did not alter nighttime sleep.
We have now added nighttime sleep data for both pan-glial and pan-neuronal domeless knockdowns in the revised manuscript (Fig. 2a). Glial knockdown increased nighttime sleep, similar to EEC-specific upd2/3 knockdown, while neuronal knockdown had no effect. These results further support the glial cells’ being the relevant target of gut-derived Unpaired signaling.
(8) The authors only used one method to induce oxidative stress (hydrogen peroxide feeding). It would strengthen their argument to test multiple methods of inducing oxidative stress, such as lipopolysaccharide (LPS) feeding. In addition, it would be useful to use a direct bacterial infection to confirm that in flies, the infection promotes sleep. Additionally, flies deficient in Dome in the BBB and infected should not be affected in their sleep by the infection. These experiments would provide direct support for the mechanism proposed. Finally, the authors should add a primary reference for using ROS as a model of bacterial infection and justify their choice better.
We agree that directly comparing different models of intestinal stress, such as bacterial infection or LPS feeding, would provide valuable insight into how gut-derived signals influence sleep in response to infection. As noted in our detailed responses above, we now include an expanded rationale for our use of H<sub>2</sub>O<sub>2</sub> feeding as a controlled and well-established method for inducing intestinal ROS – one of the key physiological responses to enteric infection and inflammation. In the revised Discussion, we explicitly acknowledge that pathogenic infections – which trigger both intestinal ROS and additional immune pathways – may engage distinct or complementary mechanisms compared to chemically induced oxidative stress. We emphasize the importance of future studies aimed at dissecting these differences. In fact, we are actively pursuing this direction in ongoing work examining sleep responses to enteric infection. For the purposes of the present study, however, we chose to focus on a tractable and specific model of ROS-induced stress to define the contribution of Unpaired cytokine signaling to gut-brain communication and sleep regulation. This approach allowed us to isolate the effect of oxidative stress from other confounding immune stimuli and identify a glia-mediated signaling mechanism linking gut epithelial stress to changes in sleep behavior.
(9) To confirm that animals lacking EEC Unpaired signaling are not more susceptible to ROS-induced damage, the authors assessed the survival of upd2 and upd3 knockdowns on 1% H2O2 and concluded they display no additional sensitivity to oxidative stress compared to controls. It may be useful to include other tests of sensitivity to oxidative stress, in addition to survival.
We appreciate the reviewer’s suggestion. In our view, survival is a highly informative and stringent readout, as it reflects the overall physiological capacity of the animal to withstand oxidative stress. Importantly, our data show that animals lacking EEC-derived Unpaired signaling do not exhibit reduced survival following H<sub>2</sub>O<sub>2</sub> exposure, indicating that their oxidative stress resistance is not compromised. Furthermore, we previously confirmed that feeding behavior is unaffected in these animals, suggesting that their ability to ingest food (and thus the stressor) is not impaired. As a molecular complement to these assays in response to this point and others, we have also performed an assessment of neuronal apoptosis (a TUNEL assay, Fig. S3f,g). This assay did not identify an increase in cell death in the brains of animals fed peroxide-containing medium. Thus, gross neurological health, behavior, and overall survival appear to be resilient to the environmental treatment regime we apply here, suggesting that the outcomes we observe arise from signaling per se.
(10) The authors confirmed that animals lacking EEC-derived upd3 displayed sleep suppression similar to controls in response to starvation. These results led the authors to conclude that there is a specific requirement for EEC-derived Unpaired signaling in responding to intestinal oxidative stress. However, they previously showed that EEC-specific knockdown of upd3 and upd2 led to increased daytime sleep under normal feeding conditions. Their interpretations of their data are inconsistent.
We appreciate the reviewer’s comment. While animals lacking EEC-derived Unpaired signaling show increased baseline sleep under normal feeding conditions, they still exhibit a robust reduction in sleep when subjected to starvation – comparable to that of control animals (Fig. S3h–j). This demonstrates that they retain the capacity to appropriately modulate sleep in response to metabolic stress. Thus, the sleep-promoting phenotype under normal conditions does not reflect a generalized inability to adjust sleep behavior. Rather, it highlights a specific role for Unpaired signaling in mediating sleep responses to intestinal oxidative stress, not in broadly regulating all sleep-modulating stimuli.
(11) The authors report a significant increase in JAK-STAT activity in surface glial cells at ZT0 in animals fed 1% H2O2-containing food for 20 hours. This response was abolished in animals with EECspecific knockdown of upd2 or upd3. The authors confirmed there were no unintended neuronal effects on upd2 or upd3 expression in the heads. They also observed an upregulation of dome transcript levels in the heads of animals with EEC-specific knockdown of upd3 fed 1% H2O2-containing food for 15 hours, which they interpret to be a compensatory mechanism in response to low levels of the ligand. This assay is inconsistent with previous experiments in which animals were fed hydrogen peroxide for 20 hours.
We thank the reviewer for identifying this discrepancy. The inconsistency arose from a labeling error in the manuscript. Both the JAK-STAT reporter assays in glial cells and the dome expression measurements were performed following 15 hours of H<sub>2</sub>O<sub>2</sub> feeding, not 20 hours as previously stated. We have now corrected this in the revised manuscript.
(12) The authors show that animals with glia-specific dome knockdown did not have decreased survival on H2O2-containing food, and displayed normal rebound sleep in the morning following sleep deprivation. These results potentially undermine the significance of the paper. If the normal sleep response to oxidative stress is an important protective mechanism, why would oxidative stress not decrease survival in dome knockdown flies (that don't have the normal sleep response to oxidative stress)? This suggests that the proposed mechanism is not important for survival. The authors conclude that Dome-mediated JAK-STAT signaling in the glial cells specifically regulates ROS-induced sleep responses, which their results support.
We agree that our survival data show that glial dome knockdown does not reduce survival under continuous oxidative stress. However, we believe this does not undermine the importance of the sleep response as an adaptive mechanism. In our survival assay, animals were continuously exposed to 1% H<sub>2</sub>O<sub>2</sub> without the opportunity to recover. In contrast, under natural conditions, oxidative stress is likely to be intermittent, and the ability to mount a sleep response may be particularly important for promoting recovery and maintaining homeostasis during or after transient stress episodes. Thus, while the JAK-STAT-mediated sleep response may not directly enhance survival under constant oxidative challenge, it likely plays a critical role in adaptive recovery under natural conditions.
(13) Altogether, the authors conclude that enteric oxidative stress induces the release of Unpaired cytokines which activate the JAK-STAT pathway in subperineurial glia of the BBB, which leads to the glial downregulation of receptors for AstA, which is a wake-promoting factor also released by EECs. This mechanism is supported by their results, however, this research raises some intriguing questions, such as the role of upd2 versus upd3, the role of AstA-R1 versus AstA-R2, the importance of this mechanism in terms of survival, the sex-specific nature of this mechanism, and the role that nutritional availability plays in the dual functionality of Unpaired cytokine signaling in regards to sleep.
We thank the reviewer for highlighting these important questions. Our data suggest that Upd2 and Upd3, while often considered partially redundant, both contribute to sleep regulation, with stronger effects observed for Upd3. This is consistent with prior studies indicating overlapping but non-identical roles for these cytokines. Similarly, although AstA-R1 and AstA-R2 can both be activated by AstA, knockdown of AstA-R2 consistently produces more robust sleep phenotypes, suggesting a predominant role in mediating this effect. The possibility of sex-specific regulation is indeed compelling. While our study focused on females, many gut hormones show sex-dependent activity, and we recognize this as an important avenue for future research. Finally, we have included new data in the revised manuscript showing that gut-derived AstA is downregulated under oxidative stress, further supporting our model in which Unpaired signaling suppresses arousal pathways during intestinal stress
(14)Data Availability: It is indicated that: "Reasonable data requests will be fulfilled by the lead author". However, eLife's guidelines for data sharing require that all data associated with an article to be made freely and widely available.
We thank the reviewer for pointing this out. We have revised the Data Availability section of the manuscript to clarify that all data will be made freely available from the lead contact without restriction, in accordance with eLife’s open data policy.
References
(1) Li, Y., Zhou, X., Cheng, C., Ding, G., Zhao, P., Tan, K., Chen, L., Perrimon, N., Veenstra, J.A., Zhang, L., and Song, W. (2023). Gut AstA mediates sleep deprivaPon-induced energy wasPng in Drosophila. Cell Discov 9, 49. 10.1038/s41421-023-00541-3. (2) Ahrentlov, N., Kubrak, O., Lassen, M., Malita, A., Koyama, T., Frederiksen, A.S., Sigvardsen, C.M., John, A., Madsen, P., Halberg, K.A., et al. (2025). Protein-responsive gut hormone Tachykinin directs food choice and impacts lifespan. Nature Metabolism. 10.1038/s42255-025-01267-0.
(3) Li, H., Janssens, J., De Waegeneer, M., Kolluru, S.S., Davie, K., Gardeux, V., Saelens, W., David, F.P.A., Brbic, M., Spanier, K., et al. (2022). Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly. Science 375, eabk2432. 10.1126/science.abk2432.
(4) Kubrak, O., Koyama, T., Ahrentlov, N., Jensen, L., Malita, A., Naseem, M.T., Lassen, M., Nagy, S., Texada, M.J., Halberg, K.V., and Rewitz, K. (2022). The gut hormone AllatostaPn C/SomatostaPn regulates food intake and metabolic homeostasis under nutrient stress. Nature communicaPons 13, 692. 10.1038/s41467-022-28268-x.
(5) Malita, A., Kubrak, O., Koyama, T., Ahrentlov, N., Texada, M.J., Nagy, S., Halberg, K.V., and Rewitz, K. (2022). A gut-derived hormone suppresses sugar appePte and regulates food choice in Drosophila. Nature Metabolism 4, 1532-1550. 10.1038/s42255-022-00672-z.
LA PARTE AMBIENTAL REPRESENTA UN RETO COGNITIVO COMPLEJO DADO EL ASPECTO INSTERDISCIPLINARIO
ESGO Y RENTABILIDAD4. Un paseo aleatorio por Wall Street5. CAPM: Capital Assets Princing Model (Un Modelo de Valorizaciónde Activos de Capital)6. El Costo del CapitalParte IV LA DEUDA Y LOS DIVIDENDOS7. La Política de Endeudamiento de la Empresa:Proposiciones de Miller y Modigliani (MM)8. La Política de Dividendos de la EmpresaParte V ANALISIS Y PLANIFICACION9. El Análisis de los Estados Financieros10. El Valor Económico Agreg
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El propósito de este trabajo fue explorar el contenido ético presente en las Filosofías de Gestión (1994 y 1997) y en el Plan de Desarrollo Estratégico de la Universidad del Zulia (LUZ).La metodología utilizada consistió fundamentalmente en la revisión de esos documentos, así como actas, gacetas oficiales, resoluciones y disposiciones del Consejo Universitario. Los resultados revelan la construcción de filosofías de gestión y plan de desarrollo estratégico sin discusión abierta y plural de la comunidad universitaria, a pesar de que la participación constituye una herramienta social privilegiada para el intercambio de ideas, opiniones, experiencias en pro de consecución de resultados consensuados. Estas macropolíticas contienen principios éticos con compromiso social, tales como: excelencia, democracia, autonomía, identidad, legalidad, responsabilidad, liderazgo, lealtad y solidaridad, los cuales no fueron discutidos ni internalizados por sus miembros, constituyéndose en letra muerta.Enconclusión, la responsabilidad compartida, uno de los valores fundamentales de la ética, no estuvo plasmada en la elaboración de las macropolíticas, por haber sido concebidas de manera cupular.
La planeación estrategia es muy importante en cualquier organización ya que es la base para orientar un proyecto que establezca los objetivos y metas a lograr y con ello las comunidades orientan su esfuerzo en cada acción para alcanzar lo establecido alineado a la visión y misión de la institución
Revisar la ortografía del resumen y ponerlo en fuente arial de 12 puntos.
La necesidad de pensar en la transformación de la universidad venezolana, y transformarla para constituirlaen acompañante de los procesos de emancipación de los pueblos, no sólo se hace evidente sino que ademásalcanza la condición de imperativo ante las circunstancias de un sector que, siendo estratégico para el país,
vamos hacia una Venezuela
ecnológico Nacional de México, ofreció la conferencia Justicia Social desde la perspectiva educativa, donde abordó los orígenes de la justicia social y sus componentes, los cuales son la Redistribución, que consiste en asegurar la atención a todos los reclamos que persiguen una distribución justa; un Recono
ME PARECE INTERESANTE EL ABORDAJE DE LA JUSTICIA SOCIAL, PERO HAY QUE PRECISAR EL TERMINO, BAJO QUE PERSPECTIVA IDEOLOGICA SE REFIERE, YA QUE EN OTROS DOCUMENTOS DEL SISTEMA TAMBIÉN SE HACE REFERENCIA A ESTOS TERMINO, SIN EMARGO SE ALEJARON DE ESTE OBJETIVO EN LA PRACTICA, DADO LA PERSPECTIVA SOCIOECONOMICA
el director general del Tecnológico, ofreció la conferencia Humanismo Mexicano, en donde dijo que el modelo está basado en una nueva corriente de pensamiento.
Este comentario es acorde al modelo educativo que incluye ejes centrales educativos basados en el pensamiento critico
l humanismo mexicano va orientado hacia el beneficio de la colectividad
no lo creo considero que va enfocado a otros aspectos menos la colectividad.
Proyectos Transversales,
Los proyectos transversales son necesarios en la Educación con la finalidad de fortalecer las competencias de los alumnos.
Adriana Castillo Rosas
La Doctora Adriana Castillo Rosas es una investigadora del CIIDET
fundamentos legales para la creación del modelo educativo
Definir el Marco Normativo sobre el decreto presidencial del 2015 que da sustento a la consolidación del TecNM
Una de las mayores razones del cambio en el modelo, es reiterar la soberanía e independencia de México, con una política educativa apegada al desarrollo económico y social del país, dijo.
A pesar de los Lucas Alamán y los maximilianos.
De entrada, mencionó que este modelo, responde a un ideario educativo general, el cual tiene una identidad compartida y enfatiza la necesidad de que la organización, a través de las normas, administración, currícula, docente, etcétera, trabaje por una meta común.
es importante entender una meta común pues ello le daría sentido a cada actividad que realizamos
ultural de cada región, diversifica los campos formativos, promueve el desarrollo de nuevos conocimientos, participa en la democracia y el estado de derecho, y fomenta la innovación científic
Estoy de acuerdo en identificar la identidad y cultura de cada región, para el éxito en el profesionalismo
La razón principal de que tenga el nombre de Humanismo, se debe a que tiene un sentido totalmente distinto al anterior modelo neoliberal, que no estaba enfocado en favor de las personas, sino en el de las empresas”
Tambien el modelo para el Siglo XXI, está basado en el SER HUMANO.
Humanismo Mexicano
El humanismo mexicano es un faro de esperanza y resiliencia que refleja la profunda riqueza cultural, la solidaridad comunitaria y la capacidad de transformar adversidad en creatividad.
Posteriormente explicó la estructura del Modelo Educativo, el cual se compone de tres ejes fundamentales: Pertinencia Social, Mejora Continua, Equidad y Excelencia. Adicionalmente, el Modelo cuenta con seis ejes transversales: Interculturalidad, Inclusión y Equidad; Interdisciplinariedades; Responsabilidad Social; Innovación y Vanguardia; Conciencia Ambiental.
Esta parte del texto, es claro y conciso sobre los tres ejes fundamentales
Arturo Chávez López,
Arturo Chávez es un docente muy calificado, fue el pionero de la educación tecnológico en México.
El presente artículo pretende provocar las reflexiones en torno a laproblematización desde una perspectiva dialéctico materialista tomando comoreferencia las diversas definiciones y conceptualizaciones relacionadas con latemática. Se abordan categorías como problema científico, objeto de estudio,hipótesis, entre otras, que resultan indispensables a la hora de planificar ydesarrollar una investigación, sobre todo en el campo educacional en el que losfenómenos resultan tan multifacéticos y complejos. Se hace énfasis en elcarácter científico de los problemas que se resuelven mediante la investigación,independientemente del nivel de generalidad de los mismos. Para ello seconcibe la teoría científica como un sistema de conocimientos en construcción
Es un artículo materialista dialéctico.
La materia está conformada por partículas;z Estas partículas tienen movimiento;z Entre las partículas existen espacios vacíos;z La conservación de la materia;z Modelización;z Estados de agregación.
Estos son los puntos y conocimientos clave para el avance en el conocimiento de la química
Él plantea que para ubicar el conocimiento que se desarrolla en las mentes de los profesores, habríaque distinguir tres tipos de éste, uno de los cuales es el conocimiento pedagógico del contenido(CPC), “el tema de la materia para la enseñanza
Esta información es importante debido a que se debe de considerar este en el conocimiento básico del estudiante
Compte Rendu Détaillé : Anna Baldy sur les Effets Psychologiques de TikTok sur les Mineurs
Introduction et Contexte
Son témoignage offre une perspective nuancée sur les impacts de TikTok, mêlant expérience personnelle, analyse critique et considérations éthiques.
Ayant commencé à produire du contenu sur TikTok il y a un an, elle a rapidement atteint 160 000 abonnés, générant des revenus basés sur le nombre de vues de ses vidéos (entre 900 et 1400 € mensuels pour environ 4 heures de travail quotidien sur la création de vidéos).
Elle déclare recevoir des revenus directement de TikTok, basés sur les vues, ce qui soulève des questions sur le modèle économique de la plateforme.
Son objectif en tant que créatrice est "d'amener un peu plus de contradictoire dans le paysage informationnel des réseaux sociaux", en s'appuyant sur ses études en sciences politiques pour étayer ses propos par des sources d'experts ou de chercheurs.
Thèmes Majeurs et Idées Clés
TikTok : Un Outil à Double Tranchant (Accessibilité vs. Inquiétudes)
Points Positifs :Accessibilité de l'information et pluralisme : TikTok est perçu comme un "lieu d'accessibilité de l'information gratuite" et un "rare espace médiatique que les jeunes peuvent investir pour faire entendre leurs paroles".
Il permet la survie d'un "contre-discours fort" et donne la parole à ceux dont les idées ou l'identité ne conviennent pas aux médias classiques, considérés comme parfois élitistes ou influencés par des agendas politiques (propriété de millionnaires à l'agenda politique "d'extrême droite" assumé). * Éducation et découverte : En tant qu'utilisatrice, Anna Baldy souligne que TikTok l'a "éduque aussi énormément", lui donnant accès à des "réflexions de militants" et des "recommandations de livres" qu'elle n'aurait pas trouvées autrement. * Liberté et indépendance pour les créateurs : Pour elle, TikTok offre la possibilité de "garder une pleine indépendance" et de "trouver une audience" plus facilement que sur d'autres plateformes. * Inquiétudes et Points Négatifs :Propagation de fausses informations et polarisation : "Je m'inquiète quant à la propagation d'informations fausses ou simplifiées sorties de leur contexte souvent clivantes et détournées pour faire peur pour polariser en jouant des biais cognitifs et rhétoriques que se manipuler par les plus grands propagandistes de nos heures sombres." Elle s'inquiète particulièrement de l'accès à ces informations pour des "enfants de 13 ans". * Bulles algorithmiques et impact psychologique : L'algorithme peut entraîner les utilisateurs dans "une spirale de pensée mauvaise pour nous-mêmes mauvaise pour les autres".
Elle a elle-même dû "supprimer TikTok de mon téléphone tellement je passais de temps dessus" et a désinstallé l'application à plusieurs reprises, notamment pendant l'adolescence, car "TikTok peut avoir tendance toujours dans cette ce fonctionnement des bulles algorithmiques à jouer sur le mal-être et le désespoir ressenti par tous mais surtout ressenti par les adolescents". * Contenu inapproprié et impunité : Elle déplore la "totale impunité qui entoure des comptes vidéos et des commentaires relevant d'un racisme qui n'a rien de tacite et d'une misogynie rampante".
Elle cite des exemples concrets de commentaires violents, sexistes et racistes reçus sous ses propres vidéos, craignant qu'ils soient écrits par des mineurs et que la violence réelle subie par d'autres jeunes femmes soit bien pire. * Opacité de l'algorithme : L'algorithme est "très opaque" et les créateurs n'ont pas une connaissance précise de son fonctionnement, ce qui est "volontaire puisque pour qu'on puisse pas jouer de ces outils nous-mêmes". Elle observe que les 10 à 20 premières secondes des vidéos sont cruciales pour l'engagement.
Modération et Responsabilité des Plateformes
Pour elle, "c'est une réaction que je trouve très facile et c'est le jeu que font les plateformes aujourd'hui [...] pour se déresponsabiliser et tirer tous les profits de cette situation".
Algorithme et Modèle Économique : Un Incitant aux Contenus Choc
Mineurs et Régulation : Éducation vs. Interdiction
Elle est "d'accord" sur le fait qu'un enfant "doit être protégée" et qu'un "principe de précaution" pourrait s'appliquer pour les plus jeunes, non armés face à des contenus "extrêmement inappropriés". * Fiabilité des statistiques d'âge : Elle note l'étrangeté des statistiques d'audience de TikTok, qui indiquent seulement 0,5% de mineurs pour son contenu, ce qui la surprend au vu des commentaires qu'elle reçoit de lycéens. Elle-même a déclaré son vrai âge (15 ans) lors de sa première inscription.
Conclusion
La plateforme est à la fois un espace de liberté d'expression, de pluralisme informationnel et d'apprentissage, et un vecteur de désinformation, de haine et de harcèlement, exacerbé par un algorithme opaque et un modèle économique qui incite à la production de contenus sensationnels.
Elle souligne la tension entre la volonté de protéger les mineurs et la réalité de leur capacité à contourner les interdictions, plaidant pour une approche équilibrée combinant protection, régulation et surtout éducation critique aux médias et à l'information.
Son approche "gramscienne" de l'occupation des espaces culturels témoigne d'une volonté d'agir de l'intérieur pour contrer les dérives.
Compte Rendu Détaillé : Défis de l'Environnement Numérique et de l'IA pour la Démocratie
Ce document de synthèse est une revue des principales thématiques, idées et faits marquants abordés lors de la table ronde "Vincent Champain, Gilles Babinet et Laurent Guimier sur les défis du nouvel environnement numérique", qui a eu lieu le 5 février. Les intervenants,
Vincent Champain (Observatoire du long terme),
I. Constat alarmant : La fragilisation de la démocratie à l'ère numérique et de l'IA
Les intervenants s'accordent sur un constat préoccupant : la démocratie est fragilisée dans ses fondements mêmes par le nouvel environnement numérique et l'intelligence artificielle.
Laurent Guimier évoque "un moment atomique" où "nous sommes tous collectivement en capacité d'avoir entre les mains un moyen de destruction massive de la démocratie". * Impact sur le contrat démocratique et la cohésion sociale : * Le principe "une personne, une voix" est mis en danger par des systèmes où ceux qui paient ou trichent ont plus de poids. * La polarisation affaiblit la cohésion sociale, menant à "une société de plus en plus fracturée" et un "débat public de moins en moins rationnel". * Perte de confiance dans les institutions et augmentation des confrontations violentes, loin de l'échange d'idées apaisé que la démocratie est censée promouvoir. * Ingérences étrangères et guerre informationnelle : * Vincent Champain souligne "une multiplication des cas en France à l'étranger" et une "aptitude de plus en plus passive des grandes plateformes" face aux manipulations. * Gilles Babinet fait référence à la "doctrine Guerassimov", selon laquelle "pour le coût de un coût insignifiant le coût d'un tank je suis capable de déstabiliser un pays". Il met en garde contre une "2e génération de système de désinformation" avec des "systèmes automatisés, des robots", qui, si rien n'est fait, nous "submergeront". * Des exemples concrets sont cités par Michel Herbillon : l'annulation d'élections en Roumanie suite à une campagne d'ingérence russe, et la cyberattaque contre la campagne d'Emmanuel Macron en 2017 attribuée aux services de renseignement russes. * Vulnérabilités et défis de la régulation : * Le débat public est marqué par des "biais forts", et les algorithmes amplifient ce qui est "inquiétant, étonnant, outrancié". * Augmentation des dépressions, notamment chez les jeunes femmes. * Affaiblissement des médias d'information traditionnels et amplification de la production d'information trompeuse. * La régulation est complexe en raison de l'évolution rapide de la technologie, du caractère extraterritorial des plateformes et du modèle économique basé sur la "captation et la marchandisation de l'attention". Laurent Guimier souligne que "la régulation dans les réseaux sociaux elle est faite par des gens et des algorithmes dont l'intérêt n'est pas de faciliter le débat". * Difficulté à sanctionner les délits sans préjudice quantifiable (ex: diffusion de fake news) et à traiter les préjudices diffus. * L'anonymat, bien que parfois bénéfique pour la liberté d'expression (ex: Printemps Arabe), profite également aux "robots y compris à des rebots manipulés par des agents de l'étranger". * La cybercriminalité est un secteur à 1000 milliards d'euros de revenus, fournissant des "moyens logistiques aux manipulateurs" de désinformation.
II. Les causes profondes de la fragilisation
Les intervenants identifient plusieurs causes structurelles à cette fragilisation :
III. Pistes de solutions et recommandations
Le rapport appelle à une "prise de conscience urgente" et propose de nombreuses pistes pour "reformer la démocratie" :
Repenser l'espace public numérique et renforcer la résilience démocratique :
Remettre les citoyens au cœur du jeu démocratique, informés et armés :
Adapter les institutions et les outils juridiques :
Développer la souveraineté technologique européenne :
IV. Conclusion des intervenants
Les intervenants concluent sur l'urgence d'agir et la nécessité d'une vision collective à long terme :
Vincent Champain : Le débat est à ses débuts, et il est crucial pour la France et l'Europe "d'essayer d'être un peu en avance et de poser un peu les fagnons de la démocratie libérale avant que les trolls se soient emparés de la place".
Laurent Guimier : Bien qu'il soit "trop tard pour notre génération", la responsabilité est de travailler pour les "trois prochaines générations" en les aidant à sortir de l'"immaturité logique" face à cette révolution. Cela passe par l'éducation et la confiance générée par la coopération entre médias et institutions.
Gilles Babinet : Les systèmes institutionnels ne pourront pas ne pas évoluer face à l'IA. Il faut réfléchir à de "nouvelles formes d'institution de nouvelles formes de régulation", notamment en créant des "corps intermédiaires" de citoyens chargés de développer les algorithmes pour l'État. Il souligne que "les défendants ont un avantage sur les attaquants" car ils partagent l'information, ce qui leur permet de se renforcer plus vite.
Enfin, il insiste sur la nécessité d'une "approche offensive" face à la désinformation.
Synthèse Détaillée : Adaptation de l'Aménagement des Territoires au Changement Climatique en France
Il met en lumière les retards accumulés par la France en matière d'adaptation, l'insuffisance des politiques actuelles et la nécessité urgente d'une stratégie plus ambitieuse, coordonnée et financée pour faire face aux impacts croissants du dérèglement climatique.
1. Le Contexte et l'Urgence de l'Adaptation
Le rapport s'inscrit dans un contexte marqué par la publication du PNACC 3 (3e Plan National d'Adaptation au Changement Climatique) en mars dernier.
Si les politiques d'atténuation (réduction des émissions de gaz à effet de serre) restent "indispensables pour limiter l'ampleur du changement climatique", elles doivent désormais être "complétées par une politique d'adaptation ambitieuse" face aux impacts déjà visibles et futurs.
Constat alarmant : La France a pris un "important retard" en matière d'adaptation.
Les réponses actuelles sont "insuffisantes", les "coûts de l'inaction s'alourdissent", et les politiques publiques peinent à traduire "l'urgence climatique en action concrète".
Prévisions climatiques : "La trajectoire actuelle de réchauffement climatique pourrait conduire les températures à augmenter d'ici 2100 en France de 3 à 4°C supplémentaire par rapport à l'ère pré-industrielle".
L'ampleur et la vitesse de ces évolutions sont "inédites", affectant les sols, la disponibilité de l'eau, les rendements agricoles, et augmentant la récurrence d'événements extrêmes (inondations, sécheresses).
Impacts sur le quotidien des Français : "Disparition d'habitations et d'infrastructures littorales ou de montagne, baisse de la productivité économique lors des fortes chaleurs, dommages considérables du retrait gonflement des argiles sur les maisons individuelles, coupure récurrente de route, impossibilité d'assurer certains biens."
2. Atténuation vs. Adaptation : Deux Piliers
Complémentaires et Complexes La lutte contre le changement climatique repose sur deux piliers :
Atténuation : Agit sur les "sources du réchauffement climatique".
Adaptation : Agit sur les "conséquences de ce dernier".
Ces deux démarches sont "complémentaires" (ex: végétalisation des villes atténue et adapte).
Cependant, elles peuvent aussi s'opposer si l'adaptation conduit à des "maladaptations" (ex: "répondre aux vagues de chaleur en installant des millions de climatiseurs").
L'adaptation est "plus complexe que celle de l'atténuation" car elle nécessite de "réduire les vulnérabilités physiques et de trouver des moyens d'adaptation fonctionnels selon des méthodes qui diffèrent en fonction des territoires des secteurs d'activité et des choix opérés par les élus et la population". Il n'existe pas de "politique d'adaptation unique".
Contrairement à l'atténuation, qui dépend d'un effort mondial, "les politiques d'adaptation ne dépendent que de nous".
L'action est urgente car "l'adaptation doit être pensée et mise en œuvre dès maintenant pour éviter la maladaptation et des coûts futurs importants".
3. Le Coût de l'Inaction et les Bénéfices de l'Adaptation
Bien qu'il n'existe pas d'"évaluation globale du coût de l'inadaptation", ce coût est "important" et "exponentiel".
4. Recommandations Clés pour une Politique d'Adaptation Efficace
Le rapport formule une centaine de propositions. Les plus importantes incluent :
4.1. Renforcement du Cadre Juridique et Stratégique
4.2. Financement et Ressources
4.3. Ingénierie Territoriale et Culture du Risque
4.4. Adaptation de l'Aménagement et du Droit
4.5. Enjeux Spécifiques et Cas Particuliers
5. L'Adaptation comme Levier de Résilience et de Développement
6. Débats et Points de Tension Plusieurs points de discussion ont émergé :
Compte Rendu Détaillé – Audition du Directeur de la Sécurité Sociale sur l'Organisation du Système et les Difficultés d'Accès aux Soins Date: Réunion de la commission des comptes de la sécurité sociale (la veille de l'audition).
Source: Extraits de "🔴Le directeur de la Sécurité sociale sur l’organisation du système & les difficultés d’accès au soin"
I. Thèmes Principaux * Cette audition du Directeur de la Sécurité Sociale aborde principalement la situation financière préoccupante de la Sécurité Sociale, en particulier de la branche maladie, la nécessité impérative d'un retour à l'équilibre financier, et les leviers possibles pour y parvenir.
La discussion s'est également étendue à la tarification à l'activité des hôpitaux, aux coûts des dispositifs médicaux, à la prise en charge des affections de longue durée (ALD) et des transports sanitaires, ainsi qu'à la question plus large de l'efficience du système de santé et de la structure de financement de la protection sociale.
II. Idées et Faits Importants
A. Situation Financière de la Sécurité Sociale
B. Nécessité d'un Retour à l'Équilibre et ses Implications
C. Leviers pour le Retour à l'Équilibre
Le Directeur identifie trois grandes familles de leviers pour ramener la branche maladie vers l'équilibre, reconnaissant que l'ampleur de l'effort nécessitera une combinaison de tous ces leviers:
Agir sur l'efficience du système de santé:
D. Tarification à l'Activité (T2A) et Gains de Productivité
E. Efficience des Dépenses de Santé et Coûts Spécifiques
F. Sujets Spécifiques de Prise en Charge
G. Enjeux Structurels du Financement
III. Citations Clés
Compte rendu détaillé : La formation des professionnels de santé et son impact sur l'accès aux soins
Ce document synthétise les points clés soulevés lors de l'audition concernant l'organisation du système de santé et l'accès aux soins, avec un accent particulier sur la formation des professionnels de santé.
Les discussions ont mis en lumière les défis actuels, les réformes en cours et les pistes de réflexion pour l'avenir.
1. Le constat des difficultés d'accès aux soins
2. Le rôle central de la formation
La formation des professionnels de santé, relevant en grande partie du ministère de l'enseignement supérieur, est identifiée comme un levier majeur pour améliorer la situation sanitaire. Les enjeux liés à la formation sont de trois ordres :
3. Les leviers d'action et réformes en cours
Plusieurs mesures sont mises en œuvre ou envisagées pour améliorer la situation :
4. Débats et pistes de réflexion complémentaires
Plusieurs points ont fait l'objet de discussions approfondies :
5. Coût de la formation
Le ministre n'est pas en mesure de donner le coût précis de la formation d'un médecin. Le coût moyen d'un étudiant toutes filières confondues est d'environ 12 000 euros par an, mais il précise que les étudiants en santé contribuent aussi directement au système de soins via leurs stages.
En conclusion, la France est engagée dans une série de réformes visant à résoudre la crise de l'accès aux soins par une refonte profonde de la formation des professionnels de santé, axée sur la décentralisation, la diversification des parcours, l'intégration de la pratique et une meilleure anticipation des besoins territoriaux et des compétences futures.
Compte Rendu Détaillé de l'Audition du Ministre de la Santé et de l'Accès aux Soins
Introduction et Contexte
Cette audition, menée par une commission d'enquête parlementaire, vise à échanger sur le système de santé français et l'accès aux soins, ainsi que sur les évolutions possibles pour améliorer son efficience et sa soutenabilité.
Le Ministre de la Santé et de l'Accès aux Soins, Yannick Neuder, a souligné que l'accès aux soins est "le plus grand et le plus important défi de notre système de santé", une priorité mais aussi une "préoccupation" pour les Français confrontés à "des délais inacceptables, à des distances infranchissables, à des découragements silencieux".
Il a insisté sur la nécessité de mobiliser "tous les leviers dans une action globale qui mobilise tous les acteurs" et de "rebâtir les organisations à la hauteur des défis et de notre époque" (vieillissement de la population, maladies chroniques, nouvelles attentes).
I. La Formation des Professionnels de Santé : Un Axe Majeur de la Politique du Ministre
La formation est placée au "centre de gravité" de l'action du Ministre, avec le triptyque "former plus, former mieux, former partout".
Augmentation des Capacités de Formation :
L'objectif est d'adapter les besoins en formation aux réalités territoriales et aux capacités des universités, tout en renforçant les moyens alloués à la formation. * Retour des étudiants français formés à l'étranger : Le Ministre estime qu'environ 10 000 étudiants français partis en Belgique, Pologne, Roumanie pourraient être réintégrés.
Il s'interroge sur la "souveraineté sanitaire" de la France, 7ème puissance mondiale, dépendante d'autres pays pour la formation de ses soignants (ex: 54% des dentistes inscrits en France sont formés à l'étranger).
Adaptation des Cursus et Compétences :
L'objectif est de familiariser les futurs médecins avec les réalités des territoires ruraux dès le début de leurs études.
De même, les doubles cursus (ingénieur-médecin) sont encouragés face à l'évolution technologique (IA, radiologie, biophysique). * Causes Historiques des "Déserts Médicaux" : Le Ministre attribue la pénurie actuelle à plusieurs facteurs : * Un numerus clausus trop bas (3 500 en 1993), n'ayant pas anticipé l'augmentation de la population (+15 millions d'habitants depuis les années 70, avec un nombre de formations similaire). * Le vieillissement de la population et l'émergence des maladies chroniques (ex: 10% de diabétiques à La Réunion). * Le changement du rapport au travail des professionnels de santé (impact des 35 heures, nécessité de 2,3 médecins pour remplacer un généraliste partant à la retraite). Le Ministre cite: "il est préférable de demander peu à beaucoup de médecins que demander beaucoup à très peu".
II. Le Rôle et la Gouvernance des Agences Régionales de Santé (ARS)
Le rôle des ARS est au cœur des discussions, avec des perceptions contrastées.
III. Financement du Système de Santé et Lutte Contre la Fraude
IV. Gouvernance des Hôpitaux
Le rapporteur suggère de transférer le règlement des conflits aux conseils de l'ordre départementaux.
Le Ministre reconnaît une différence de traitement entre PH (CNG) et HU (juridiction universitaire). * Pénurie de Médecins et Pouvoir : La pénurie de médecins est reconnue comme ayant inversé le rapport de pouvoir, donnant aux médecins une influence significative et la possibilité de "chantage" en raison de la facilité de départ. * GHT (Groupements Hospitaliers de Territoire) : Les GHT sont vus comme une solution pour les petits hôpitaux, souvent plus "médico-dépendants" et confrontés à des problématiques différentes des CHU.
Une "deuxième génération de GHT" pourrait favoriser la collaboration territoriale et réduire les tensions.
V. Relations Public-Privé et Médecine de Ville
Il souligne que dans certains territoires, l'offre d'hospitalisation est uniquement privée, et l'hôpital public ne pourrait pas absorber le flux si elle disparaissait. Il souhaite réunir les PCME et directeurs, y compris du privé, pour envisager des projets de territoire. * Permanence des Soins (PDSES) : Le Ministre appelle à la vigilance sur la permanence des soins, notamment la nuit profonde, pour éviter de mobiliser des moyens disproportionnés par rapport aux besoins réels. Il souligne également les limites à la mutualisation des équipes et du matériel entre le public et le privé pour des actes techniques d'urgence, où la sécurité du patient prime. Il juge que "sur le papier ça fonctionne mais dans la réalité c'est pas ça". * Accès aux Médicaments : La pénurie de médicaments est un "enjeu qui dépasse largement la problématique française", nécessitant une solution à l'échelle européenne.
La relocalisation des entreprises pharmaceutiques est cruciale, mais la dépendance aux principes actifs produits en Asie ou aux États-Unis est une réalité.
Le Ministre a pris des mesures nationales (substitution, interdiction d'export, préparation magistrale, dispensation à l'unité), mais leur efficacité est limitée sans la "souveraineté de production". * Dispensation à l'Unité : Le Ministre réaffirme que la dispensation à l'unité en cas de pénurie est autorisée par la loi et qu'aucun pharmacien ne devrait être sanctionné pour son application. Il a cependant clarifié un cas spécifique où des pharmaciens ont été sanctionnés pour des motifs disciplinaires plus larges et non pour cette pratique.
VI. Points Divers et Transversaux
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review)>
Summary:
This research group has consistently performed cutting-edge research aiming to understand the role of hormones in the control of social behaviors, specifically by utilizing the genetically tractable teleost fish, medaka, and the current work is no exception. The overall claim they make, that estrogens modulate social behaviors in males and females is supported, with important caveats. For one, there is no evidence these estrogens are generated by "neurons" as would be assumed by their main claim that it is NEUROestrogens that drive this effect. While indeed the aromatase they have investigated is expressed solely in the brain, in most teleosts, brain aromatase is only present in glial cells (astrocytes, radial glia). The authors should change this description so as not to mislead the reader. Below I detail more specific strengths and weaknesses of this manuscript.
We thank the reviewer for this very positive evaluation of our work and greatly appreciate their helpful comments and suggestions for improving the manuscript. We agree with the comment that the term “neuroestrogens” is misleading. Therefore, we have replaced “neuroestrogens” with “brain-derived estrogens” or “brain estrogens” throughout the manuscript, including the title.
In the following sections, “neuroestrogens” has been revised to align with the surrounding context.
Line 21: “in the brain, also known as neuroestrogens,” → “in the brain.”
Line 28: “neuroestrogens” → “these estrogens.”
Line 30: “mechanism of action of neuroestrogens” → “mode of action of brain-derived estrogens.”
Line 43: “brain-derived estrogens, also called neuroestrogens,” → “estrogens.”
Line 74: “neuroestrogen synthesis is selectively impaired while gonadal estrogen synthesis remains intact” → “estrogen synthesis in the brain is selectively impaired while that in the gonads remains intact.”
Line 77: “neuroestrogens” → “these estrogens.”
Line 335: “levels of neuroestrogens” → “brain estrogen levels.”
Line 338: “neuroestrogens” → “these estrogens.”
Line 351: “neuroestrogens” → “these estrogens.”
Line 357: “neuroestrogen action” → “the action of brain-derived estrogens.”
Line 359: “neuroestrogens” → “estrogen synthesis in the brain.”
Line 390: “active synthesis of neuroestrogens” → “active estrogen synthesis in the brain.”
Line 431: “neuroestrogens” → “estrogens in the brain.”
Line 431: “neuroestrogen action” → “the action of brain-derived estrogens.”
Line 433: “neuroestrogen action” → “their action.”
Strengths:
Excellent use of the medaka model to disentangle the control of social behavior by sex steroid hormones.
The findings are strong for the most part because deficits in the mutants are restored by the molecule (estrogens) that was no longer present due to the mutation.
Presentation of the approach and findings are clear, allowing the reader to make their own inferences and compare them with the authors'.
Includes multiple follow-up experiments, which lead to tests of internal replication and an impactful mechanistic proposal.
Findings are provocative not just for teleost researchers, but for other species since, as the authors point out, the data suggest mechanisms of estrogenic control of social behaviors may be evolutionarily ancient.
We again thank the reviewer for their positive evaluation of our work.
Weaknesses:
(1) As stated in the summary, the authors attribute the estrogen source to neurons and there isn't evidence this is the case. The impact of the findings doesn't rest on this either.
As noted in Response to reviewer #1’s summary comment, we have replaced “neuroestrogens” with “brain-derived estrogens” or “brain estrogens” throughout the manuscript.
Line 63: We have also added the text “In teleost brains, including those of medaka, aromatase is exclusively localized in radial glial cells, in contrast to its neuronal localization in rodent brains (18– 20).” Following this addition, “This observation suggests” in the subsequent sentence has been replaced with “These observations suggest.”
The following references (#18–20), cited in the newly added text above, have been included in the reference list, with other references renumbered accordingly:
P. M. Forlano, D. L. Deitcher, D. A. Myers, A. H. Bass, Anatomical distribution and cellular basis for high levels of aromatase activity in the brain of teleost fish: aromatase enzyme and mRNA expression identify glia as source. J. Neurosci. 21, 8943–8955 (2001).
N. Diotel, Y. Le Page, K. Mouriec, S. K. Tong, E. Pellegrini, C. Vaillant, I. Anglade, F. Brion, F. Pakdel, B. C. Chung, O. Kah, Aromatase in the brain of teleost fish: expression, regulation and putative functions. Front. Neuroendocrinol. 31, 172–192 (2010).
A. Takeuchi, K. Okubo, Post-proliferative immature radial glial cells female-specifically express aromatase in the medaka optic tectum. PLoS One 8, e73663 (2013).
(2) The d4 versus d8 esr2a mutants showed different results for aggression. The meaning and implications of this finding are not discussed, leaving the reader wondering.
Line 282: As the reviewer correctly noted, circles were significantly reduced in mutant males of the Δ8 line, whereas no significant reduction was observed in those of the Δ4 line. However, a tendency toward reduction was evident in the Δ4 line (P = 0.1512), and both lines showed significant differences in fin displays. Based on these findings, we believe our conclusion that esr2a<sup>−/−</sup> males exhibit reduced aggression remains valid. To clarify this point and address potential reader concerns, we have revised the text as follows: “esr2a<sup>−/−</sup> males from both the Δ8 and Δ4 lines exhibited significantly fewer fin displays than their wildtype siblings (P = 0.0461 and 0.0293, respectively). Circles followed a similar pattern, with a significant reduction in the Δ8 line (P = 0.0446) and a comparable but non-significant decrease in the Δ4 line (P = 0.1512) (Fig. 5L; Fig. S8E), showing less aggression.”
(3) Lack of attribution of previously published work from other research groups that would provide the proper context of the present study.
In response to this and other comments from this reviewer, we have revised the Introduction and Discussion sections as follows.
Line 56: “solely responsible” in the Introduction has been modified to “largely responsible”.
Line 57: “This is consistent with the recent finding in medaka fish (Oryzias latipes) that estrogens act through the ESR subtype Esr2b to prevent females from engaging in male-typical courtship (10)” has been revised to “This is consistent with recent observations in a few teleost species that genetic ablation of AR severely impairs male-typical behaviors (13–16) and with findings in medaka fish (Oryzias latipes) that estrogens act through the ESR subtype Esr2b to prevent females from engaging in maletypical courtship (12)” to include previous studies on the behavior of AR mutant fish (Yong et al., 2017; Alward et al., 2020; Ogino et al., 2023; Nishiike and Okubo, 2024) in the Introduction.
Line 65: “It is worth mentioning that systemic administration of estrogens and an aromatase inhibitor increased and decreased male aggression, respectively, in several teleost species, potentially reflecting the behavioral effects of brain-derived estrogens (21–24)” has been added to the Introduction. This addition provides an overview of previous studies on the effects of estrogens and aromatase on male fish aggression (Hallgren et al., 2006; O’Connell and Hofmann, 2012; Huffman et al., 2013; Jalabert et al., 2015).
Line 367: “treatment of males with an aromatase inhibitor reduces their male-typical behaviors (31– 33)” has been edited to read “treatment of males with an aromatase inhibitor reduces their male-typical behaviors, while estrogens exert the opposite effect (21–24).”
After the revisions described above, the following references (#13, 14, and 22) have been added to the reference list, with other references renumbered accordingly:
L. Yong, Z. Thet, Y. Zhu, Genetic editing of the androgen receptor contributes to impaired male courtship behavior in zebrafish. J. Exp. Biol. 220, 3017–3021 (2017).
B. A. Alward, V. A. Laud, C. J. Skalnik, R. A. York, S. A. Juntti, R. D. Fernald, Modular genetic control of social status in a cichlid fish. Proc. Natl. Acad. Sci. U.S.A. 117, 28167–28174 (2020).
L. A. O’Connell, H. A. Hofmann, Social status predicts how sex steroid receptors regulate complex behavior across levels of biological organization. Endocrinology 153, 1341–1351 (2012).
(4) There are a surprising number of citations not included; some of the ones not included argue against the authors' claims that their findings were "contrary to expectation".
Line 68: As detailed in Response to reviewer #1’s comment 3 on weaknesses, we have cited previous studies on the effects of estrogens and aromatase on male fish aggression (Hallgren et al., 2006; O’Connell and Hofmann, 2012; Huffman et al., 2013; Jalabert et al., 2015) in the Introduction.
The following revisions have also been made to avoid phrases such as “contrary to expectation” and “unexpected.”
Line 76: “Contrary to our expectations” → “Remarkably.”
Line 109: “Contrary to this expectation, however” → “Nevertheless.”
Line 135: “Again, contrary to our expectation, cyp19a1b<sup>−/−</sup> males” → “cyp19a1b<sup>−/−</sup> males.”
Line 333: “unexpected” → “noteworthy.”
Line 337: “unexpected” → “notable.”
(5) The experimental design for studying aggression in males has flaws. A standard test like a resident intruder test should be used.
We agree that the resident-intruder test is the most commonly used method for assessing aggression. However, medaka form shoals and lack strong territoriality, and even slight dominance differences between the resident and the intruder can increase variability in the results, compromising data consistency. Therefore, in this study, we adopted an alternative approach: placing four unfamiliar males together in a tank and quantifying aggressive interactions in total. This method allows for the assessment of aggression regardless of territorial tendencies, making it more appropriate for our investigation.
(6) While they investigate males and females, there are fewer experiments and explanations for the female results, making it feel like a small addition or an aside.
We agree that the data and discussion for females are less extensive than for males. However, we have previously elucidated the mechanism by which estrogen/Esr2b signaling promotes female mating behavior (Nishiike et al., 2021, Curr Biol, 1699–1710). Accordingly, it follows that the new insights into female behavior gained from the cyp19a1b knockout model are more limited than those for males. Nevertheless, when combined with our prior findings, the female data in this study offer valuable insights, and the overall mechanism through which estrogens promote female mating behavior is becoming clearer. Therefore, we do not consider the female data in this study to be incomplete or merely supplementary.
(7) The statistics comparing "experimental to experimental" and "control to experimental" aren't appropriate.
The reviewer raises concerns about the statistical analysis used for Figures 4C and 4E, suggesting that Bonferroni’s test should be used instead of Dunnett’s test. However, Dunnett’s test is commonly used to compare treatment groups to a reference group that receives no treatment, as in our study. Since we do not compare the treated groups with each other, we believe Dunnett’s test is the most appropriate choice.
Line 619: The reviewer’s concern may have arisen from the phrase “comparisons between control and experimental groups” in the Materials and Methods. We have revised it to “comparisons between untreated and E2-treated groups in Fig. 4, C and D” for clarity.
Reviewer #2 (Public Review):
Summary:
The novelty of this study stems from the observations that neuro-estrogens appear to interact with brain androgen receptors to support male-typical behaviors. The study provides a step forward in clarifying the somewhat contradictory findings that, in teleosts and unlike other vertebrates, androgens regulate male-typical behaviors without requiring aromatization, but at the same time estrogens appear to also be involved in regulating male-typical behaviors. They manipulate the expression of one aromatase isoform, cyp19a1b, that is purported to be brain-specific in teleosts. Their findings are important in that brain estrogen content is sensitive to the brain-specific cyp19a1b deficiency, leading to alterations in both sexual behavior and aggressive behavior. Interestingly, these males have relatively intact fertility rates, despite the effects on the brain.
We thank this reviewer for their positive evaluation of our work and constructive comments, which we found very helpful in improving the manuscript.
That said, the framing of the study, the relevant context, and several aspects of the methods and results raise concerns. Two interpretations need to be addressed/tempered:
(1) that the rescue of cyp19a1b deficiency by tank-applied estradiol is not necessarily a brain/neuroestrogen mode of action, and
Line 155: cyp19a1b-deficient males exhibited a severe reduction in brain E2 levels, yet their peripheral E2 levels remained comparable to those in wild-type males. Given this hormonal milieu and the lack of behavioral change in wild-type males following E2 treatment, the observed recovery of mating behavior in cyp19a1b-deficient males following E2 treatment can be best explained by the restoration of brain E2 levels. However, as the reviewer pointed out, we cannot rule out the possibility that bath-immersed E2 influenced behavior through an indirect peripheral mechanism. To address this concern, we have modified the text as follows: “These results suggest that reduced E2 in the brain is the primary cause of the mating defects, highlighting a pivotal role of brain-derived estrogens in male mating behavior. However, caution is warranted, as an indirect peripheral effect of bath-immersed E2 on behavior cannot be ruled out, although this is unlikely given the comparable peripheral E2 levels in cyp19a1b-deficient and wild-type males. In contrast to mating.”
(2) the large increases in peripheral and brain androgen levels in the cyp19a1b deficient animals imply some indirect/compensatory effects of lifelong cyp19a1b deficiency.
As stated in line 151, androgen/AR signaling has a strong facilitative effect on male-typical behaviors in teleosts. If increased androgen levels in the periphery and brain affected behavior, the expected effect would be facilitative. However, cyp19a1b-deficient males exhibited impaired male-typical behaviors, suggesting that elevated androgen levels were unlikely to be responsible. Although chronic androgen elevation could cause androgen receptor desensitization, which could lead to behavioral suppression, our long-term androgen treatments have consistently promoted, rather than inhibited, male-typical behaviors (e.g., Nishiike et al., Proc Natl Acad Sci USA 121:e2316459121). Hence, this possibility is also highly unlikely.
Reviewer #3 (Public Review):
Summary:
Taking advantage of the existence in fish of two genes coding for estrogen synthase, the enzyme aromatase, one mostly expressed in the brain (Cyp19a1b) and the other mostly found in the gonads (Cyp19a1a), this study investigates the role of neuro-estrogens in the control of sexual and aggressive behavior in teleost fish. The constitutive deletion of Cyp19a1b reduced brain estrogen content by 87% in males and about 50% in females. It led to reduced sexual and aggressive behavior in males and reduced sexual behavior in females. These effects are reversed by adult treatment with estradiol thus indicating that they are activational in nature. The deletion of Cyp19a1b is associated with a reduced expression of the genes coding for the two androgen receptors, ara, and arb, in brain regions involved in the regulation of social behavior. The analysis of the gene expression and behavior of mutants of estrogen receptors indicates that these effects are likely mediated by the activation of the esr1 and esr2a isoforms. These results provide valuable insight into the role of neuro-estrogens in social behavior in the most abundant vertebrate taxa. While estrogens are involved in the organization of the brain and behavior of some birds and rodents, neuro-estrogens appear to play an activational role in fish through a facilitatory action of androgen signaling.
We thank this reviewer for their positive evaluation of our work and comments that have improved the manuscript.
Strengths:
Evaluation of the role of brain "specific" Cyp19a1 in male teleost fish, which as a taxa are more abundant and yet proportionally less studied than the most common birds and rodents. Therefore, evaluating the generalizability of results from higher vertebrates is important. This approach also offers great potential to study the role of brain estrogen production in females, an understudied question in all taxa.
Results obtained from multiple mutant lines converge to show that estrogen signaling drives aspects of male sexual behavior.
The comparative discussion of the age-dependent abundance of brain aromatase in fish vs mammals and its role in organization vs activation is important beyond the study of the targeted species.
We again thank the reviewer for their positive evaluation of our work.
Weaknesses:
(1) The new transgenic lines are under-characterized. There is no evaluation of the mRNA and protein products of Cyp19a1b and ESR2a.
We did not directly assess the function of cyp19a1b and esr2a in our mutant fish. However, the observed reduction in brain E2 levels, with no change in peripheral E2 levels, in cyp19a1b-deficient fish strongly supports the loss of cyp19a1b function. This is stated in the Results section (line 97) as follows: “These results show that cyp19a1b-deficient fish have reduced estrogen levels coupled with increased androgen levels in the brain, confirming the loss of cyp19a1b function.”
Line 473: A previous study reported that female medaka lacking esr2a fail to release eggs due to oviduct atresia (Kayo et al., 2019, Sci Rep 9:8868). Similarly, in this study, some esr2a-deficient females exhibited spawning behavior but were unable to release eggs, although the sample size was limited (Δ8 line: 2/3; Δ4 line: 1/1). In contrast, this was not observed in wild-type females (Δ8 line: 0/12; Δ4 line: 0/11). These results support the effective loss of esr2a function. To incorporate this information into the manuscript, the following text has been added to the Materials and Methods: “A previous study reported that esr2a-deficient female medaka cannot release eggs due to oviduct atresia (59). Likewise, some esr2a-deficient females generated in this study, despite the limited sample size, exhibited spawning behavior but were unable to release eggs (Δ8 line: 2/3; Δ4 line: 1/1), while such failure was not observed in wild-type females (Δ8 line: 0/12; Δ4 line: 0/11). These results support the effective loss of esr2a function.”
The following reference (#59), cited in the newly added text above, have been included in the reference list:
D. Kayo, B. Zempo, S. Tomihara, Y. Oka, S. Kanda, Gene knockout analysis reveals essentiality of estrogen receptor β1 (Esr2a) for female reproduction in medaka. Sci. Rep. 9, 8868 (2019).
(2) The stereotypic sequence of sexual behavior is poorly described, in particular, the part played by the two sexual partners, such that the conclusions are not easily understandable, notably with regards to the distinction between motivation and performance.
Line 103: To provide a more detailed description of medaka mating behavior, we have revised the text from “The mating behavior of medaka follows a stereotypical pattern, wherein a series of followings, courtship displays, and wrappings by the male leads to spawning” to “The mating behavior of medaka follows a stereotypical sequence. It begins with the male approaching and closely following the female (following). The male then performs a courtship display, rapidly swimming in a circular pattern in front of the female. If the female is receptive, the male grasps her with his fins (wrapping), culminating in the simultaneous release of eggs and sperm (spawning).”
(3) The behavior of females is only assessed from the perspective of the male, which raises questions about the interpretation of the reduced behavior of the males.
In medaka, female mating behavior is largely passive, except for rejecting courtship attempts and releasing eggs. Therefore, its analysis relies on measuring the latency to receive following, courtship displays, or wrappings from the male and the frequency of courtship rejection or wrapping refusal. We understand the reviewer’s perspective that cyp19a1b-deficient females might not be less receptive but instead less attractive to males, potentially leading to reduced male mating efforts. However, since these females are approached and followed by males at levels comparable to wild-type females, this possibility appears unlikely. Moreover, cyp19a1b-deficient females tend to avoid males and exhibit a slightly female-oriented sexual preference. While these traits are closely associated with reduced sexual receptivity, they do not readily align with reduced sexual attractiveness. Therefore, it is more plausible to conclude that these females have decreased receptivity rather than being less attractive to males.
(4) At no point do the authors seem to consider that a reduced behavior of one sex could result from a reduced sensory perception from this sex or a reduced attractivity or sensory communication from the other sex.
Line 112: As noted above, the impaired mating behavior of cyp19a1b-deficient females is unlikely to be due to reduced attractiveness to males. Similarly, mating behavior tests using esr2b-deficient females as stimulus females suggest that the impaired mating behavior of cyp19a1b-deficient males cannot be attributed to reduced attractiveness to females. However, the possibility that their impaired mating behavior could be attributed to altered cognition or sexual preference cannot be ruled out. To reflect this in the manuscript, we have revised the text “, suggesting that they are less motivated to mate” to “. These results suggest that they are less motivated to mate, though an alternative interpretation that their cognition or sexual preference may be altered cannot be dismissed.”
(5) Aspects of the methods are not detailed enough to allow proper evaluation of their quality or replication of the data.
In response to this and other specific comments from this reviewer, we have revised the Materials and Methods section to include more detailed descriptions of the methods.
Line 469: The following text has been added to describe the method for domain identification in medaka Esr2a: “The DNA- and ligand-binding domains of medaka Esr2a were identified by sequence alignment with yellow perch (Perca flavescens) Esr2a, for which these domain locations have been reported (58).”
The following reference (#58), cited in the newly added text above, have been included in the reference list:
S. G. Lynn, W. J. Birge, B. S. Shepherd, Molecular characterization and sex-specific tissue expression of estrogen receptor α (esr1), estrogen receptor βa (esr2a) and ovarian aromatase (cyp19a1a) in yellow perch (Perca flavescens). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 149, 126–147 (2008).
Line 540: The text “, and the total area of signal in each brain nucleus was calculated using Olyvia software (Olympus)” has been revised to include additional details on the single ISH method as follows: “. The total area of signal across all relevant sections, including both hemispheres, was calculated for each brain nucleus using Olyvia software (Olympus). Images were converted to a 256-level intensity scale, and pixels with intensities from 161 to 256 were considered signals. All sections used for comparison were processed in the same batch, without corrections between samples.”
Line 596: The following text has been added to include additional details on the double ISH method: “Cells were identified as coexpressing the two genes when Alexa Fluor 555 and fluorescein signals were clearly observed in the cytoplasm surrounding DAPI-stained nuclei, with intensities markedly stronger than the background noise.”
(6) It seems very dangerous to use the response to a mutant abnormal behavior (ESR2-KO females) as a test, given that it is not clear what is the cause of the disrupted behavior.
esr2b-deficient females have fully developed ovaries, a normal sex steroid milieu, and sexual attractiveness to males comparable to wild-type females, yet they are completely unreceptive to male courtship (Nishiike et al., 2021, Curr Biol, 1699–1710). Although, as the reviewer noted, the detailed mechanisms underlying this phenotype remain unclear, it is evident that the loss of estrogen/Esr2b signaling in the brain severely impairs sexual receptivity. Therefore, using esr2b-deficient females as stimulus females in the mating behavior test eliminates the influence of female sexual receptivity and male attractiveness to females, enabling the exclusive assessment of male mating motivation. This rationale is already presented in the Results section (lines 116–120), and we believe this experimental design offers a robust framework for assessing male mating motivation.
Additionally, the mating behavior test with esr2b-deficient females complemented the test with wildtype females, and its results were not the sole basis for our discussion of the male mating behavior phenotype. The results of both tests were largely concordant, and we believe that the conclusions drawn from them are highly reliable.
Meanwhile, in the test with esr2b-deficient females, cyp19a1b-deficient males were courted more frequently by these females than wild-type males. As the reviewer noted, this may suggest an anomaly in the test. Accordingly, we have confined our discussion to the possibility that “Perhaps cyp19a1b<sup>−/−</sup> males are misidentified as females by esr2b-deficient females because they are reluctant to court or they exhibit some female-like behavior” (line 131).
(7) Most experiments are weakly powered (low sample size) and analyzed by multiple T-tests while 2 way ANOVA could have been used in several instances. No mention of T or F values, or degrees of freedom.
Histological analysis was conducted with a relatively small sample size, as our previous experience suggested that interindividual variability in the results would not be substantial. As significant differences were detected in many analyses, further increasing the sample size is unnecessary.
Although two-way ANOVA could be used instead of multiple T-tests for analyzing the data in Figures 4D, 4F, 6D, S4A, and S4B, we applied the Bonferroni–Dunn correction to control for multiple pairwise comparisons in multiple T-tests. As this comparison method is equivalent to the post hoc test following two-way ANOVA, the statistical results are identical regardless of whether T-tests or two-way ANOVA are used.
For the data in Figures 4D, 4F, S4A, and S4B, the primary focus is on whether relative luciferase activity differs between E2-treated and untreated conditions for each mutant construct. Therefore, two-way ANOVA is not particularly relevant, as assessing the main effect of construct type or its interaction with E2 treatment does not provide meaningful insights. Similarly, in Figure 6D, the focus is solely on whether wild-type and mutant females differ in time spent at each distance. Given this, two-way ANOVA is unnecessary, as analyzing the main effect of distance is not meaningful.
Accordingly, two-way ANOVA was not employed in this study, and therefore, its corresponding F values were not included. As the figure legends specify the sample sizes for all analyses, specifying degrees of freedom separately was deemed unnecessary.
(8) The variability of the mRNA content for the same target gene between experiments (genotype comparison vs E2 treatment comparison) raises questions about the reproducibility of the data (apparent disappearance of genotype effect).
As the reviewer pointed out, the overall area of ara expression is larger in Figure 2J than in Figure 2F. However, the relative area ratios of ara expression among brain nuclei are consistent between the two figures, indicating the reproducibility of the results. Thus, this difference is unlikely to affect the conclusions of this study.
Additionally, the differences in ara expression in pPPp and arb expression in aPPp between wild-type and cyp19a1b-deficient males appear less pronounced in Figures 2J and 2K than in Figures 2F and 2H. This is likely attributable to the smaller sample size used in the experiments for Figures 2J and 2K, resulting in less distinct differences. However, as the same genotype-dependent trends are observed in both sets of figures, the conclusion that ara and arb expression is reduced in cyp19a1b-deficient male brains remains valid.
(9) The discussion confuses the effects of estrogens on sexual differentiation (developmental programming = permanent) and activation (= reversible activation of brain circuits in adulthood) of the brain and behavior. Whether sex differences in the circuits underlying social behaviors exist is not clear.
We recognize that the effects of adult steroids are sometimes not considered to be sexual differentiation, as they do not differentiate the neural substrate, but rather transiently activate the already masculinized or feminized substrate. Arnold (2017, J Neurosci Res 95:291–300) contends that all factors that cause sex differences, including the transient effects of adult steroids, should be incorporated into a theory of sexual differentiation, and indeed, these effects may be the most potent proximate factors that make males and females different. We concur with this perspective and have adopted it as a foundation for our manuscript.
In teleosts, early developmental exposure to steroids has minimal impact, and sexual differentiation relies primarily on steroid action in adulthood (Okubo et al., 2022, Spectrum of Sex, pp. 111–133). This is evidenced by the effective reversal of sex-typical behaviors through experimental hormonal manipulation in adult teleosts and the absence of transient early-life steroid surges observed in mammals and birds. Accordingly, our discussion on brain sexual differentiation, including the statement in line 347, “This variation among species may represent the activation of neuroestrogen synthesis at life stages critical for sexual differentiation of behavior that are unique to each species”, remains well-supported. Additionally, given these considerations, while sex differences in neural circuit activation are evident in teleosts, substantial structural differences in these circuits are unlikely.
(10) Overall, the claims regarding the activational role of neuro-estrogens on male sexual behavior are supported by converging evidence from multiple mutant lines. The role of neuroestrogens on gene expression in the brain is mostly solid too. The data for females are comparatively weaker. Conclusions regarding sexual differentiation should be considered carefully.
We agree that the data for females are less extensive than for males. However, we have previously elucidated the mechanism by which estrogen/Esr2b signaling promotes female mating behavior (Nishiike et al., 2021). Accordingly, it follows that the new insights into female behavior gained from the cyp19a1b knockout model are more limited than those for males. Nevertheless, when integrated with our prior findings, the data on females in this study provide significant insights, and the overall mechanism through which estrogens promote female mating behavior is becoming clearer. Therefore, we do not consider the female data in this study to be incomplete or merely supplementary.
Recommendations For The Authors:
Reviewer #1 (Recommendations For The Authors):
The authors set out to answer an intriguing question regarding the hormonal control of innate social behaviors in medaka. Specifically, they wanted to test the effects of cyp19a1b mutation on mating and aggression in males. They also test these effects in females. Their approach takes them down several distinct experimental pathways, including one investigating how cyp19a1a function is related to androgen receptor expression and how estrogens themselves may act on the androgen receptor to modulate its expression, as well as how different esr genes may be involved. The study and its results are valuable and a clear, general conclusion of a pathway from brain aromatase>estrogens>esr genes> androgen receptor can be made. This is important, novel, and impactful. However, there are issues with how the study logic is set up, the approach for assessing certain behaviors, the statistics used, the interpretation of findings, and placing the findings in the proper context based on previous work, which manifests as a general issue where previous work is not properly attributed to.
Thank you for your thoughtful review. We have carefully addressed each specific comment, as detailed below.
Major comments:
(1) The background for the rationale of the current study is misleading and lacks proper context. The authors root the logic of their experiment in determining whether estrogens regulate male-typical behaviors because the current assumption is androgens are "solely responsible" for male-typical behaviors in teleosts. This is not the case. Previous studies have shown aromatase/estrogens are involved in male-typical aggression in teleosts. For example, to name a couple:
Huffman, L. S., O'Connell, L. A., & Hofmann, H. A. (2013). Aromatase regulates aggression in the African cichlid fish Astatotilapia burtoni. Physiology & behavior, 112, 77-83.
O'Connell, L. A., & Hofmann, H. A. (2012). Social status predicts how sex steroid receptors regulate complex behavior across levels of biological organization. Endocrinology, 153(3), 1341-1351.
And even a recent paper sheds light on a possible AR>aromatase.estradiol hypothesis of male typical behaviors:
Lopez, M. S., & Alward, B. A. (2024). Androgen receptor deficiency is associated with reduced aromatase expression in the ventromedial hypothalamus of male cichlids. Annals of the New York Academy of Sciences.
Interestingly, the authors cite Hufmann et al in the discussion, so I don't understand why they make the claims they do about estrogens and male-typical behavior.
Related to this, is an issue of proper attribution to published work. Indeed, missing are key references from lab groups using AR mutant teleosts. Here are a couple:
Yong, L., Thet, Z., & Zhu, Y. (2017). Genetic editing of the androgen receptor contributes to impaired male courtship behavior in zebrafish. Journal of Experimental Biology, 220(17), 3017-3021.
Alward, B. A., Laud, V. A., Skalnik, C. J., York, R. A., Juntti, S. A., & Fernald, R. D. (2020). Modular genetic control of social status in a cichlid fish. Proceedings of the National Academy of Sciences, 117(45), 28167-28174.
Ogino, Y., Ansai, S., Watanabe, E., Yasugi, M., Katayama, Y., Sakamoto, H., ... & Iguchi, T. (2023). Evolutionary differentiation of androgen receptor is responsible for sexual characteristic development in a teleost fish. Nature communications, 14(1), 1428.
As noted in Response to reviewer #1’s comment 3 on weaknesses, we have revised the Introduction and Discussion sections as follows.
Line 56: “solely responsible” in the Introduction has been modified to “largely responsible”.
Line 57: The text “This is consistent with the recent finding in medaka fish (Oryzias latipes) that estrogens act through the ESR subtype Esr2b to prevent females from engaging in male-typical courtship (10)” has been revised to “This is consistent with recent observations in a few teleost species that genetic ablation of AR severely impairs male-typical behaviors (13–16) and with findings in medaka fish (Oryzias latipes) that estrogens act through the ESR subtype Esr2b to prevent females from engaging in male-typical courtship (12)” to include previous studies on the behavior of AR mutant fish (Yong et al., 2017; Alward et al., 2020; Ogino et al., 2023; Nishiike and Okubo, 2024) in the Introduction.
Line 65: “It is worth mentioning that systemic administration of estrogens and an aromatase inhibitor increased and decreased male aggression, respectively, in several teleost species, potentially reflecting the behavioral effects of brain-derived estrogens (21–24)” has been added to the Introduction, providing an overview of previous studies on the effects of estrogens and aromatase on male fish aggression (Hallgren et al., 2006; O’Connell and Hofmann, 2012; Huffman et al., 2013; Jalabert et al., 2015).
Line 367: “treatment of males with an aromatase inhibitor reduces their male-typical behaviors (31– 33)” has been edited to read “treatment of males with an aromatase inhibitor reduces their male-typical behaviors, while estrogens exert the opposite effect (21–24).”
After the revisions described above, the following references (#13, 14, and 22) have been added to the reference list:
L. Yong, Z. Thet, Y. Zhu, Genetic editing of the androgen receptor contributes to impaired male courtship behavior in zebrafish. J. Exp. Biol. 220, 3017–3021 (2017).
B. A. Alward, V. A. Laud, C. J. Skalnik, R. A. York, S. A. Juntti, R. D. Fernald, Modular genetic control of social status in a cichlid fish. Proc. Natl. Acad. Sci. U.S.A. 117, 28167–28174 (2020).
L. A. O’Connell, H. A. Hofmann, Social status predicts how sex steroid receptors regulate complex behavior across levels of biological organization. Endocrinology 153, 1341–1351 (2012).
While Lopez and Alward (2024) provide valuable insights into the regulation of cyp19a1b expression by androgens, our study focuses specifically on the functional aspects of cyp19a1b. Expanding the discussion to include expression regulation would divert from the primary focus of our manuscript. For this reason, we have opted not to cite this reference.
(2) As it is now, the authors are only citing a book chapter/review from their own group. This is a serious issue as it does not provide the proper context for the work. The authors need to fix their issues of attribution to previously published work and the proper interpretation of the work that they are aware of as it pertains to ideas proposed on the roles of androgens and estrogens in the control of male-typical behaviors. This is also important to get the citations right because the common use of "contrary to expectations" when describing their results is actually not correct. Many of the observations are expected to a degree. However, this doesn't take away from a generally stellar experimental design and mostly clear results. The authors do not need to rely on enhancing the impact of their paper by making false claims of unexpected findings. The depth and clarity of your findings are where the impact of your work is.
As detailed in Response to reviewer #1’s comment 3 on weaknesses, we have cited previous studies on the effects of estrogens and aromatase on male fish aggression (Hallgren et al., 2006; O’Connell and Hofmann, 2012; Huffman et al., 2013; Jalabert et al., 2015) in the Introduction.
Additionally, as noted in Response to reviewer #1’s comment 4 on weaknesses, we have made the following revisions to avoid phrases such as “contrary to expectation” and “unexpected.”
Line 76: “Contrary to our expectations” → “Remarkably.”
Line 109: “Contrary to this expectation, however” → “Nevertheless.”
Line 135: “Again, contrary to our expectation, cyp19a1b<sup>−/−</sup> males” → “cyp19a1b<sup>−/−</sup> males.”
Line 333: “unexpected” → “noteworthy.”
Line 337: “unexpected” → “notable.”
(3) The experimental design for studying aggression in males has flaws. A standard test like a residentintruder test should be used. An assay in which only male mutants are housed together? I do not understand the logic there and the logic for the approach isn't even explained. Too many confounds that are not controlled for. It makes it seem like an aspect of the study that was thrown in as an aside.
As noted in Response to reviewer #1’s comment 5 on weaknesses, medaka form shoals and lack strong territoriality. As a result, even slight differences in dominance between the resident and intruder can substantially impact the outcomes of the resident-intruder test. Therefore, we adopted an alternative approach in this study.
(4) Hormonal differences in the mutants seem to vary based on sex, and the meaning of these differences, or how they affect interpreting the findings, wasn't discussed. There was no acknowledegment of the fact that female central E2 was still at 50%, meaning the "rescue" experiments using peripheral injections are not given the proper context. For example, this is different than giving a fish with only 16% of their normal central E2 an E2 injection. Missing as well is a clear hypothesis for why E2 injections did not rescue aggression deficits in cyp19a1b mutant males.
Line 385: As the reviewer pointed out, the degree of brain estrogen reduction in cyp19a1b-deficient fish differs greatly between males and females. This is likely because females receive a large supply of estrogens from the ovaries. Given that estrogen levels in cyp19a1b-deficient females were 50% of those in wild-type females, it can be inferred that half of their brain estrogens are synthesized locally, while the other half originates from the ovaries. This is an important finding, and we have already noted in the Discussion that “females have higher brain levels of estrogens, half of which are synthesized locally in the brain (i.e., neuroestrogens)” However, as this explanation was not sufficiently clear, we have revised it to “females have higher brain levels of estrogens, with half being synthesized locally and the other half supplied by the ovaries.”
The reviewer raised a concern that conducting the estrogen rescue experiment in females, where 50% of brain estrogens remain, might be inappropriate. However, as this experiment was conducted exclusively in males, this concern is not applicable.
Line 377: As noted in the reviewer’s subsequent comment, the failure of aggression recovery in E2treated cyp19a1b-deficient males could be due to insufficient induction of ara/arb expression in aggression-relevant brain regions. To address this concern, we have inserted the following statement into the Discussion after “the development of male behaviors may require moderate neuroestrogen levels that are sufficient to induce the expression of ara and arb, but not esr2b, in the underlying neural circuitry”: “This may account for the lack of aggression recovery in E2-treated cyp19a1b-deficient males in this study.”
(5) In relation to that, the "null" results may have some of the most interesting implications, but they are barely discussed. For example, what does it mean that E2 didn't restore aggression in male cyp19 mutants? Is this a brain region factor? Could this relate to findings from Lopez et al NYAS, where male and female Ara mutants show different effects on brain-region-specific aromatase expression? And maybe this relates to the different impact of estrogens on ar expression. Were the different effects impacted in aggression areas? Maybe this is why E2 injection didn't retore aggression in males. You could make the argument that: (1) E2 doesn't restore ar expression in aggression regions and that's why there was no rescue. Or (2) that the circuits in adulthood that regulate aggression are NOT dependent on aggression but in early development they are. Another null finding not expanded on is why the two esr2a mutant lines showed differences. There is no reason to trust one line over the other, meaning we still don't know whether esr2a is required for latency to follow.
As stated in our response to the previous comment, we have added the following text to the Discussion (line 377): “This may account for the lack of aggression recovery in E2-treated cyp19a1b-deficient males in this study.” Meanwhile, as discussed in lines 341–342, it is highly unlikely that the neural circuits regulating aggression are primarily influenced by early-life estrogen exposure, because androgen administration in adulthood alone is sufficient to induce high levels of aggression in both sexes. This notion is further supported by previous observations that cyp19a1b expression in the brain is minimal during embryonic development (Okubo et al., 2011, J Neuroendocrinol, 23:412–423).
The findings of Lopez and Alward (2024) pertain to the regulation of cyp19a1b expression by androgen receptors. While this represents an important aspect of neuroendocrine regulation, it does not appear to be directly relevant to our discussion on cyp19a1b-mediated regulation of androgen receptor expression.
To ensure the reliability of behavioral analyses in mutant fish, we consider a phenotype valid only when it is consistently observed in two independent mutant lines. In the mating behavior test examining esr2adeficient males using esr2b-deficient females as stimulus females, Δ8 line males exhibited a shorter latency to initiate following than wild-type males, whereas Δ4 line males did not. This discrepancy led us to refrain from drawing conclusions about the role of esr2a in mating behavior, even though the mating behavior test using wild-type females as stimulus females yielded consistent results in the Δ8 and Δ4 lines. Therefore, we do not consider the reviewer’s concern to be a significant issue.
(6) Not sure what's going on with the statistics, but it is not appropriate here to treat a "control" group as special. All groups are "experimental" groups. There is nothing special about the control group in this context. all should be Bonferroni post-hoc tests.
Line 619: As detailed in Response to reviewer #1’s comment 7 on weaknesses, we consider Dunnett’s test the most appropriate choice for the experiments presented in Figures 4C and 4E. We acknowledge that the reviewer’s concern may stem from the phrase “comparisons between control and experimental groups” in the Materials and Methods section. To clarify this point, we have revised it to “comparisons between untreated and E2-treated groups in Fig. 4, C and D” for clarity.
Minor comments:
Line 47: then how can you say the aromatization hypothesis is "correct"? it only applies to a few species so far. Need to change the framing, not state so strongly such a vague thing as a hypothesis being "correct".
Line 45: To address this concern, we have modified “widely accepted as correct” to “widely acknowledged”, ensuring a more precise characterization.
Figure 1: looks like a dosage effect in males but not females. this should be discussed at some point, even if just to mention a dosage effect exists and put it in context.
Line 91: We have revised the sentence “In males, brain E2 in heterozygotes (cyp19a1b+/−) was also reduced to 45% of the level in wild-type siblings (P = 0.0284) (Fig. 1A)” by adding “, indicating a dosage effect of cyp19a1b mutation” to make this point explicit.
Were male cyp19 KO aggressive towards females?
We have not observed cyp19a1b-deficient males exhibiting aggressive behavior towards females in our experiments. Therefore, we do not consider them aggressive toward females.
Please explain how infertility would lead to reduced mating.
Line 142: As the reviewer has questioned, even if cyp19a1b-deficient males exhibit infertility due to efferent duct obstruction, it is difficult to imagine that this directly leads to reduced mating. However, the inability to release sperm could indirectly affect behavior. To address this, we have added “, possibly due to the perception of impaired sperm release” after “If this is also the case in medaka, the observed behavioral defects might be secondary to infertility.”
Describe something about the timing of the treatment here. How can peripheral E2 injections restore it when peripheral levels are normal? Did these injections restore central levels? This needs to be shown experimentally.
Line 517: As described in the Materials and Methods, E2 treatment was conducted by immersing fish in E2-containing water for 4 days. However, we had not explicitly stated that the water was changed daily to maintain the nominal concentration. To clarify this and address reviewer #2’s comment 9, we have revised “males were treated with 1 ng/ml of E2 (Fujifilm Wako Pure Chemical, Osaka, Japan) or vehicle (ethanol) alone by immersion in water for 4 days” to “males were treated with 1 ng/ml of E2 (Fujifilm Wako Pure Chemical, Osaka, Japan), which was first dissolved in 100% ethanol (vehicle), or with the vehicle alone by immersion in water for 4 days, with daily water changes to maintain the nominal concentration.”
Line 522: The treatment effectively restored mating activity and ara/arb expression in the brain, suggesting a sufficient increase in brain E2 levels. However, we did not measure the actual increase, and its extent remains uncertain. To reflect this in the manuscript, we have now added the following sentence: “Although the exact increase in brain E2 levels following E2 treatment was not quantified, the observed positive effects on behavior and gene expression suggest that it was sufficient.”
I know the nomenclature differs among those who study teleosts, but it's ARa and then gene is ar1 (as an example; arb would be ar2). You're recommended the following citation to remain consistent:
Munley, K. M., Hoadley, A. P., & Alward, B. A. (2023). A phylogenetics-based nomenclature system for steroid receptors in teleost fishes. General and Comparative Endocrinology, 114436.
Paralogous genes resulting from the third round of whole-genome duplication in teleosts are typically designated by adding the suffixes “a” and “b” to their gene symbols. This convention also applies to the two androgen receptor genes, commonly referred to as ara and arb. While the alternative names ar1 and ar2 may gain broader acceptance in the future, ara and arb remain more widely used at present. Therefore, we have chosen to retain ara and arb in this manuscript.
Line 268: how is this "suggesting" less aggression? They literally showed fewer aggressive displays, so it doesn't suggest it - it literally shows it.
Line 285: Following this thoughtful suggestion, we have changed “suggesting less aggression” to “showing less aggression.”
Line 317: how can you still call it the primary driver?
The stimulatory effects of aromatase/estrogens on male-typical behaviors are exerted through the potentiation of androgen/AR signaling. Thus, we still believe that androgens—specifically 11KT in teleosts—serve as the primary drivers of these behaviors.
Line 318: not all deficits, like aggression, were rescued.
Line 334: To address this comment, “These behavioral deficits were rescued by estrogen administration, indicating that reduced levels of neuroestrogens are the primary cause of the observed phenotypes: in other words, neuroestrogens are pivotal for male-typical behaviors in teleosts” has been modified and now reads “Deficits in mating were rescued by estrogen administration, indicating that reduced brain estrogen levels are the primary cause of the observed mating impairment; in other words, brain-derived estrogens are pivotal at least for male-typical mating behaviors in teleosts.”
Line 324: what do you mean by "sufficient"? To show that, you'd have to castrate the male and only give estrogen back. the authors continue to overstate virtually every aspect of their study, seemingly in an unnecessary manner.
Line 341: Our intention was to convey that brain-derived estrogens early in life are not essential for the expression of male-typical behaviors in teleosts. However, we recognize that the term “sufficient” could be misinterpreted as implying that estrogens alone are adequate, without contributions from other factors such as androgens. To clarify this, we have revised the text from “neuroestrogen activity in adulthood is sufficient for the execution of male-typical behaviors, while that in early in life is not requisite. Thus, while” to “brain-derived estrogens early in life is not essential for the execution of male-typical behaviors. While.”
Line 329: so? in adult mice, amygdala aromatase neurons still regulate aggression. The amount in adulthood seems less important compared to site-specific functions.
Line 346: We do not intend to suggest that brain aromatase activity in adulthood plays a negligible role in male behaviors in rodents, as we have already acknowledged its necessity in the Introduction (lines 42–43). To enhance clarity and prevent misinterpretation, we have added “, although it remains important for male behavior in adulthood” to the end of the sentence: “brain aromatase activity in rodents reaches its peak during the perinatal period and thereafter declines with age.”
Line 351: This contradicts what you all have been saying.
Line 65: As mentioned in Response to reviewer #1’s comment 3 on weaknesses, the following text has been added to the Introduction: “It is worth mentioning that systemic administration of estrogens and an aromatase inhibitor increased and decreased male aggression, respectively, in several teleost species, potentially reflecting the behavioral effects of brain-derived estrogens (21–24)”, providing an overview of previous studies on the effects of estrogens and aromatase on male fish aggression (Hallgren et al., 2006; O’Connell and Hofmann, 2012; Huffman et al., 2013; Jalabert et al., 2015). With this revision, we believe the inconsistency has been addressed.
Line 367: Additionally, we have revised the sentence from “treatment of males with an aromatase inhibitor reduces their male-typical behaviors (31–33)” to “treatment of males with an aromatase inhibitor reduces their male-typical behaviors, while estrogens exert the opposite effect (21–24).”
Line 360: change to "...possibility that is not mutually exclusive,"
Line 378: We have revised the phrase as suggested from “Another possibility, not mutually exclusive,” to “Another possibility that is not mutually exclusive.”
Line 363: but it didn't rescue aggression
Line 381: In response, we have revised the sentence from “This possibility is supported by the present observation that estrogen treatment facilitated mating behavior in cyp19a1b-deficient males but not in their wild-type siblings” to “This possibility is at least likely for mating behavior, as estrogen treatment facilitated mating behavior in cyp19a1b-deficient males but not in their wild-type siblings.”
Line 367: on average
To explain the sex differences in the role of aromatase, what about the downstream molecular or neural targets? In mammals, hodology is related to sex differences. there could be convergent sex differences in regulating the same type of behaviors as well.
Our findings demonstrate that brain-derived estrogens promote the expression of ara, arb, and their downstream target genes vt and gal in males, while enhancing the expression of npba, a downstream target of Esr2b signaling, in females. The identity of additional target genes and their roles in specific neural circuits remain to be elucidated, and we aim to address these in future research.
Lines 378-382: this doesn't logically follow. pgf2a could be the target of estrogens which in the intact animal do regulate female sexual receptivity. And how can you say this given that your lab has shown in esr2b mutants females don't mate?
We agree that PGF2α signaling may be activated by estrogen signaling, as stated in lines 404–407: “the present finding provides a likely explanation for this apparent contradiction, namely, that neuroestrogens, rather than or in addition to ovarian-derived circulating estrogens, may function upstream of PGF2α signaling to mediate female receptivity.” The observation that esr2b-deficient females do not accept male courtship is also stated in lines 401–403: “we recently challenged it by showing that female medaka deficient for esr2b are completely unreceptive to males, and thus estrogens play a critical role in female receptivity.”
Line 396-397: or the remaining estrogens are enough to activate esr2b-dependent female-typical mating behaviors.
We agree that cyp19a1b deficiency did not completely preclude female mating behavior, most likely because residual estrogens in the brains of cyp19a1b-deficient females enable weak activation of Esr2b signaling. However, the relevant section in the Discussion is not focused on examining why mating behavior persisted, but rather on considering the implications of this finding for the neural circuits regulating mating behavior. Therefore, incorporating the suggested explanation here would shift the focus and would not be appropriate.
Line 420-421: this is a lot of variation. Was age controlled for?
The time required for medaka to reach sexual maturity varies with rearing density and food availability. Due to space constraints, we adjust these parameters as needed, which led to variation in the ages of the experimental fish. However, since all experiments were conducted using sibling fish of the same age that had just reached sexual maturity, we believe this does not affect our conclusions.
Line 457: have these kits been validated in medaka?
Although we have not directly validated its applicability in medaka, its extensive use in this species suggests that it us unlikely to pose any issues (e.g., Ussery et al., 2018, Aquat Toxicol, 205:58–65; Lee et al., 2019, Ecotoxicol Environ Saf, 173:174–181; Kayo et al., 2020, Gen Comp Endocrinol, 285:113272; Fischer et al., 2021, Aquat Toxicol, 236:105873; Royan et al., 2023, Endocrinology, 164:bqad030).
Line 589, re fish that spawned: how many times did this happen? Please note it is based on genotype and experiment. This could be important.
Line 627: In response to this comment, we have added the following details: “Specifically, 7/18 cyp19a1b<sup>+/+</sup>, 11/18 cyp19a1b<sup>+/−</sup>, and 6/18 cyp19a1b<sup>−/−</sup> males were excluded in Fig. 1D; 6/10 cyp19a1b<sup>+/+</sup>, 3/10 cyp19a1b<sup>+/−</sup>, and 6/10 cyp19a1b<sup>−/−</sup> females were excluded in Fig. 6B; 2/23 esr1+/+ and 5/24 esr1−/− males were excluded in Fig. S7; 2/24 esr2a+/+ and 3/23 esr2a<sup>−/−</sup> males were excluded in Fig. S8A; 0/23 esr2a+/+ and 0/23 esr2a<sup>−/−</sup> males were excluded in Fig. S8B.”
Reviewer #2 (Recommendations For The Authors):
Abstract:
(A1) The framing of neuroestrogens being important for male-typical rodents, and not for other vertebrate lineages, does not account for other groups (birds) in which this is true (the authors can consult their cited work by Balthazart (Reference 6) for extensive accounting of this). This makes the novelty clause in the abstract "indicating that neuro-estrogens are pivotal for male-typical behaviors even in nonrodents" less surprising and should be acknowledged by the authors by amending or omitting this novelty clause. The findings regarding androgen receptor transcription (next sentence) are more important and pertinent.
Line 27: We recognize that the aromatization hypothesis applies to some birds, including zebra finches, as stated in the Introduction (lines 48–49) and Discussion (lines 432–433). However, this was not reflected in the Abstract. Following the reviewer’s suggestion, we have changed “in non-rodents” to “in teleosts.”
(A2) The medaka line that has been engineered to have aromatase absent in the brain is presented briefly in the abstract, but can be misinterpreted as naturally occurring. This should be amended, by including something like "engineered" or "directed mutant" before 'male medaka fish'.
Line 24: We have added “mutagenesis-derived” before “male medaka fish” in response to this comment.
Introduction:
(I1) The paragraph on teleost brain aromatase should acknowledge that while the capacity for estrogen synthesis in the brain is 100-1000 fold higher in teleosts as compared to rodents and other vertebrates, the majority of this derives from glial and not neural sources. This can be confusing for readers since the term 'neuroestrogens' often refers to the neuronal origin and signalling. And this observation includes the exclusive radial glial expression of cyp19a1b in medaka (Diotel et al., 2010), and first discovered in midshipman (Forlano et al., 2001), each of which should also be cited here. In addition, the authors expend much text comparing teleosts and rodents, but it is worth expanding these kinds of comparisons, especially by pointing out that parts of the primate brain are found to densely express aromatase (see work by Ei Terasawa and others).
In response to this comment and a similar comment from reviewer #1, we have replaced “neuroestrogens” with “brain-derived estrogens” or “brain estrogens” throughout the manuscript.
Line 63: We have also added the text “In teleost brains, including those of medaka, aromatase is exclusively localized in radial glial cells, in contrast to its neuronal localization in rodent brains (18– 20).” As a result of this addition, we have changed “This observation suggests” to “These observations suggest” in the subsequent sentence.
Line 51: Additionally, to include information on aromatase in the primate brain, we have added the following text: “In primates, the hypothalamic aromatization of androgens to estrogens plays a central role in female gametogenesis (10) but is not essential for male behaviors (7, 8).”
The following references (#10 and 18–20), cited in the newly added text above, have been included in the reference list, with other references renumbered accordingly:
E. Terasawa, Neuroestradiol in regulation of GnRH release. Horm. Behav. 104, 138–145 (2018).
P. M. Forlano, D. L. Deitcher, D. A. Myers, A. H. Bass, Anatomical distribution and cellular basis for high levels of aromatase activity in the brain of teleost fish: aromatase enzyme and mRNA expression identify glia as source. J. Neurosci. 21, 8943–8955 (2001).
N. Diotel, Y. Le Page, K. Mouriec, S. K. Tong, E. Pellegrini, C. Vaillant, I. Anglade, F. Brion, F. Pakdel, B. C. Chung, O. Kah, Aromatase in the brain of teleost fish: expression, regulation and putative functions. Front. Neuroendocrinol. 31, 172–192 (2010).
A. Takeuchi, K. Okubo, Post-proliferative immature radial glial cells female-specifically express aromatase in the medaka optic tectum. PLoS One 8, e73663 (2013).
(I2) It is difficult to resolve from the introduction and work cited how restricted cyp19a1b is to the medaka brain. Important for the results of this study, it is not clear whether it is more of a bias in the brain vs other tissues, or if the cyp19a1b deficiency is restricted to the brain, and gonadal/peripheral cyp19 expression persists. The authors need to improve their consideration of the alternatives, i.e., that this manipulation is not somehow affecting: 1) peripheral aromatase expression (either cyp19a1a or cyp19a1b) in the gonad or elsewhere, 2) compensatory processes, such as other steroidogenic genes (are androgen synthesizing enzymes increasing?).
Our previous study demonstrated that cyp19a1b is expressed in the gonads, but at levels tens to hundreds of times lower than those in the brain (Okubo et al., 2011, J Neuroendocrinol 23:412–423). Additionally, a separate study in medaka reported that cyp19a1b expression in the ovary is considerably lower than that of cyp19a1a (Nakamoto et al., 2018, Mol Cell Endocrinol 460:104–122). Given these observations, any potential effect of cyp19a1b knockout on peripheral estrogen synthesis is likely negligible. Indeed, Figures S1C and S1D confirm that cyp19a1b knockout does not alter peripheral E2 levels.
Line 72: To incorporate this information into the Introduction and address the following comment, we have added the following text: “In medaka, cyp19a1b is also expressed in the gonads, but only at a level tens to hundreds of times lower than in the brain and substantially lower than that of cyp19a1a (26, 27).”
The following references (#26 and 27), cited in the newly added text above, have been included in the reference list, with other references renumbered accordingly:
K. Okubo, A. Takeuchi, R. Chaube, B. Paul-Prasanth, S. Kanda, Y. Oka, Y. Nagahama, Sex differences in aromatase gene expression in the medaka brain. J. Neuroendocrinol. 23, 412–423 (2011).
M. Nakamoto, Y. Shibata, K. Ohno, T. Usami, Y. Kamei, Y. Taniguchi, T. Todo, T. Sakamoto, G. Young, P. Swanson, K. Naruse, Y. Nagahama, Ovarian aromatase loss-of-function mutant medaka undergo ovary degeneration and partial female-to-male sex reversal after puberty. Mol. Cell. Endocrinol. 460, 104–122 (2018).
We have not assessed whether the expression of other steroidogenic enzymes is altered in cyp19a1bdeficient fish, and this may be investigated in future studies.
(I3) Related, there are documented sex differences in the brain expression of cyp19a1b especially in adulthood (Okubo et al 2011) and this study should be cited here for context.
Line 72: As stated in our previous response, we have cited Okubo et al. (2011) by adding the following sentence: “In medaka, cyp19a1b is also expressed in the gonads, but only at a level tens to hundreds of times lower than in the brain and substantially lower than that of cyp19a1a (26, 27).”
Methods
(M1) The rationale is unclear as presented for using mutagen screening for cype19a1b while using CRISPR for esr2a. Are there methodological/biochemical reasons why the authors chose to not use the same method for both?
At the time we generated the cyp19a1b knockouts, genome editing was not yet available, and the TILLING-based screening was the only method for obtaining mutants in medaka. In contrast, by the time we generated the esr2a knockouts, CRISPR/Cas9 had become available, enabling a more efficient and convenient generation of knockout lines. This is why the two knockout lines were generated using different methods.
(M2) Measurement of steroids in biological matrices is not straightforward, and it is good that the authors use multiple extraction steps (organic followed by C18 columns) before loading samples on the ELISA plates, which are notoriously sensitive. Even though these methods have been published before by this group of authors previously, the quality control and ELISA performance values (recovery, parallelism, etc.) should be presented for readers to evaluate.
Thank you for appreciating our sample purification method. Unfortunately, we have not evaluated the recovery rate or parallelism, but we recognize this a subject for future studies.
(M3) Mating behavior - E2 treated males were not co-housed with social partners for the full 24 hr before testing, but instead a few hours (?) prior to testing. The rationale for this should be spelled out explicitly.
Line 494: In response to this comment, we have added “to ensure the efficacy of E2 treatment” to the end of the sentence “The set-up was modified for E2-treated males, which were kept on E2 treatment and not introduced to the test tanks until the day of testing.”
(M4) The E2 treatment is listed as 1ng/ml vs. vehicle (ethanol). Is the E2 dissolved in 100% ethanol for administration to the tank water? Clarification is needed.
Line 517: As the reviewer correctly assumed, E2 was first dissolved in 100% ethanol before being added to the tank water. To provide this information and address reviewer #1’s minor comment 5, we have revised “males were treated with 1 ng/ml of E2 (Fujifilm Wako Pure Chemical, Osaka, Japan) or vehicle (ethanol) alone by immersion in water for 4 days” to “males were treated with 1 ng/ml of E2 (Fujifilm Wako Pure Chemical, Osaka, Japan), which was first dissolved in 100% ethanol (vehicle), or with the vehicle alone by immersion in water for 4 days, with daily water changes to maintain the nominal concentration.”
(M5) The authors exclude fish from the analysis of courtship display behavior for those individuals that spawned immediately at the start of the testing (and therefore it was impossible to register courtship display behaviors). How often did fish in the various treatment groups exhibit this "fast spawning" behavior? Was the occurrence rate different by treatment group? It is unlikely that these omissions from the data set drove large-scale patterns, but an indication of how often this occurred would be reassuring.
Line 627: In response to this comment, we have included the following details: “Specifically, 7/18 cyp19a1b<sup>+/+</sup>, 11/18 cyp19a1b<sup+/−</sup>, and 6/18 cyp19a1b<sup>−/−</sup> males were excluded in Fig. 1D; 6/10 cyp19a1b+/+, 3/10 cyp19a1b+/−, and 6/10 cyp19a1b<sup>−/−</sup> females were excluded in Fig. 6B; 2/23 esr1+/+ and 5/24 esr1−/− males were excluded in Fig. S7; 2/24 esr2a+/+ and 3/23 esr2a<sup>−/−</sup> males were excluded in Fig. S8A; 0/23 esr2a+/+ and 0/23 esr2a<sup>−/−</sup> males were excluded in Fig. S8B.” These data indicate that the proportion of excluded males is nearly constant within each trial and is independent of the genotype of the focal fish.
Results
(R1) It is striking to see the genetic-'dose' dependent suppression of brain E2 content by heterozygous and homozygous cyp19a1b deficiency, indicating that, as the authors point out, the majority of E2 in the male medaka brain (and 1/2 in the female brain) have a brain-derived origin. It is important also for the interpretation that there are large compensatory increases in brain levels of androgens, when E2 levels drop in the cyp19a1b mutant homozygotes. This latter point should receive more attention.
Also, there are large increases in peripheral androgen levels in the homozygote mutants for cyp19a1b in both males and females. This indicates a peripheral effect in addition to the clear brain knockdown of E2 synthesis. These nuances need to be addressed.
In response to this comment, we have revised the Results section as follows:
Line 91: “, indicating a dosage effect of cyp19a1b mutation” has been added to the end of the sentence “In males, brain E2 in heterozygotes (cyp19a1b<sup>+/−</sup>) was also reduced to 45% of the level in wild-type siblings (P = 0.0284) (Fig. 1A).”
Line 94: To draw more attention to the increase in brain androgen levels caused by cyp19a1b deficiency, “Brain levels of testosterone” has been modified to “Strikingly, brain levels of testosterone.”
Line 100: “Their peripheral 11KT levels also increased 3.7- and 1.8-fold, respectively (P = 0.0789, males; P = 0.0118, females) (Fig. S1, C and D)” has been modified and now reads “In addition, peripheral 11KT levels in cyp19a1b<sup>−/−</sup> males and females increased 3.7- and 1.8-fold, respectively (P = 0.0789, males; P = 0.0118, females) (Fig. S1, C and D), indicating peripheral influence in addition to central effects.”
(R2) The interpretation on page 4 that cyp19a1b deficient males are 'less motivated' to mate is premature, given the behavioral measures used in this study. There are several competing explanations for these findings (e.g., alterations in motivation, sensory discrimination, preference, etc.) that could be followed up in future work, but the current results are not able to distinguish among these possibilities.
Line 112: We agree that the possibility of altered cognition or sexual preference cannot be dismissed. To incorporate this perspective, we have revised the text “, suggesting that they are less motivated to mate” to “These results suggest that they are less motivated to mate, though an alternative interpretation that their cognition or sexual preference may be altered cannot be dismissed.”
(R3) On page 5, the authors present that peripheral E2 manipulation (delivery to the fish tank) restores courtship behavior in males, and then go on to erroneously conclude that this demonstrates "that reduced E2 in the brain was the primary cause of the mating defects, indicating a pivotal role of neuroestrogens in male mating behavior." Because this is a peripheral E2 treatment, there can be manifold effects on gonadal physiology or other endocrine events that can have indirect effects on the brain and behavior. Without manipulation of E2 directly to the brain to 'rescue' the cyp19a1b deficiency, the authors cannot conclude that these effects are directly on the central nervous system. Tellingly, the tank E2 treatment did not rescue aggressive behavior, suggestive of the potential for indirect effects.
Line 155: As detailed in Response to reviewer #2’s specific comment 1, we have revised the text from “These results demonstrated that reduced E2 in the brain was the primary cause of the mating defects, indicating a pivotal role of neuroestrogens in male mating behavior. In contrast” to “These results suggest that reduced E2 in the brain is the primary cause of the mating defects, highlighting a pivotal role of brain-derived estrogens in male mating behavior. However, caution is warranted, as an indirect peripheral effect of bath-immersed E2 on behavior cannot be ruled out, although this is unlikely given the comparable peripheral E2 levels in cyp19a1b-deficient and wild-type males. In contrast to mating.”
(R4) The downregulation of androgen-dependent gene expression (vasotocin in pNVT and galanin in pPMp) in the cyp19a1b deficient males (Figure 3) could be due to exceedingly high levels of brain androgens in the cyp19a1b deficient males. The best way to test the idea that estrogens can restore the expression to be more wild-type directly (like what is happening for ara and arb) is to look at these same markers (vasotocin and galanin) in these same brain areas in the brains of E2-treated males. The authors should have these brains from Figure 2. Unless I missed something, those experiments were not performed/reported here. It is clear that the ara and arb receptors have EREs and are 'rescued' by E2 treatment, but in principle, there could be indirect actions for reasons stated above for the behavior due to the peripheral E2 tank application.
Thank you for your insightful comment. We agree that the current results cannot exclude the possibility that excessive androgen levels caused the downregulation of vt and gal. However, our previous studies showed that excessive 11KT administration to gonadectomized males and females increased the expression of these genes to levels comparable to wild-type males (Yamashita et al., 2020, eLife, 9:e59470; Kawabata-Sakata et al., 2024, Mol Cell Endocrinol 580:112101), making this scenario unlikely. That said, testing whether estrogen treatment restores vt and gal expression in cyp19a1bdeficient males would be informative, and we see this as an important direction for future research.
Discussion
(D1) The authors need to clarify whether EREs are found in other vertebrate AR introns, or is this unique to the teleost genome duplication?
We have identified multiple ERE-like sequences within intron 1 of the mouse AR gene. However, sequence data alone do not provide sufficient evidence of their functionality, rendering this information of limited relevance. Therefore, we have chosen not to include this discussion in the current paper.
Reviewer #3 (Recommendations For The Authors):
(1) The authors are strongly encouraged to report information regarding the effect of Cyp19a1b deletion on the brain content of aromatase protein (ideally both isoforms investigated separately) as the two isoforms are mostly but not completely brain vs gonad specific. The analysis of other tissues would also strengthen the characterization of this model.
We agree that measuring aromatase protein levels in the brain of our fish would be valuable for confirming the loss of cyp19a1b function. However, as no suitable method is currently available, this issue will need to be addressed in future studies. While this constitutes indirect evidence, the observed reduction in brain E2 levels, with no change in peripheral E2 levels, in cyp19a1b-deficient fish strongly suggests the loss of cyp19a1b function, as noted in Response to reviewer #3’s comment 1 on weaknesses.
(2) As presented, this study reads as niche work. A better description of the behavior and reproductive significance of the different aspects of the behavioral sequence would allow a better understanding of the results and would thus allow the non-specialist to appreciate the significance of the observations.
Line 103: In response to this comment and Reviewer #3’s comment 2 on weaknesses, we have revised the sentence from “The mating behavior of medaka follows a stereotypical pattern, wherein a series of followings, courtship displays, and wrappings by the male leads to spawning” to “The mating behavior of medaka follows a stereotypical sequence. It begins with the male approaching and closely following the female (following). The male then performs a courtship display, rapidly swimming in a circular pattern in front of the female. If the female is receptive, the male grasps her with his fins (wrapping), culminating in the simultaneous release of eggs and sperm (spawning)” in order to provide a more detailed description of medaka mating behavior.
(3) The data regarding female behavior are limited and incomplete. It is suggested to keep this for another manuscript unless data on the behavior of the female herself is added. Indeed, analyzing female's behavior from the male's perspective complicates the interpretation of the results while a description of what the females do would provide valuable and interpretable information.
We thank the reviewer for this thoughtful suggestion and agree that the data and discussion for females are less extensive than for males. However, we have previously elucidated the mechanism by which estrogen/Esr2b signaling promotes female mating behavior (Nishiike et al., 2021). Accordingly, it follows that the new insights into female behavior gained from the cyp19a1b knockout model are more limited than those for males. Nevertheless, when combined with our prior findings, the female data in this study offer valuable insights, and the overall mechanism through which estrogens promote female mating behavior is becoming clearer. Therefore, we do not consider the female data in this study to be incomplete or merely supplementary.
(4) In Figure 2, the validity to run multiple T-tests rather than a two-way ANOVA comparing TRT and genotype is questionable. Moreover, why are the absolute values in CTL higher than in the initial experiment comparing genotypes for ara in PPa, pPPp, and NVT as well as for arb in aPPp. More importantly, these graphs do not seem to reproduce the genotype effects for ara in pPPp and NVT and for arb in aPPp.
The data in Figures 2J and 2K were analyzed with an exclusive focus on the difference between vehicletreated and E2-treated males, without considering genotype differences. Therefore, the use of T-tests for significance testing is appropriate.
As the reviewer noted, the overall ara expression area is larger in Figure 2J than in Figure 2F. However, as detailed in Response to reviewer #3’s comment 8 on weaknesses, the relative area ratios of ara expression among brain nuclei are consistent between the two figures, indicating the reproducibility of the results. Thus, we consider this difference unlikely to affect the conclusions of this study.
Additionally, the differences in ara expression in pPPp and arb expression in aPPp between wild-type and cyp19a1b-deficient males appear smaller in Figures 2J and 2K compared to Figures 2F and 2H. This is likely due to the smaller sample size used in the experiments for Figures 2J and 2K, which makes the differences less distinct. However, since the same genotype-dependent trends are observed in both sets of figures, the conclusion that ara and arb expression is reduced in cyp19a1b-deficient male brains remains valid.
(5) More information is required regarding the analysis of single ISH - How was the positive signal selected from the background in the single ISH analyses? How was this measure standardized across animals? How many sections were imaged per region? Do the values represent unilateral or bilateral analysis?
Line 540: Following this comment, we have provided additional details on the single ISH method in the manuscript. Specifically, “, and the total area of signal in each brain nucleus was calculated using Olyvia software (Olympus)” has been revised to “The total area of signal across all relevant sections, including both hemispheres, was calculated for each brain nucleus using Olyvia software (Olympus). Images were converted to a 256-level intensity scale, and pixels with intensities from 161 to 256 were considered signals. All sections used for comparison were processed in the same batch, without corrections between samples.”
(6) More information should be provided in the methods regarding the image analysis of double ISH. In particular, what were the criteria to consider a cell as labeled are not clear. This is not clear either from the representative images.
Line 596: To provide additional details on the single ISH method in the manuscript, we have added the following sentence: “Cells were identified as coexpressing the two genes when Alexa Fluor 555 and fluorescein signals were clearly observed in the cytoplasm surrounding DAPI-stained nuclei, with intensities markedly stronger than the background noise.”
(7) There is no description of the in silico analyses run on ESR2a in the methods.
The method for identifying estrogen-responsive element-like sequences in the esr2a locus is described in line 549: “Each nucleotide sequence of the 5′-flanking region of ara and arb was retrieved from the Ensembl medaka genome assembly and analyzed for potential canonical ERE-like sequences using Jaspar (version 5.0_alpha) and Match (public version 1.0) with default settings.”
However, the method for domain identification in Esr2a was not described. Therefore, we have added the following text in line 469: “The DNA- and ligand-binding domains of medaka Esr2a were identified by sequence alignment with yellow perch (Perca flavescens) Esr2a, for which these domain locations have been reported (58).”
The following reference (#58), cited in the newly added text above, have been included in the reference: S. G. Lynn, W. J. Birge, B. S. Shepherd, Molecular characterization and sex-specific tissue expression of estrogen receptor α (esr1), estrogen receptor βa (esr2a) and ovarian aromatase (cyp19a1a) in yellow perch (Perca flavescens). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 149, 126–147 (2008).
(8) Information about the validation steps of the EIA that were carried out as well as the specificity of the antibody the steroids and the extraction efficacy should be provided.
We have not directly validated the applicability of the EIA kit, but its extensive use in medaka suggests that it us unlikely to pose any issues (e.g., Ussery et al., 2018, Aquat Toxicol, 205:58–65; Lee et al., 2019, Ecotoxicol Environ Saf, 173:174–181; Kayo et al., 2020, Gen Comp Endocrinol, 285:113272; Fischer et al., 2021, Aquat Toxicol, 236:105873; Royan et al., 2023, Endocrinology, 164:bqad030).
The specificity (cross-reactivity) of the antibodies is detailed as follows.
(1) Estradiol ELISA kits: estradiol, 100%; estrone, 1.38%; estriol, 1.0%; 5α-dihydrotestosterone, 0.04%; androstenediol, 0.03%; testosterone, 0.03%; aldosterone, <0.01%; cortisol, <0.01%; progesterone, <0.01%.
(2) Testosterone ELISA kits: testosterone, 100%; 5α-dihydrotestosterone, 27.4%; androstenedione, 3.7%; 11-ketotestosterone, 2.2%; androstenediol, 0.51%; progesterone, 0.14%; androsterone, 0.05%; estradiol, <0.01%.
(3) 11-Keto Testosterone ELISA kits: 11-ketotestosterone, 100%; adrenosterone, 2.9%; testosterone, <0.01%.
As this information is publicly available on the manufacturer’s website, we deemed it unnecessary to include it in the manuscript.
Unfortunately, we have not evaluated the extraction efficacy of the samples, but we recognize this a subject for future studies.
(9) I wonder whether the evaluation of the impact of the mutation by comparing the behavior of a group of wild-type males to a group of mutated males is the most appropriate. Justifying this approach against testing the behavior of one mutated male facing one or several wild-type males would be appreciated.
We agree that the resident-intruder test, in which a single focal resident is confronted with one or more stimulus intruders, is the most commonly used method for assessing aggression. However, medaka form shoals and lack strong territoriality, and even slight dominance differences between the resident and the intruder can increase variability in the results, compromising data consistency. Therefore, in this study, we adopted an alternative approach: placing four unfamiliar males together in a tank and quantifying aggressive interactions in total. This method allows for the assessment of aggression regardless of territorial tendencies, making it more appropriate for our investigation.
(10) Lines 329-331: this sentence should be rephrased as it contributes to the confusion between sexual differentiation and activation of circuits. The restoration of sexual behavior by adult estrogen treatment pleads in favor of an activational role of neuro-estrogens on behavior rather than an organizational role. Therefore, referring to sexual differentiation is misleading, even more so that the study never compares sexes.
As detailed in Response to reviewer #3’s comment 9 on weaknesses, we consider that all factors that cause sex differences, including the transient effects of adult steroids, need to be incorporated into a theory of sexual differentiation. In teleosts, since steroids during early development have little effect and sexual differentiation primarily relies on steroid action in adulthood, our discussion on brain sexual differentiation remains valid, including the statement in line 347: “This variation among species may represent the activation of neuroestrogen synthesis at life stages critical for sexual differentiation of behavior that are unique to each species.”
(11) Lines 384-386: I may have missed something but I do not see data supporting the notion that neuroestrogens may function upstream of PGF2a signaling to mediate female receptivity.
Line 403: We acknowledge that our explanation was insufficient and apologize for any confusion. To clarify this point, “Given that estrogen/Esr2b signaling feminizes the neural substrates that mediate mating behavior, while PGF2α signaling triggers female sexual receptivity,” has been added before the sentence “The present finding provides a likely explanation for this apparent contradiction, namely, that neuroestrogens, rather than or in addition to ovarian-derived circulating estrogens, may function upstream of PGF2α signaling to mediate female receptivity.”
Additional alteration
Reference list (line 682): a preprint article has now been published in a peer-reviewed journal, and the information has been updated accordingly as follows: “bioRxiv doi: 10.1101/2024.01.10.574747 (2024)” to “Proc. Natl. Acad. Sci. U.S.A. 121, e2316459121 (2024).”
For ICILS 2023, exploratory factor analysis (EFA) supports a two-factor structure distinguishing between general and specialized digital self-efficacy (DSE).
Esto no debería estar aquí. No se mencioa EFA en la estrategia.
Statistical analyses will address missing data using Full Information Maximum Likelihood (FIML) during confirmatory analyses
Sobre el tratamiento de missing. Me parece que debería ser más claro y separado de la estimación factorial. Explicar que se considera missing en cada caso y luego mencion ar lo de FIML.
Partial match (same intent)
En la tabla debería haber una columna para la fase de cada item, y en una columna aparte un comentario sobre el match
Note de synthèse de l'audition de Isac Mayembo (Alex Hitchens) par la commission d'enquête sur TikTok
Date de l'audition : [Non spécifiée, mais contexte indique "aujourd'hui" et "cet après-midi"]
Personne auditionnée : Isac Mayembo, créateur de contenu sous le pseudonyme Alex Hitchens.
1. Contexte de l'audition et déclaration d'intérêts
2. Évolution et impact perçu de TikTok
3. Parcours sur TikTok et stratégies de contenu
Il n'avait même pas connecté sa carte bancaire car la vente de formations était beaucoup plus rentable.
La monétisation TikTok était "bien moins avantageuse" en 2021/début 2022. Actuellement, il laisse même la monétisation à ses collaborateurs qui créent du contenu pour lui.
4. La communauté et l'interaction
5. Gestion des comptes et responsabilité
Il pense qu'il n'est pas obligé de le notifier si la promotion n'est pas faite "dans la vidéo même" mais par un lien en description.
Il reconnaît qu'il le ferait si la promotion était directe dans la vidéo. La rapporteur indique que la question sera examinée.
6. Contenus controversés, modération et désinformation
Il accuse la commission de "désinformation" en isolant ses propos : "Vous êtes en train d'isoler mon propos Donc vous avez pris 10 secondes d'un propos de 8 10 minutes où j'expliquais justement parce queon va rentrer en détail parce qu'apparemment on est en train d'en parler donc pourquoi pas J'expliquais que le gouvernement était responsable de la sécurité J'ai n'était pas normal qu'une femme en 2025 ne puisse pas sortir dehors euh le soir très tard sur Paris Et justement après je parlais du fait que dans ce monde dans ce monde qui est dur que fait une femme dehors après 22h et je poursuivais ensuite il est préférable de sortir avec une amie ou avec deux amis ou avec un homme Vous vous avez pris 10 secondes de vos propos vous l'avez isolé et vous venez de me le balancer à la gueule."
7. Incident et suspension de l'audition
Ya
Providing numbers without references is not enough
Document de Synthèse : Audition d'Adrien Laurent par la Commission d'Enquête sur TikTok
Date de l'audition : Non spécifiée, mais fait suite à plus d'une centaine d'auditions. Intervenants principaux :
1. Contexte et Objectifs de l'Audition
L'objectif est de faire la lumière sur la production de contenus et la régulation. L'audition d'Adrien Laurent fait suite à une consultation citoyenne ayant recueilli plus de 30 000 réponses et à des témoignages, son nom étant "revenu très souvent" parmi les influenceurs ou créateurs de contenu jugés "problématiques" par les jeunes.
Le Président de la commission a rappelé qu'il ne s'agit pas d'un tribunal mais d'un travail de compréhension, avec des pouvoirs étendus, sans se substituer à la justice. L'audition est médiatisée, mais l'objectif premier reste la prise en compte des victimes.
Adrien Laurent a été invité à déclarer tout intérêt public ou privé pouvant influencer ses déclarations et à détailler la nature de ses revenus liés aux plateformes. Il a prêté serment de "dire la vérité, toute la vérité, rien que la vérité".
2. Présentation et Défense d'Adrien Laurent
Il se présente comme son "propre producteur". Parallèlement, il est depuis deux ans créateur de contenu pour adultes, vidéos commercialisées exclusivement sur des plateformes réservées aux majeurs et "ne sont pas présentes sur TikTok". Il travaille avec des "actrices professionnelles majeures qui ont elles-mêmes leur compte sur ces plateformes".
Ses arguments principaux :
Il affirme n'avoir "aucun contenu érotique sur TikTok". * Twitter/X : Reconnaît que Twitter autorise les contenus pour adultes mais en restreint l'accès aux majeurs via des réglages. * Accusations de sexisme/violence : Affirme que son contenu n'est "ni sexiste ni misogyne ni masculiniste" et qu'il n'a "jamais été violent". Il évoque sa vie d'acteur de contenu pour adultes "de manière assumée mais transparente et respectueuse", sans "dénigrer leurs envies ni leurs pratiques ni les rabaisser". * Public mineur et responsabilité : "Je n'ai jamais encouragé un public mineur à consommer du contenu inadapté." Il insiste sur le fait que "la sexualité ne s'apprend pas à travers le contenu pour adultes". Avec près de 2 millions d'abonnés sur TikTok, il a diffusé des messages de prévention sur le dépistage et le port du préservatif. * Accès des CM2 à TikTok : "Les CM2 ne peuvent pas avoir accès à TikTok. La plateforme est interdite au moins de 13 ans." Si des enfants de cet âge y accèdent, il s'agit d'un "problème de contrôle parental et de responsabilité de TikTok, pas de la mienne." Il refuse "d'endosser une responsabilité qui ne [lui] appartient pas".
3. Relations avec TikTok et Revenus
Si nous ne pouvons pas confirmer ton âge, tu ne pourras passer en live et enfin si nous confirmons que tu as moins de 18 ans tu ne pourras pas accéder au live."
Il réfute les accusations de la ministre Aurore Berger concernant des "lives sexuels avec des jeunes femmes masquées" douteuses quant à leur majorité et consentement, affirmant qu'aucun contenu sexuel n'était diffusé et que les participants étaient "majeurs et consentants".
4. Modération et Algorithme
5. Échanges avec les Députés
La commission cherche à comprendre "pourquoi vous êtes allé sur TikTok", "comment vous gérez vos contenus" face à cette communauté jeune, et la modération.
Bannissements d'autres plateformes : Adrien Laurent confirme avoir été banni de Snapchat et Instagram en raison de signalements massifs, sans toujours avoir d'explication claire.
Choix de TikTok : S'est mis sur TikTok "très tard" sur conseil de son frère, y voyant une opportunité de visibilité grâce à l'application "la plus téléchargée en France et même dans le monde". Il appréciait les vidéos "très rapides avec beaucoup d'engagement".
Diversité de sa communauté : Sur ses 2 millions d'abonnés, "il n'y a pas 2 millions de personnes qui me suivent par rapport au contenu pour adultes". Beaucoup le suivent pour la téléréalité ou l'aspect "positif" et "bienveillant" de ses lives.
Il met en parallèle la vente d'alcool en magasin, où la responsabilité n'incombe pas au vendeur si un mineur contourne l'interdiction d'achat.
Il se considère comme un "simple usager de la plateforme qui respecte les règles". * Comparaison avec Alex Chen : Il se dit "totalement mais sur tous les points opposés à ce monsieur", affirmant ne pas être "ni sexiste ni misogyne ni masculiniste". Il défend la "liberté sexuelle à égalité" et met la femme "sur un piédestal tout le temps". * Promotion croisée et contenu pour adultes : Il confirme rediriger sa communauté TikTok vers d'autres plateformes (Snap, Insta, Twitch, YouTube) pour "alimenter [ses] autres plateformes", mais insiste sur le fait que son contenu est "totalement différent" et adapté à chaque plateforme. Pour ses contenus privés (Mim, OnlyFans), il affirme une "double authentification" qui empêche l'accès aux mineurs, qualifiant son porno d'"éthique". * Lives avec des mineurs : Confronté à une vidéo de live avec un mineur très jeune, Adrien Laurent explique qu'il prend "énormément de gens tous les soirs" et que si une personne de moins de 18 ans réussit à contourner les règles, ce n'est pas son problème. Il affirme cependant avoir une "responsabilité" en énonçant un "propos responsable" même dans ces cas-là (ex: "il est tard faut que tu ailles te coucher"). * "Pranks" et provocation : Décrit ses "pranks" comme de l'"autodérision" et du "fun", citant l'exemple de la "trompe d'éléphant" ou "viser le trou" au basket. Sa mère, professeur, le soutient. * Accusation de transphobie/humiliation : Réfute l'accusation d'avoir humilié une personne transgenre en live. Il pose des questions par "intérêt", et "n'humilie jamais personne". * Outils de restriction d'audience : Questionné par M. Vogeta sur l'utilisation d'outils de restriction d'audience pour exclure les mineurs sur ses contenus "classiques" (non pornographiques), Adrien Laurent répond que sur Twitter/X, il faut modifier les réglages pour voir du contenu sensible, ce qui protège le compte vierge. * Agents OnlyFans/MIM : Il n'a pas d'agent mais une "équipe qui bosse avec [lui] justement pour sécuriser un petit peu [son] travail". Il insiste sur le fait que c'est un "boulot" qui demande "beaucoup de travail". Il se considère comme un "artiste" et demande le respect de sa "liberté de création artistique". * Recrutement pour la pornographie via TikTok : Il indique que certaines abonnées peuvent exprimer leur intérêt pour des collaborations dans le milieu du contenu pour adultes, mais il les redirige alors vers un "email professionnel" et "coupe le lien" sur TikTok, ne souhaitant pas en parler sur la plateforme. * Impact sur les enfants : Questionné en tant que députée et mère sur l'impact de ses contenus sur un enfant de 13 ans, il répond qu'il ne "choisit pas sa communauté". En tant que futur père, il ferait une éducation basée sur la "communication" et laisserait à sa fille, une fois majeure, le choix de son chemin professionnel. * Contenus sexualisants en live : La rapporteure constate, après un test avec un compte mineur, que les lives sont accessibles aux mineurs, malgré les affirmations d'Adrien Laurent. Elle cite des propos "sexualisants" (ex: "Ça va ma puce il y a du monde au balcon") et "hypersexualisants" (taille des poitrines) tenus en live. * Comparaison avec des artistes musicaux : Adrien Laurent compare ses propos à des paroles de chansons d'artistes comme Michel Sardou, Bruno Mars ou Orelsan (citant des extraits explicites), se plaignant d'une "indignation à géométrie" variable, ce que la rapporteure qualifie d'"œuvres à caractère artistique" et non de lives soumis à des conditions d'utilisation différentes. * Restriction des lives : La rapporteure précise que TikTok permet aux hôtes de "restreindre" leurs lives, option qu'Adrien Laurent n'active pas car il considère ses lives comme "bon enfant". Il reconnaît qu'il n'est pas un "technicien TikTok" mais s'efforce de "se conformer au maximum aux règles communautaires". * Modération interne : Il a une équipe de "modérateurs modératrices" qui "bannissent tous les commentaires à caractère négatif les insultes". Il note l'opacité de l'algorithme et la nécessité de contourner certains mots pour éviter le "shadow ban" (ex: "viol" par "ool", "pute" par "pu pute").
6. Conclusions de l'Audition
Adrien Laurent insiste sur sa professionnalisation dans le contenu pour adultes éthique et son respect des règles (en théorie) de TikTok, tout en reconnaissant les défis de la modération et de l'accès des mineurs aux plateformes.
La commission insiste sur la réalité de la présence de jeunes mineurs sur TikTok et la nécessité de trouver des solutions législatives pour protéger les enfants.
La question de l'accessibilité réelle des lives aux mineurs et la responsabilité des créateurs et de la plateforme reste un point de divergence majeur.
Reviewer #2 (Public review):
The authors of the present study are responsible for a previous study, which also showed that in response to DNA damage, Werner syndrome protein WRN, WRN interacting protein WRNIP1, and Rev1 assemble together with Y-family Pols (Polη, Polι, or Polκ), and that they are indispensable for Trans-Lesion-Synthesis (TLS) (Genes Dev 2024). They also identified a role of WRN's 3'→5' exonuclease activity in the high in vivo fidelity of TLS by Y-family, through UV-induced CPDs by Polη, through N6 ethenodeoxyadenosine (εdA) by Polι, through thymine glycol by Polκ, and through UV-induced (6-4) photoproducts by Polη and Polι. Thus, by removing nucleotides misinserted opposite DNA lesions by the Y-family Pols, WRN's 3'→5' exonuclease activity improves the fidelity of TLS by these Pols. The present work, which follows up on this previous work, reports the crucial role also of the ATPase activities of WRN and WRNIP1 in raising the fidelity of TLS by Y family Pols, in addition to the exonuclease activity, with an entirely different mechanism, which normally consists in unwinding of DNA containing secondary structures.
By using adequate cell line models and methodologies, notably DNA fiber, TLS, and mutation analyses assays, as well as specific ATPase point mutations, they found that progression of the replication forks through UV lesions was not affected in cells lacking the WRN exonuclease activity as well as the WRN and WRNIP1 ATPase activities, but occurs with a vast increase in error-prone TLS, notably through CPDs by Polη, with differential impacts on the nature of mutations between WRN ATPase and WRNIP1 ATPase. The relative contributions of these activities (exonuclease and ATPase) to the fidelity of TLS Pols, however, vary, depending upon the DNA lesion and the TLS Pol involved. Additionally, defects in these ATPase activities cause mutational hot spot formation in different sequence contexts. The authors provide evidence that the combined action of WRN and WRNIP1 ATPases, along with WRN 3' to 5' exonuclease, confers an enormous rise in the fidelity of TLS by Y-family Pols. They identify the means by which these otherwise highly error-prone TLS Pols have been adapted to function in an error-free manner. They suggest that WRNIP1 ATPases prevent misincorporations while WRN exonuclease removes misinserted nucleotides. This combination confers a vast increase in the fidelity of Y-family Pols, essential for genome stability.
Overall, this is a comprehensive and thoughtful manuscript, and all the findings reported are convincing and well supported. The data cannot be considered as entirely novel, as they follow-up on the recent 2024 publication by the same authors who unveiled that the exonuclease activity of WRN and WRNIP1 confers accuracy of TLS. The experimental methods are multiple and rigorous.
Reviewer #3 (Public review):
Summary:
Replication through DNA lesions such as UV-induced pyrimidine dimers is mainly performed by Y-family pols. These translesion synthesis (TLS) pols are intrinsically error-prone. However, in living cells, TLS must be conducted in an error-free manner. This manuscript demonstrated that WRN and WRNIP1 ATPases play an important role in addition to WRN 3'>5' exonuclease in human cells.
Strengths:
The authors made use of WT human fibroblasts and WRN-deficient cell line for TLS assays in human cells and siRNA knock-down experiments to analyze TLS efficiency. For the cII mutation assay, the big blue mouse embryonic fibroblasts were used. These materials, as well as other Materials and Methods, had already been well established by this group or other groups. The authors used Pol eta, iota, kappa, and theta as TLS pols, and used UV-induced CPD, (6-4)PP, epsilon dA, and thymine glycol as DNA lesions. Thus, the authors examined the generality of their results in terms of TLS pols and DNA lesions.
Weaknesses:
Although the main part of this manuscript is the impact of the deficiencies of WRN and WRNIP1 ATPases on TLS by Y-family DNA polymerases, especially on TLS efficiency and mutation spectrum, many readers would be interested in how these ATPases could change molecular structure of Pol eta, because the structure of it have been studied for some time.
NHPRR, Boston, MA)
DOI: 10.3389/fimmu.2025.1587688
Resource: Nonhuman Primate Reagent Resource (RRID:SCR_012986)
Curator: @giovanni.decastro
SciCrunch record: RRID:SCR_012986
RRID:SCR_002798
DOI: 10.3389/fphar.2025.1590688
Resource: GraphPad Prism (RRID:SCR_002798)
Curator: @scibot
SciCrunch record: RRID:SCR_002798
RRID:SCR_007037
DOI: 10.1155/da/2739947
Resource: SPM (RRID:SCR_007037)
Curator: @scibot
SciCrunch record: RRID:SCR_007037
RRID:SCR_004757
DOI: 10.1155/da/2739947
Resource: ANTS - Advanced Normalization ToolS (RRID:SCR_004757)
Curator: @scibot
SciCrunch record: RRID:SCR_004757
RRID:SCR_001905
DOI: 10.1155/da/2739947
Resource: R Project for Statistical Computing (RRID:SCR_001905)
Curator: @scibot
SciCrunch record: RRID:SCR_001905
RRID:AB_2314509
DOI: 10.1016/j.cub.2018.12.027
Resource: (DSHB Cat# 40-1a, RRID:AB_528100)
Curator: @scibot
SciCrunch record: RRID:AB_528100
Addgene_12259
DOI: 10.1016/j.celrep.2025.115826
Resource: RRID:Addgene_12259
Curator: @scibot
SciCrunch record: RRID:Addgene_12259
Compte-rendu de l'Audition de Manon et Julien Tanti par la Commission d'Enquête sur TikTok
I. Préambule et Positionnement des Influenceurs
Manon et Julien Tanti ont été convoqués en tant qu'influenceurs pour s'exprimer sur leur modèle économique, leurs revenus via TikTok et d'autres plateformes, ainsi que sur l'impact de TikTok sur les mineurs.
Réaction de Manon Tanti face aux accusations : Manon Tanti exprime un profond regret concernant la préparation publique de l'audition et les déclarations du président l'accusant d'être une "influenceuse à contenu problématique" et d'humilier ou dégrader l'image de ses propres enfants. Elle déclare : "Je ne suis pas venue ici pour fuir mes responsabilités. J'ai conscience que lorsque l'on s'expose publiquement, surtout sur des plateformes comme TikTok, on a un devoir d'exemplarité, d'autant plus quand on est suivi par des jeunes. Mais je veux poser les choses clairement dès le départ : je regrette profondément deux choses dans la manière dont cette audition a été préparée publiquement. Je regrette vos déclarations Monsieur le Président, je ne suis pas une influenceuse à contenu problématique, du moins je ne pense pas, et du coup quelle décision de justice vous fondez-vous pour affirmer cela ? Ce genre de jugement lancé dans les médias avant toute audition ne révèle pas d'un travail d'enquête objectif, mais d'une condamnation sans débat. La seconde, plus grave à mes yeux, ce sont ces propos affirmant que j'humilierai et que je dégraderai l'image de mes propres enfants. Ces accusations sont violentes, injustes et totalement déplacées. Je suis mère avant tout et jamais je ne tolérerais qu'on prête des intentions aussi graves sans preuve ni dialogue." Elle précise avoir été "profondément attristée" par la diffusion de ces accusations sur des chaînes comme TF1 et BFM, alors qu'elle était initialement "flattée d'avoir été convoquée" pour aider à comprendre le fonctionnement de TikTok.
Positionnement professionnel : Julien Tanti se définit principalement comme une "personne qui fait de la téléréalité" depuis 14 ans, et non comme un influenceur. Il affirme : "Moi travail principal les tournages, je fais de la télé ça fait 14 ans que je fais de la télé et le reste c'est des hobis parce que ben je suis à Dubaï (...)." Il ajoute : "je n'influence personne je suis sur les réseaux parce que j'aime ça parce que c'est pour moi c'est un divertissement c'est quelque chose que je kiffe et je n'influence personne à faire quoi que ce soit je ne suis pas un influenceur." Manon Tanti soutient cette idée, expliquant qu'ils sont des "personnes qui font de la téléréalité" et que les réseaux sociaux sont une continuité de cette exposition de leur vie.
II. Modèle Économique et Sources de Revenus
Les deux influenceurs détaillent leurs différentes sources de revenus, insistant sur le fait que TikTok n'est pas leur source principale.
Revenus de Julien Tanti :
Revenus de Manon Tanti :
Motivation de l'exposition des enfants : Manon Tanti explique que l'exposition de ses enfants est une "continuité de ce qu'on fait en télé" car ils sont des "personnages publics".
Elle insiste sur le caractère "naturel" de cette exposition dans leur quotidien : "nous sommes des personnages publics, nos enfants aussi".
Elle affirme ne pas les "surexposer" et qu'ils sont "consentants" et même demandeurs d'apparaître dans les vidéos.
Elle cite l'exemple de son fils qui "aimerait avoir une chaîne par exemple", comme des "enfants youtubeurs" célèbres.
Elle défend une vidéo où les enfants ont la "tête dans une espèce de bassine d'eau", expliquant que c'était une "trend" (tendance) et que les enfants étaient "morts de rire".
Elle reconnaît cependant que "peut-être que d'autres parents le le reproduirait de d'une mauvaise manière" mais insiste : "sur cette vidéo je ne trouve absolument pas la vidéo humiliante puisque mes enfants sont ils sont morts de rire parce que c'est vraiment dans une bonne c'est bon enfant encore une fois je je je suis pas du tout en train de noyer mes enfants".
Non-rémunération directe des enfants : Manon affirme ne pas utiliser ses enfants pour la rémunération : "je me sers pas de mes enfants pour la rémunération parce que dans tous les cas moi avec ou mes ou mes sans mes enfants je gagne ma vie".
La députée mentionne l'absence de "contrat de manquina" et de versement des revenus sur un "compte sous séquestre" comme l'exige la loi sur l'influence commerciale.
Manon Tanti reconnaît : "premièrement déjà ça je ne savais pas deuxièmement encore une fois que cette marque là et cette vidéo là en l'occurrence que vous pouvez me citer et troisièmement au rabora en fait j'ai dû le faire une cinquantaine de je je n'ai pas besoin de ma fille pour vendre en fait".
Un autre exemple est soulevé : une collaboration commerciale récente avec la marque "Shane" (vêtements) où les enfants apparaissent, sans mention de "collaboration commerciale" et sans respect du cadre légal.
Manon se défend en disant : "j'ai appris avec vous il y a donc 10 minutes qu'à partir du moment où il y avait écrit collaboration mes enfants ne pouvaient pas apparaître je l'ai appris ben c'est vous qui me l'avez appris".
Elle ajoute avoir déjà été contrôlée par la DGCCRF et avoir payé une amende, mais que "lorsque que Julien a eu son amende par exemple ça n'a pas été mentionné dans les choses qu'on avait mal faites donc si on l'avait su bah forcément on aurait arrêté".
IV. Impact de TikTok sur les Mineurs et Propositions
Les influenceurs sont interrogés sur leur perception de l'âge de leur communauté et leur avis sur la protection des mineurs.
Elle est catégorique : "mes enfants n'auront pas de compte TikTok et n'auront pas accès à TikTok".
De même, pour Instagram, leurs enfants ont un compte géré par elle en tant que mère, mais "n'auront pas accès à ces réseaux sociaux là on en parlera quand ils seront plus grands mais je pense au moins avant leur 14 voir 16 ans".
Conclusion
Les Tanti se positionnent comme des personnalités de téléréalité avant d'être des influenceurs, gérant leurs réseaux sociaux comme un prolongement de leur vie médiatisée. Ils reconnaissent certaines lacunes dans leur connaissance de la loi (notamment sur les contrats de mannequins enfants) mais affirment leur volonté de se conformer aux réglementations une fois informés.
L'échange souligne également le fossé entre la perception de l'influenceur sur son propre contenu ("bon enfant") et la réception par le public ou l'interprétation par la loi ("humiliant", "illégal").
Compte rendu détaillé de l'audition de Nasser Sari (Nasdas) par la commission d'enquête sur TikTok
Personne auditionnée : Nasser Sari, alias Nasdas, influenceur avec 3,7 millions de followers sur TikTok et plus de 9 millions sur Snapchat.
Contexte : La commission d'enquête de l'Assemblée nationale vise à comprendre les mécanismes de TikTok et des réseaux sociaux en général, ainsi qu'à élaborer une meilleure régulation pour protéger les mineurs des contenus choquants.
L'audition fait suite à une consultation citoyenne et des signalements.
Thèmes principaux et idées/faits importants :
1. Rôle et rémunération sur TikTok vs. Snapchat :
2. Utilisation de TikTok comme "tremplin" et viralité :
3. Audience et présence de mineurs :
4. Les jeunes fugueurs et la responsabilité :
5. La santé mentale des créateurs de contenu et la pression :
6. Évolution de la ligne éditoriale et accusation de contenus choquants :
7. Recommandations et auto-critique :
En conclusion, il conseille à ceux qui veulent se lancer sur les réseaux : "vous lancez pas sur les réseaux."
Questions en suspens / Points d'attention :
Voici un compte-rendu détaillé des principaux thèmes et idées importants des sources fournies, incluant des citations pertinentes :
Synthèse du Rapport de la Commission d’Experts sur l’Impact de l’Exposition des Jeunes aux Écrans
Ce document de briefing synthétise les points clés soulevés lors de l'audition de deux experts, * Madame Mouton (neurologue) et * Monsieur Benjamina (neurophysiologiste), co-présidents d'une commission antérieure sur l'impact des écrans.
L'audition se concentre sur l'impact des réseaux sociaux, en particulier TikTok, sur la santé mentale et physique des jeunes.
1. La Détérioration de la Santé Mentale des Jeunes et le Rôle des Réseaux Sociaux
Les experts soulignent une chute de la santé mentale des moins de 25 ans depuis les années 2010, antérieure à la pandémie de COVID-19, qui a cependant accentué cette tendance.
Parallèlement, l'usage des réseaux sociaux s'est massivement répandu, soulevant des questions sur leur implication dans cette détérioration.
Problème global des réseaux sociaux : Bien que la commission se concentre sur TikTok, les experts insistent sur le fait que "les réseaux sociaux posent globalement aujourd'hui tous les mêmes problèmes de design non éthique". TikTok est "peut-être particulièrement efficace pour capter et retenir l'attention des usagers", mais d'autres réseaux ne sont pas exempts de ces problèmes.
Modèle économique et captation de l'attention : Le problème fondamental réside dans le "design de TikTok et des réseaux sociaux qui étant basé sur l'économie de l'attention, la captation des données qui vont ensuite être monnayées à des fins de publicité ciblées". L'objectif est de "maintenir les usagers en ligne le plus longtemps possible de les faire venir en ligne le plus souvent possible et également de leur faire des achats en ligne".
2. Conséquences Négatives sur la Santé Physique et Mentale
L'usage excessif des écrans et des réseaux sociaux entraîne de multiples effets délétères, même indépendamment des contenus.
3. La Question de l'Addiction aux Écrans et Réseaux Sociaux
La discussion autour de l'addiction est nuancée, soulignant une réalité clinique distincte des classifications académiques.
Ces problématiques sont prises en charge "à l'instar des produits comme le cannabis la cocaïne les extasiies ou l'alcool".
Les dommages incluent des "effets métaboliques ou bien somatiques", ainsi que des "problématiques associées de type psychiatrique ou psychologique anxiété insomnie dépression difficulté l'adaptation relationnelle environnementale".
Classification académique : Sur le plan académique, l'addiction aux écrans ou aux réseaux sociaux n'est pas encore classée comme telle dans les classifications internationales (OMS, Association Américaine de Psychiatrie), à l'exception du jeu pathologique. Cependant, il est probable que cela évoluera à l'avenir.
TikTok, un produit "dépendogène" : TikTok est considéré comme "extrêmement accrocheur addictogène" en raison de son algorithme "extrêmement développé" et de la "fugacité du contenu" (vidéos courtes et répétitives), qui stimulent de manière intense le système de récompense. "Ces deux éléments ne sont pas là par le fait du hasard".
4. Responsabilité des Plateformes et Nécessité de Régulation
Les experts estiment que la responsabilité première du "mésusage" ou "surutilisation" des plateformes incombe aux industriels.
Design non éthique : Le modèle économique de TikTok et autres réseaux sociaux est délibérément conçu pour maximiser le temps passé en ligne, sans considération éthique pour la santé des utilisateurs. "Aucune éthique évidemment n'est convoquée puisque on a des contenus absolument scandaleux".
Manque de bonne foi des plateformes : Les plateformes sont réticentes à mettre en place des contraintes sans obligation légale. Elles mettent en avant "responsabilité liberté" et renvoient la "minorité" vers la "responsabilité de ses parents".
Le Professeur Benjamina déclare : "Il faut pas s'attendre que les plateformes s'exécutent s'il n'y a pas de contrainte parce qu'elles ont les capacités à évidemment mettre en place des choses éthiques".
5. Prise de Conscience Sociétale et Mesures Recommandées
Il y a une prise de conscience sociétale croissante, mais des efforts significatifs sont encore nécessaires.
Écart de connaissance : Il existe un "gouffre entre peut-être des parents ou des professionnels de santé qui sont sensibles au sujet et qui sont bien informés et qui peuvent déjà avoir cette connaissance de l'impact des réseaux sociaux sur la santé des jeunes mais qu'il y a aussi toute une frange de la population aujourd'hui qui ignore totalement ses effets".
Communication massive et ciblée : Une "communication qui soit extrêmement massive sur ce sujet" est nécessaire, ainsi qu'une "communication plus ciblée aussi sur les professionnels de santé" qui n'ont pas toujours le réflexe d'interroger les jeunes sur leur usage des réseaux sociaux.
Formation des soignants : La formation des soignants sur l'usage des écrans est "très hétérogène aujourd'hui" et nécessite une formation "massive" des professions en lien avec la petite enfance et les adolescents.
Alternatives et réinvestissement des espaces physiques : La question "si on nous retire les réseaux sociaux qu'est-ce que vous nous mettez à la place" est comprise.
Les jeunes trouvent dans ces plateformes un "refuge de divertissement". Il est essentiel de "proposer des alternatives aujourd'hui suffisamment puissantes pour les extraire de cette attraction très très forte de l'univers numérique".
L'exemple des terrains vagues aménagés en espaces d'activités physiques montre l'efficacité de "choses qui n'ont pas demandé beaucoup de ni de temps ni d'énergie ni surtout d'argent et qui les ont finalement motivés à faire autre chose".
Le concept de "réseau social éthique" implique un design qui ne vise pas la captation à tout prix, mais le bien-être de l'utilisateur. Cependant, la définition et l'application d'un tel âge limite se heurtent à la complexité de la vérification de l'âge et à la mauvaise foi des plateformes.
L'idée de 15 ans se cale sur la "majorité numérique" et la "majorité sexuelle". La question est posée si cet âge ne devrait pas être plus élevé (18 ans), étant donné la vulnérabilité des adolescents et le fait que le cerveau continue de mûrir jusqu'à 25 ans.
6. La Résistance des Jeunes et le Conflit de Génération
Les jeunes rejettent souvent l'idée de la dangerosité des plateformes, considérant les adultes comme des "boomers" qui "n'ont rien compris".
Les experts insistent sur le fait que "oui on a le choix" de développer des modèles différents et de réguler.
Information insuffisante : "L'information qu'un produit est néfaste pour la santé suffisait à changer les comportements depuis le temps qu'on fait la prévention en santé publique on le saurait". L'information seule ne suffit pas ; une "régulation extrêmement" forte est nécessaire pour les produits addictifs ou "addictif-like".
Temps de la science vs. Évolution technologique : La reconnaissance scientifique des addictions prend du temps (90-100 ans pour le tabac), mais la technologie évolue "toutes les semaines" avec de nouveaux produits, plaçant la science "des années de retard".
7. Approche Clinique et Traitement des Addictions aux Écrans
La prise en charge clinique ne dépend pas d'une classification officielle mais des dommages constatés.
Transfert de dépendance : Le fait d'arrêter une consommation d'écrans n'est pas "à l'origine d'un transfert" vers d'autres substances. La réalité est plutôt celle de "polyconsommateur" et "polyxpérimentateur" où l'offre de drogues est variée.
En conclusion, l'audition met en lumière l'urgence d'une prise de conscience collective et d'une action politique ferme face à l'impact délétère des réseaux sociaux, dont le modèle économique est intrinsèquement problématique pour la santé des jeunes.
La régulation des plateformes, la formation des professionnels de santé et le développement d'alternatives concrètes sont des pistes essentielles pour inverser la tendance.
Document d'information détaillé : "Le pavillon des irresponsables"
Ce document d'information analyse les thèmes principaux, les idées essentielles et les faits marquants des extraits du documentaire "Le pavillon des irresponsables".
Il se concentre sur la vie des patients déclarés pénalement irresponsables et internés en Unités pour Malades Difficiles (UMD), en France.
1. La Catégorie des "Irresponsables Pénaux" : Définition et Contexte
Le documentaire s'ouvre sur la présentation des "irresponsables pénaux", des individus ayant commis des crimes (meurtre, agressions graves) mais échappant à la prison en raison de leur état mental au moment des faits. Ils sont internés dans des UMD.
2. Le Quotidien et les Défis de la Vie en UMD
Le documentaire offre un aperçu du quotidien des patients internés, soulignant les contraintes de l'environnement, mais aussi les efforts pour créer un espace de vie et de soin.
3. Les Histoires Individuelles et la Complexité des Cas
Le documentaire met en lumière des cas spécifiques qui illustrent la nature des maladies mentales et les défis de leur prise en charge à long terme.
4. Les Commissions de Suivi Médical et la Question de la Sortie
Les commissions sont un élément central de la vie en UMD, déterminant le maintien ou le transfert des patients.
Dilemmes de la Sortie :
Conclusion : Une Réflexion sur l'Équilibre
Le documentaire met en lumière la complexité de la prise en charge des patients pénalement irresponsables. Entre la nécessité de protéger la société, de soigner des individus atteints de pathologies lourdes et de préserver leurs droits, les UMD représentent un équilibre délicat.
La question de la durée d'internement, de la conscience de la maladie par les patients, et de leur potentiel de réinsertion, même minime, reste au cœur des débats et des préoccupations des professionnels.
Tour Bình Hưng Nha Trang 1 ngày là một lựa chọn du lịch hấp dẫn do Nha Trang Travel tổ chức, mang đến trải nghiệm nghỉ ngơi và thư giãn trong khung cảnh thiên nhiên tuyệt đẹp của đảo Bình Hưng, hòn đảo được ví như “Maldives Việt Nam” với diện tích nhỏ nhưng sở hữu vẻ đẹp quyến rũ. Trong tour, du khách sẽ được khám phá các địa điểm nổi tiếng như Bãi Kinh/Cây Me, Bãi Đá Trứng, Lăng Ông Nam Hải, đình làng Bình Hưng, Miếu Bà, Đồng Cừu Suối Tiên. Tham gia vào các hoạt động tắm biển, lặn ngắm san hô hấp dẫn. Du khách sẽ được di chuyển bằng cano cao tốc, thưởng thức ẩm thực đặc sản miền biển với thực đơn hải sản phong phú như mực hấp, cá chiên, tôm rim, sò nướng,…Tour du lịch Đảo Bình Hưng hấp dẫn tại Nha Trang TravelTour có lịch trình chi tiết từ 7h30 đến 15h00, bao gồm xe đưa đón, bữa trưa, vé tham quan, cano, xe điện, hướng dẫn viên và bảo hiểm du lịch, với chính sách giá ưu đãi cho trẻ em theo chiều cao và lưu ý về việc tour ghép đoàn, không áp dụng cho khách nước ngoài và ngày lễ Tết, cùng những kinh nghiệm hữu ích để chuyến đi thêm trọn vẹn.
Tour Bình Hưng Nha Trang 1 ngày là một lựa chọn du lịch hấp dẫn do Nha Trang Travel tổ chức, mang đến trải nghiệm nghỉ ngơi và thư giãn trong khung cảnh thiên nhiên tuyệt đẹp của đảo Bình Hưng, hòn đảo được ví như “Maldives Việt Nam” với diện tích nhỏ nhưng sở hữu vẻ đẹp quyến rũ. Trong tour, du khách sẽ được khám phá các địa điểm nổi tiếng như Bãi Kinh/Cây Me, Bãi Đá Trứng, Lăng Ông Nam Hải, đình làng Bình Hưng, Miếu Bà, Đồng Cừu Suối Tiên. Tham gia vào các hoạt động tắm biển, lặn ngắm san hô hấp dẫn. Du khách sẽ được di chuyển bằng cano cao tốc, thưởng thức ẩm thực đặc sản miền biển với thực đơn hải sản phong phú như mực hấp, cá chiên, tôm rim, sò nướng,…

Chào mọi người,Gia đình nhỏ của mình gồm hai vợ chồng và một bé trai 4 tuổi vừa kết thúc chuyến đi Nha Trang. Vấn đề nan giải nhất khi đi du lịch cùng con nhỏ chính là tìm được một hoạt động vừa vui cho con, vừa "nhàn" cho bố mẹ. Sau khi cân nhắc, nhà mình đã quyết định đặt tour Bình Hưng 1 ngày của Nha Trang Travel, và thật sự đây là một quyết định vô cùng đúng đắn.Mình xin chia sẻ lại trải nghiệm chân thực nhất để các gia đình có con nhỏ khác tham khảo nhé!Những Nỗi Lo Của Bố Mẹ Và Lời Giải Đáp Từ Tour1. Vấn đề di chuyển có an toàn và tiện lợi không?Đây là điều mình lo lắng nhất. Nhưng tour đã giải quyết rất tốt: Xe đưa đón tận nơi: Cả nhà không phải vất vả tự lái xe hay tìm đường. Đúng giờ là xe đến đón, bé nhà mình có thể ngủ thêm một giấc trên xe. Cano cao tốc: Mình đã lo con sẽ sợ, nhưng không ngờ bé lại rất phấn khích! Cano chạy êm, an toàn và quan trọng là có đầy đủ áo phao cho cả người lớn và trẻ em. Anh hướng dẫn viên cũng rất tâm lý, luôn để mắt và dặn dò các gia đình có con nhỏ. Xe điện trên đảo: Đây chính là "cứu tinh"! Thay vì phải bế con đi bộ dưới trời nắng, cả nhà được ngồi xe điện tham quan rất thảnh thơi. Bé nhà mình rất thích thú khi được ngồi xe điện vi vu ngắm cảnh. 2. Lịch trình có quá sức với trẻ nhỏ không?Lịch trình được sắp xếp rất hợp lý, không hề dồn dập. Thời gian di chuyển và tham quan xen kẽ, có đủ thời gian nghỉ ngơi nên bé nhà mình không hề bị mệt hay quấy khóc.
Chào mọi người,
Gia đình nhỏ của mình gồm hai vợ chồng và một bé trai 4 tuổi vừa kết thúc chuyến đi Nha Trang. Vấn đề nan giải nhất khi đi du lịch cùng con nhỏ chính là tìm được một hoạt động vừa vui cho con, vừa "nhàn" cho bố mẹ. Sau khi cân nhắc, nhà mình đã quyết định đặt tour Bình Hưng 1 ngày của Nha Trang Travel, và thật sự đây là một quyết định vô cùng đúng đắn.
Mình xin chia sẻ lại trải nghiệm chân thực nhất để các gia đình có con nhỏ khác tham khảo nhé!

Tụi mình là nhóm 6 đứa vừa có chuyến "chạy trốn deadline" ở Nha Trang, và một trong những quyết định đúng đắn nhất của cả đám là đặt tour Bình Hưng 1 ngày của Nha Trang Travel. Đi chơi theo nhóm mà tự túc thì kiểu gì cũng có một đứa phải đứng ra lo hết mọi thứ, nên lần này tụi mình quyết định phó thác cho tour và chỉ việc xách mông đi chơi thôi. Kết quả là một ngày vui quên lối về, nên phải ngoi lên review liền cho nóng!
Aloha cả nhà!
Tụi mình là nhóm 6 đứa vừa có chuyến "chạy trốn deadline" ở Nha Trang, và một trong những quyết định đúng đắn nhất của cả đám là đặt tour Bình Hưng 1 ngày của Nha Trang Travel. Đi chơi theo nhóm mà tự túc thì kiểu gì cũng có một đứa phải đứng ra lo hết mọi thứ, nên lần này tụi mình quyết định phó thác cho tour và chỉ việc xách mông đi chơi thôi. Kết quả là một ngày vui quên lối về, nên phải ngoi lên review liền cho nóng!

Our finding of different expression levels of toxins in different developmental stages and adult tissues strongly suggests that venom composition changes across development and that each arsenal of toxins might have been shaped by selection for different biotic interactions. As Nematostella develops from a non-predatory, swimming larva to an adult sessile predatory polyp that is 150-fold larger than the larva (Figure 1A), its interspecific interactions vastly change across development.
This makes sense because just as humans change and develop across different stages of life, venom composition would do the same
Art. 448
OJ 411 – SDI-1. SUCESSÃO TRABALHISTA. AQUISIÇÃO DE EMPRESA PERTENCENTE A GRUPO ECONÔMICO. RESPONSABILIDADE SOLIDÁRIA DO SUCESSOR POR DÉBITOS TRABALHISTAS DE EMPRESA NÃO ADQUIRIDA. INEXISTÊNCIA. - O sucessor não responde solidariamente por débitos trabalhistas de empresa não adquirida, integrante do mesmo grupo econômico da empresa sucedida, quando, à época, a empresa devedora direta era solvente ou idônea economicamente, ressalvada a hipótese de má-fé ou fraude na sucessão.
Nesses termos, tem-se:
Empresa sucessora que adquire apenas uma empresa de grupo econômico não responde solidariamente pelos débitos trabalhistas das demais empresas do grupo não adquiridas, desde que, no momento da sucessão, a devedora direta fosse solvente ou idônea, salvo se demonstrada fraude ou má-fé.
OJ SDI-1 92. DESMEMBRAMENTO DE MUNICÍPIOS. RESPONSABILIDADE TRABALHISTA Em caso de criação de novo município, por desmembramento, cada uma das novas entidades responsabiliza-se pelos direitos trabalhistas do empregado no período em que figurarem como real empregador.
https://www.greetingstour.com/
classic large letter postcard style

Document d'information détaillé : Stratégies contre la violence scolaire : Prévention et Gestion Source : Extraits de "Stratégies Contre la Violence Scolaire : Prévention et Gestion" (Circulaire n° 2019-122 du 3-9-2019, NOR : MENE1925181C)
Ce document officiel du Ministère de l'Éducation Nationale et de la Jeunesse (MENJ) et de la Direction Générale de l'Enseignement Scolaire (DGESCO) détaille un plan d'action exhaustif visant à prévenir et gérer la violence en milieu scolaire. Il s'adresse aux recteurs d'académie, inspecteurs, directeurs académiques, chefs d'établissement et professeurs.
Thèmes principaux et idées clés :
2. Tolérance Zéro pour les Incivilités et Agressions : Un principe non négociable est établi : "Chaque agression, chaque insulte, chaque incivilité doit être signalée et sanctionnée." Cette exigence est d'autant plus forte si les actes sont dirigés contre un représentant de l'autorité publique. L'institution scolaire doit à la fois prévenir et apporter des réponses concrètes.
Signalement systématique et suivi : Chaque incident doit être signalé, faire l'objet d'un suivi, et être recensé avec la sanction appliquée. Un bilan annuel est présenté au conseil d'administration.
4. Renforcement de la Protection des Personnels : Les personnels scolaires bénéficient du statut juridique de "personne chargée d'une mission de service public", ce qui aggrave la qualification pénale des violences commises à leur encontre.
Réponse institutionnelle systématique : "Toute incivilité, atteinte ou autre fait grave commis à l'encontre d'un personnel de l'éducation nationale doit systématiquement faire l'objet d'une réponse de la part de l'institution."
5. Prise en Charge des Élèves Hautement Perturbateurs et Poly-exclus : Des dispositifs gradués sont envisagés pour les élèves dont les actes de violence sont récurrents.
Équipe mobile mixte d'intervention scolaire : Intervient en "troisième niveau", après l'échec des approches pédagogiques et éducatives et l'intervention d'autres professionnels. Cette équipe associe les familles dans le cadre d'un contrat limité dans le temps.
Le Par vise à "poursuivre un dialogue avec les personnes responsables de l'élève et de les guider, en cas de besoin, vers le service ou le dispositif de soutien le plus approprié."
Il repose sur l'alliance avec les parents, qui s'engagent à faire respecter par l'élève les obligations scolaires et à participer aux mesures d'accompagnement (aide pédagogique, activités éducatives, suivi médico-social).
6. Organisations Adaptées aux Enjeux : Le plan prévoit des structures et des rôles spécifiques pour une mise en œuvre efficace.
Cellule de lutte contre les violences scolaires et référent départemental : Chaque DASEN doit créer un comité de pilotage avec un référent dédié au suivi des questions de violence. Ce comité a pour missions le suivi des faits, l'analyse des décisions disciplinaires, le traitement des affectations après exclusion, l'élaboration de conventions Justice/Éducation nationale, la régulation des saisines pour le PAR, la promotion des initiatives innovantes, l'impulsion de formations, et le renforcement des liens avec les acteurs locaux.
Ils reposent sur les principes d'inclusion, de personnalisation et de responsabilisation, avec un encadrement éducatif renforcé et un projet pédagogique global. Le placement peut être décidé si la classe relais est insuffisante, si l'élève relève d'une mesure de protection de l'enfance et ne peut rester en famille, ou sur décision judiciaire. * Nouveaux partenariats interministériels : Une circulaire interministérielle réactivera les conventions Éducation nationale/Justice et organisera les contrats intercommunaux/locaux de sécurité (CI/LSPD) pour impulser de nouveaux partenariats avec la justice, l'intérieur, l'éducation nationale et l'agriculture. * Annexe 1 : Modifications du Régime Disciplinaire (D. 2019-906 et 2019-908) * * Simplification des procédures : Réduction des délais pour le prononcé des sanctions (3 à 2 jours) et la convocation des conseils de discipline (8 à 5 jours). * Renforcement des réponses : Allongement de la durée de conservation de certaines sanctions dans le dossier administratif de l'élève (blâme et mesure de responsabilisation : fin de l'année scolaire suivante ; exclusions temporaires : fin de la deuxième année scolaire ; exclusion définitive : fin de la scolarité au second degré). Révocation systématique du sursis en cas de nouveau manquement grave. * Mesures d'accompagnement spécifiques : Pour les élèves réintégrés après une exclusion temporaire pour des faits de violence. * Information annuelle du conseil d'administration : Bilan des décisions disciplinaires et des suites données aux demandes de saisine du conseil de discipline. * Renforcement du droit des victimes : Les témoins mineurs sont entendus en présence de leur représentant légal. * Annexe 2 : Modèle type de Protocole d'accompagnement et de responsabilisation des parents (Par)
Cette annexe fournit un modèle détaillé du PAR, incluant :
Objet du protocole : Rappel des obligations parentales et proposition de mesures d'accompagnement.
Des volets spécifiques peuvent être ajoutés (ex: accompagner l'enfant, vérifier le matériel, consulter le carnet de correspondance, participer aux réunions). * Mesures d'accompagnement : Actions pédagogiques (aide, remédiation, tutorat), éducatives (Devoirs faits, stages, activités UNSS), et médico-sociales (rencontres avec l'assistante sociale, l'infirmière, le médecin scolaire). * Suivi du dispositif : Points réguliers avec les parents, suivi par le chef d'établissement qui rend compte au DASEN de l'exécution du protocole. * Durée et modification : Le protocole a une durée déterminée, renouvelable, et peut être modifié par avenant ou résilié en cas de non-respect des engagements.
Synthèse des auditions des responsables de la modération TikTok par la commission d’enquête
1. Structure de la modération de contenu chez TikTok
TikTok emploie une approche hybride pour la modération des contenus, combinant des systèmes automatisés (algorithmes et IA) avec l'intervention humaine.
Nikessou, responsable de la sécurité juridique, a précisé que "98 % des contenus qui violent les conditions" sont supprimés de manière proactive grâce à ces systèmes automatisés.
La modération humaine, qui implique 509 modérateurs francophones, se concentre sur les contenus les plus sensibles ou contextuels. Les contenus signalés ou devenus populaires font l'objet d'examens supplémentaires.
Cependant, il y a une diminution du nombre de modérateurs humains, passant de 634 au premier semestre à 509 au deuxième semestre.
TikTok justifie cette baisse par l'amélioration de ses outils d'IA, permettant une suppression plus rapide et cohérente des contenus problématiques, et minimisant l'exposition des utilisateurs et des employés à ces contenus.
Les modérateurs reçoivent une formation initiale et des vérifications hebdomadaires et mensuelles de leur interprétation des règles. Un soutien psychologique est également mis en place pour les modérateurs exposés à des contenus difficiles.
2. Efficacité de la modération et défis liés aux contenus problématiques
TikTok affirme que "moins de 1 % du contenu ne respecte pas les lignes directrices", mais la commission a remis en question la pertinence de ce pourcentage en raison de l'algorithme de recommandation qui peut amplifier même un faible volume de contenu problématique.
Un exemple majeur de ce défi est la tendance "Skinny Talk", qui a incité à l'anorexie et aux troubles alimentaires. TikTok a expliqué avoir initialement détecté un volume faible et un faible taux de non-conformité.
Ce n'est qu'après l'augmentation du volume et l'émergence d'une communauté centrée sur de mauvaises habitudes alimentaires que des mesures, y compris le blocage du hashtag, ont été prises.
Malgré ces efforts, des contenus problématiques liés à ce thème, comme le hashtag "fearfood", persistent, soulevant des questions sur l'efficacité de la modération et la capacité de TikTok à honorer son "obligation de résultat".
La représentante de TikTok a admis que "bien sûr, nous faisons des erreurs, c'est inévitable".
La commission a également soulevé le problème des "moyens de contournement" utilisés par les jeunes, tels que l'utilisation de symboles (ex: petit zèbre pour la scarification) pour aborder des sujets sensibles sans être détectés.
Les responsables de TikTok reconnaissent cette problématique et affirment travailler à anticiper ces contournements.
3. Gestion des signalements et relations avec les organisations externes
TikTok collabore avec des organisations comme Stop Fisha, e-Enfance, Génération Numérique et Point de Contact, qui agissent comme "signaleurs de confiance". Ces organisations bénéficient d'un canal de signalement prioritaire, assurant une réponse rapide.
Cependant, la commission a fait état de divergences entre les signalements effectués par des particuliers et ceux des organisations, ces dernières entraînant des suppressions de contenu plus fréquentes. TikTok justifie cette différence par l'expertise des signaleurs de confiance, qui aident à identifier plus précisément les violations des règles communautaires.
4. Sanction des comptes et politiques de tolérance
TikTok applique une politique de tolérance variable selon la gravité des infractions.
Les violations mineures peuvent entraîner des avertissements et des opportunités de correction, tandis que les infractions graves comme les discours de haine ou la pédopornographie entraînent une "tolérance zéro" et une interdiction immédiate.
Un document public détaillant cette gradation des sanctions existe et peut être partagé.
La commission a exprimé sa préoccupation quant à la lenteur de réaction face à des comptes d'influenceurs connus, suivis par des millions de personnes, qui diffusent des contenus problématiques (ex: propos sexistes, incitation à la violence). TikTok a souligné la difficulté d'examiner l'intégralité du contenu d'un utilisateur et le caractère contextuel des violations.
5. Modération et fonctionnalités de TikTok Live
TikTok Live est un produit de diffusion en direct où les créateurs interagissent avec leur communauté. L'équipe de modération (TNS) est la même que pour les contenus préenregistrés et agit en toute indépendance.
Des modèles dédiés sont utilisés pour détecter des signaux de violation pendant les diffusions en direct, permettant d'interrompre la vidéo ou de donner des retours aux créateurs. Les menaces à la vie sont signalées aux autorités locales.
Une fonctionnalité notable est le "Live Match", où deux ou quatre créateurs s'affrontent pendant 5 minutes, accumulant des points via des cadeaux virtuels et des "J'aime" du public.
Le vainqueur est celui qui a le plus de points. Les cadeaux virtuels vont d'une "rose virtuelle" valant environ 5 centimes à plusieurs centaines d'euros. TikTok prélève 50 % de la valeur des cadeaux.
Les agences externes spécialisées dans le live streaming sont rémunérées par TikTok et peuvent recevoir des pénalités financières si leurs créateurs enfreignent les règles. Un "score de santé" est attribué aux agences, démarrant à 100 points et diminuant en cas de violation.
La commission a exprimé de vives inquiétudes quant au caractère addictif de TikTok, en particulier des Lives. Les responsables de TikTok ont souligné plusieurs mesures de protection:
Cependant, la commission a confronté TikTok à des témoignages directs de mineurs participant à des Lives et dépensant de l'argent via le compte Apple Pay de leurs parents, ou étant incités à changer leur date de naissance pour accéder à certaines fonctionnalités. La vérification de l'âge reste un défi majeur. TikTok a reconnu la persistance du problème, indiquant que "642 000 comptes" de moins de 13 ans ont été supprimés en France l'année dernière, et 6 millions par mois dans le monde.
La commission a également interrogé le modèle de rémunération des Live, où les "top créateurs" peuvent gagner "plusieurs dizaines de milliers d'euros par mois", et la nature des "Live Match" qui, selon certains membres, s'apparentent à des "mécanismes similaires à ceux des jeux d'argent". TikTok a réfuté cette assimilation, arguant qu'il n'y a pas d'espérance de gain pour les donateurs et que les jeux d'argent sont strictement interdits.
Le remerciement des donateurs par les streamers, même s'il est considéré par TikTok comme de la "politesse", est perçu par la commission comme une "forme de gratification" et d' "encouragement au don".
7. Transparence et obligations légales
TikTok publie des rapports de transparence trimestriels et des rapports dédiés sur les demandes de retrait gouvernementales, les demandes d'information, les suppressions pour propriété intellectuelle, la lutte contre les opérations d'influence et les abus sexuels sur mineurs.
La plateforme est également soumise aux obligations du DSA (Digital Services Act) et du code de pratique de lutte contre la désinformation de l'Union européenne.
Conclusion
Si TikTok a détaillé ses efforts en matière de sécurité et de conformité réglementaire, la commission a exprimé de fortes réserves quant à l'efficacité réelle de ces mesures, en particulier concernant la protection des mineurs et la persistance de contenus problématiques.
Des informations complémentaires ont été demandées à TikTok par écrit, avec la possibilité d'une reconvocation en cas de non-fourniture ou de divergence des réponses.
Compte-Rendu d'Audition des Responsables de TikTok France
Contexte de l'Audition et Objectifs * L'audition vise à examiner le modèle économique, les stratégies de contenu, les mesures de sécurité et de modération de TikTok en France, ainsi que son impact sur les mineurs. Les représentantes de TikTok, * Marlène Masure (Responsable du contenu, Europe, Moyen-Orient, Afrique) et * Marie Hugon (Responsable des enquêtes réglementaires européennes), ont prêté serment de dire la vérité.
L'objectif de la commission est d'obtenir des informations précises et d'éviter les redites avec les auditions précédentes.
I. Activités et Positionnement de TikTok en France et en Europe
1. Portée et Mission de TikTok :
2. Partenariats et Contenus Promus :
II. Engagement Réglementaire et Mesures de Sécurité
1. Conformité au DSA (Digital Services Act) et Transparence :
2. Mesures Spécifiques pour la Protection des Mineurs :
3. Gestion des Données (Projet Clover) :
III. Défis et Points de Tension
1. Contradiction entre Propos et Témoignages :
2. Fonctionnement de l'Algorithme et "Bulles de Contenu" :
3. Efficacité de la Modération et Contournement des Règles :
4. Vérification de l'Âge et Comptes de Mineurs :
5. Différences avec Douyin (version chinoise) :
6. Influenceurs Problématiques et Signalement :
IV. Conclusion et Perspectives
Author response:
The following is the authors’ response to the previous reviews.
Public Reviews:
Reviewer #1 (Public review):
Summary:
Recent work has demonstrated that the hummingbird hawkmoth, Macroglossum stellatarum, like many other flying insects, use ventrolateral optic flow cues for flight control. However, unlike other flying insects, the same stimulus presented in the dorsal visual field, elicits a directional response. Bigge et al., use behavioral flight experiments to set these two pathways in conflict in order to understand whether these two pathways (ventrolateral and dorsal) work together to direct flight and if so, how. The authors characterize the visual environment (the amount of contrast and translational optic flow) of the hawkmoth and find that different regions of the visual field are matched to relevant visual cues in their natural environment and that the integration of the two pathways reflects a prioritization for generating behavior that supports hawkmoth safety rather than the prevalence for a particular visual cue that is more prevalent in the environment.
Strengths:
This study creatively utilizes previous findings that the hawkmoth partitions their visual field as a way to examine parallel processing. The behavioral assay is well-established and the authors take the extra steps to characterize the visual ecology of the hawkmoth habitat to draw exciting conclusions about the hierarchy of each pathway as it contributes to flight control.
Reviewer #2 (Public review):
Summary
Bigge and colleagues use a sophisticated free-flight setup to study visuo-motor responses elicited in different parts of the visual field in the hummingbird hawkmoth. Hawkmoths have been previously shown to rely on translational optic flow information for flight control exclusively in the ventral and lateral parts of their visual field. Dorsally presented patterns, elicit a formerly completely unknown response - instead of using dorsal patterns to maintain straight flight paths, hawkmoths fly, more often, in a direction aligned with the main axis of the pattern presented (Bigge et al, 2021). Here, the authors go further and put ventral/lateral and dorsal visual cues into conflict. They found that the different visuomotor pathways act in parallel, and they identified a 'hierarchy': the avoidance of dorsal patterns had the strongest weight and optic flow-based speed regulation the lowest weight. The authors linked their behavioral results to visual scene statistics in the hawkmoths' natural environment. The partition of ventral and dorsal visuomotor pathways is well in line with differences in visual cue frequencies. The response hierarchy, however, seems to be dominated by dorsal features, that are less frequent, but presumably highly relevant for the animals' flight safety.
Strengths
The data are very interesting and unique. The manuscript provides a thorough analysis of free-flight behavior in a non-model organism that is extremely interesting for comparative reasons (and on its own). These data are both difficult to obtain and very valuable to the field.
Weaknesses
While the present manuscript clearly goes beyond Bigge et al, 2021, the advance could have perhaps been even stronger with a more fine-grained investigation of the visual responses in the dorsal visual field. Do hawkmoths, for example, show optomotor responses to rotational optic flow in the dorsal visual field?
I find the majority of the data, which are also the data supporting the main claims of the paper, compelling. However, the measurements of flight height are less solid than the rest and I think these data should be interpreted more carefully.
Reviewer #3 (Public review):
The authors have significantly improved the paper in revising to make its contributions distinct from their prior paper. They have also responded to my concerns about quantification and parameter dependency of the integration conclusion. While I think there is still more that could be done in this capacity, especially in terms of the temporal statistics and quantification of the conflict responses, they have a made a case for the conclusions as stated. The paper still stands as an important paper with solid evidence a bit limited by these concerns.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
The edits have significantly improved the clarity of the manuscript. A few small notes:
Figure 2B legend - describe what the orange dashed line represents
We added a description.
Figure 2B legend - references Table 1 but I believe this should reference Table S1. There are other places in the manuscript where Table 1 is referenced and it should reference S1
We changed this for all instances in the main paper and supplement, where the reference was wrong.
Figure S1 legend - some figure panel letters are in parentheses while others are not
We unified the notation to not use parentheses for any of the panel letters.
Reviewer #2 (Recommendations for the authors):
I couldn't find the l, r, d, v indications in Fig. 1a. This was just a suggestion, but since you wrote you added them, I was wondering if this is the old figure version.
We added them to what is now Fig. 2, which was originally part of Fig. 1. After restructuring, we did indeed not add an additional set to Fig. 1, which we have now adjusted.
Fig. 2: Adding 'optic flow' and 'edges' to the y-axis in panels E and F, would make it faster for me to parse the figure. Maybe also add the units for the magnitudes? Same for Figure 6B
We added 'optic flow' and 'edges' to the panels E and F in Fig. 2 and Fig. 6.
Fig. 2: Very minor - could you use the same pictograms in D and E&F (i.e. all circles for example, instead of switching to "tunnels" in EF)?
We used the tunnel pictograms, because we associated those with the short notations for the different conditions summarised in Table S1. Because we wanted to keep this consistent across the paper, we used the “tunnel” pictograms here too.
In the manuscript, you still draw lots of conclusions based on these area measurements (L132-142, L204-209 etc). This does not fully reflect what you wrote in your reply to the reviewers. If you think of these measurements as qualitative rather than quantitative, I would say so in the manuscript and not use quantitative statistics etc. My suggestion would be to be more specific about potential issues that can influence the measurement (you mentioned body size, image contrast, motion blur, pitch across conditions etc) and give that data not the same weight as the rest of the measurements.
We do express explicit caution with this measure in the methods section (l. 657-659) and the results section (l. 135-137). Nevertheless, as the trends in the data are consistent with optic flow responses in the other planes, and with responses reported in the literature, we felt that it is valuable to report the data, as well as the statistics for all readers, who can – given out cautionary statement – assess the data accordingly.
The area measurements suggest that moths fly lower with unilateral vertical gratings (Fig. S1, G1 and G2 versus the rest). If you leave the data in can you speculate why that would be? (Sorry if I missed that)
We agree, this seems quite consistent, but we do not have a good explanation for this observation. It would certainly require some additional experiments and variable conditions to understand what causes this phenomenon.
Fig.4 - is panel B somehow flipped? Shouldn't the flight paths start out further away from the grating and then be moved closer to midline (as in A). That plot shows the opposite.
Absolutely right, thank you for spotting this, it was indeed an intermediate and not the final figure which was uploaded to the manuscript. It also had outdated letter-number identifiers, which we now updated.
L198 - should be "they avoided"
Corrected.
Sin embargo, estos resultados no reflejan el comportamiento esperado. Por un lado, seanticipaba que el valor de FR disminuyera sistemáticamente al aumentar la capacidad, y porotro, que todos los valores con capacitor fueran inferiores al caso sin capacitor. Esto noocurrió
Veo que en los gráficos la figura 9 y 10 la señal de salida muestra valores negativos, imagino que midieron solo la componente AC de la señal de salida para tener una mayor resolución. Tenían que haber aclarado eso en el texto, es importante transparentar este tipo de cosas. En este caso, es importante también mostrar la señal con la componente de continua para visualizar mejor el efecto del ripple sobre la señal original.
Particularmente, como, la aproximaciónτ𝑐=10μ𝐹 = (0. 01168 ± 0. 00058)𝑠 ≫ 𝑇 = (0. 001695 ± 0. 000085)𝑠resulta adecuada y parala aproximaciónτ𝑐=100μ𝐹 = (0. 1168 ± 0. 0058) 𝑠 ≫ 𝑇 = (0. 001695 ± 0. 000085) 𝑠resulta aún mejor.
Esto queda un poco desprolijo, mejor escribir esas ecuaciones línea a línea
Si la resolución delinstrumental lo permitiera, se podrían comparar directamente con la expresión generalmediante mediciones más precisas de ΔV y Vmed.12
Esto parece ser más un problema de ruido en el circuito que de resolución de instrumental
Fig. 9
En temas de formato de la figura se aplica todos los comentarios que dije en las figuras anteriores para esta. Pero además en el titulo les quedo "nano", algo bastante desprolijo.
Fig. 8:
en la leyenda de la figura no se pone channel 1 y channel 2, se pone lo que es cada señal
Fig. 4:
Indicar en la leyenda que la barra de error es la barra de no es recomendable. Los títulos en los gráficos tampoco. Hubiese estado bien indicar que la figura tiene dos paneles (a) y (b). Que el gráfico de arriba, que es el más importante, no tenga ticks labels en el eje x no ayuda nada al lector
Compte Rendu Détaillé de l'Audition de la Présidente du Haut Conseil à l'Égalité entre les femmes et les hommes (HCE)
Il met en lumière les priorités du HCE, ses travaux récents et futurs, ainsi que les préoccupations soulevées par les parlementaires.
I. Priorités et Rapports Clés du HCE
La Présidente du HCE a souligné le rôle du Conseil comme « interlocuteur de premier plan pour les pouvoirs publics » et « moteur du débat démocratique sur les droits des femmes et les questions de genre ».
Elle a mis en avant les domaines d'intervention du HCE et les synergies possibles avec les travaux parlementaires.
A. Le Sexisme en France : Un état des lieux alarmant et polarisé
La Présidente se réjouit de l'annonce par la Ministre de l'Éducation nationale du déploiement de ce programme dès la prochaine rentrée, car 9 Français sur 10 y sont favorables, et 70 % le considèrent comme la mesure la plus efficace.
B. La Parentalité : L'« éléphant dans la pièce » des inégalités professionnelles
Il souligne l'importance de le rendre accessible à toutes les catégories sociales, car il est moins pris par les plus précaires.
C. Parité dans l'Encadrement Sportif : Un angle mort à combler
D. Lutte contre les Violences Sexuelles et Sexistes : Améliorer la prise en charge judiciaire
La commission "violences faites aux femmes" du HCE a travaillé sur le traitement judiciaire des viols et agressions sexuelles et rendra ses conclusions et recommandations publiques prochainement, visant une « meilleure prise en charge des victimes ».
II. Discussions et Questions des Parlementaires
Les parlementaires ont salué la qualité des travaux du HCE et ont soulevé des questions spécifiques, souvent en écho aux préoccupations du Conseil.
A. Parité Sportive et Accompagnement Local
B. Pénalité Parentale et Réforme des Congés
C. Diplomatie Féministe et Soutien aux Associations
D. Conséquences Genrées de la COVID et Santé Mentale des Femmes
E. Soutien aux Associations Féministes et Âgisme
F. Contraception Masculine
Elle a reconnu que c'est un sujet à regarder, car la contraception reste « à la charge aujourd'hui des femmes et ressenti comme une contrainte féminine ».
III. Perspectives et Orientations Futures du HCE
La Présidente a réaffirmé sa volonté de redonner au HCE toute sa force dans le débat public et politique.
Elle a pris l'exemple des budgets alloués au sport, souvent déséquilibrés en faveur des pratiques masculines.
En conclusion, la Présidente du HCE a réaffirmé l'engagement du Conseil à éclairer le débat public et à faire avancer l'égalité réelle, en se concentrant sur des sujets structurants comme le sexisme, la parentalité, la parité sportive et la santé des femmes, tout en cherchant à impliquer l'ensemble des acteurs de la société.
Reviewer #1 (Public review):
This study is part of an ongoing effort to clarify the effects of cochlear neural degeneration (CND) on auditory processing in listeners with normal audiograms. This effort is important because ~10% of people who seek help for hearing difficulties have normal audiograms and current hearing healthcare has nothing to offer them.
The authors identify two shortcomings in previous work that they intend to fix. The first is a lack of cross-species studies that make direct comparisons between animal models in which CND can be confirmed and humans for which CND must be inferred indirectly. The second is the low sensitivity of purely perceptual measures to subtle changes in auditory processing. To fix these shortcomings, the authors measure envelope following responses (EFRs) in gerbils and humans using the same sounds, while also performing histological analysis of the gerbil cochleae, and testing speech perception while measuring pupil size in the humans.
The study begins with a comprehensive assessment of the hearing status of the human listeners. The only differences found between the young adult (YA) and middle aged (MA) groups are in thresholds at frequencies > 10 kHz and DPOAE amplitudes at frequencies > 5 kHz. The authors then present the EFR results, first for the humans and then for the gerbils, showing that amplitudes decrease more rapidly with increasing envelope frequency for MA than for YA in both species. The histological analysis of the gerbil cochleae shows that there were, on average, 20% fewer IHC-AN synapses at the 3 kHz place in MA relative to YA, and the number of synapses per IHC was correlated with the EFR amplitude at 1024 Hz.
The study then returns to the humans to report the results of the speech perception tests and pupillometry. The correct understanding of keywords decreased more rapidly with decreasing SNR in MA than in YA, with a noticeable difference at 0 dB, while pupillary slope (a proxy for listening effort) increased more rapidly with decreasing SNR for MA than for YA, with the largest differences at SNRs between 5 and 15 dB. Finally, the authors report that a linear combination of audiometric threshold, EFR amplitude at 1024 Hz, and a few measures of pupillary slope is predictive of speech perception at 0 dB SNR.
I only have two questions/concerns about the specific methodologies used:
(1) Synapse counts were made only at the 3 kHz place on the cochlea. But the EFR sounds were presented at 85 dB SPL, which means that a rather large section of the cochlea will actually be excited. Do we know how much of the EFR actually reflects AN fibers coming from the 3 kHz place? And are we sure that this is the same for gerbils and humans given the differences in cochlear geometry, head size, etc.?
[Note added after revision: the authors have added new data, references, and discussion that have answered my initial questions].
(2) Unless I misunderstood, the predictive power of the final model was not tested on held out data. The standard way to fit and test such model would be to split the data into two segments, one for training and hyperparameter optimization, and one for testing. But it seems that the only spilt was for training and hyperparameter optimization.
[Note added after revision: the authors now make it clear in their response that the modeling tells us how much of the current data can be explained but not necessary about generalization to other datasets.]
While I find the study to be generally well executed, I am left wondering what to make of it all. The purpose of the study with respect to fixing previous methodological shortcomings was clear, but exactly how fixings these shortcomings has allowed us to advance is not. I think we can be more confident than before that EFR amplitude is sensitive to CND, and we now know that measures of listening effort may also be sensitive to CND. But where is this leading us?
I think what this line of work is eventually aiming for is to develop a clinical tool that can be used to infer someone's CND profile. That seems like a worthwhile goal but getting there will require going beyond exploratory association studies. I think we're ready to start being explicit about what properties a CND inference tool would need to be practically useful. I have no idea whether the associations reported in this study are encouraging or not because I have no idea what level of inferential power is ultimately required.
[Note added after revision: the authors have added to the Discussion to put their work into a broader perspective.]
That brings me to my final comment: there is an inappropriate emphasis on statistical significance. The sample size was chosen arbitrarily. What if the sample had been half the size? Then few, if any, of the observed effects would have been significant. What if the sample had been twice the size? Then many more of the observed effects would have been significant (particularly for the pupillometry). I hope that future studies will follow a more principled approach in which relevant effect sizes are pre-specified (ideally as the strength of association that would be practically useful) and sample sizes are determined accordingly.
[Note added after revision: my intention with this comment was not to make a philosophical or nitty-gritty point about statistics. It was more of a follow on to the previous point. Because I don't know what sort of effect size is big enough to matter (for whatever purpose), I don't find the statistical significance (or lack thereof) of the effect size observed to be informative. But I don't think there is anything more that the authors can or should do in this regard.]
So, in summary, I think this study is a valuable but limited advance. The results increase my confidence that non-invasive measures can be used to infer underlying CND, but I am unsure how much closer we are to anything that is practically useful.
Reviewer #2 (Public review):
Summary:
This paper addresses the bottom-up and top-down causes of hearing difficulties in middle-aged adults with clinically-normal audiograms using a cross-species approach (humans vs. gerbils, each with two age groups) mixing behavioral tests and electrophysiology.. The study is not only a follow-up of Parthasarathy et al (eLife 2020), since there are several important differences. Parthasarathy et al. (2020) only considered a group of young normal-hearing individuals with normal audiograms yet with high complaints for hearing in noisy situations. Here, this issue is considered specifically regarding aging, using a between-subject design comparing young NH and older NH individuals recruited from the general population, without additional criterion (i.e. no specifically high problems of hearing in noise). In addition, this is a cross-species approach, with the same physiological EFR measurements with the same stimuli deployed on gerbils.
This article is of very high quality. It is extremely clear, and the results show clearly a decrease of neural phase-locking to high modulation frequencies in both middle-aged humans and gerbils, compared to younger groups/cohorts. In addition, pupillometry measurements conducted during the QuickSIN task suggest increased listening efforts in middle-aged participants, and a statistical model including both EFRs and pupillometry features suggest that both factors contribute to reduced speech-in-noise intelligibility evidenced in middle-aged individuals, beyond their slight differences in audiometric thresholds (although they were clinically normal in both groups).
These provide strong support to the view that normal aging in humans leads to auditory nerve synaptic loss (cochlear neural degeneration - CND- or, put differently, cochlear synaptopathy) as well as increased listening effort, before any clearly visible audiometric deficits as defined in current clinical standards. This result is very important for the community, since we are still missing direct evidence that cochlear synaptopathy might likely underly a significant part of hearing difficulties in complex environments for listeners with normal thresholds, such as middle-aged and senior listeners. This paper shows that these difficulties can be reasonably well accounted for by this sensory disorder (CND), but also that listening effort, i.e. a top-down factor, further contributes to this problem. The methods are sound, well described and I would like to emphasize that they are presented concisely yet in a very precise manner, so that they can be understood very easily - even for a reader that is not familiar with the employed techniques. I believe this study will be of interest to a broad readership. I have some comments and questions which I think would make the paper even stronger once addressed.
Main comments:
(1) Presentation of EFR analyses / Interpretation of EFR differences found in both gerbils and humans
a) Could you comment further on why you think you found a significant difference only at the highest mod. frequency of 1024 Hz in your study? Indeed, previous studies employing SAM or RAM tones very similar to the ones employed here were able to show age effects already at lower modulation freqs. of ~100H; e.g. there are clear age effects reported in human studies of Vasilikov et al. (2021) or Mepani et al. (2021), and also in animals ( see Garrett et al. bioRxiv : https://www.biorxiv.org/content/biorxiv/early/2024/04/30/2020.06.09.142950.full.pdf)
Furthermore, some previous EEG experiments in humans that SAM tones with modulation freqs. of ~100Hz showed that EFRs do not exhibit a single peak, i.e. there are peaks not only at fm but also for the first harmonics (e.g. 2fm or 3fm) see e.g. Garrett et al. bioXiv https://www.biorxiv.org/content/biorxiv/early/2024/04/30/2020.06.09.142950.full.pdf
Did you try to extract EFR strength by looking at the summed amplitude of multiple peaks (Vasilikov Hear Res. 2021), in particular for the lower modulation frequencies? (Indeed, there will be no harmonics for the higher mod. freqs).
b) How the present EFR results relate to FFR results, where effects of age are already at low carrier freqs? (e.g. Märcher-Rørsted et al., Hear. Res., 2022 for pure tones with freq < 500 Hz) Do you think it could be explained by the fact that this is not the same cochlear region, and that synapses die earlier in higher compared to lower CFs. This should be discussed. Beyond the main group effect of age, there were no negative correlations of EFRs with age in your data?
(2) Size of the effects / comparing age effects between two species: Although the size of the age effect on EFRs cannot be directly compared between humans and gerbils - the comparison remains qualitative - could you a least provide references regarding the rate of synaptic loss with aging in both humans and gerbils, so that we understand that the yNH/MA difference can be compared between the two age groups used for gerbils; it would have been critical in case of a non-significant age effect in one species.
Equalization / control of stimuli differences across the two species: For measuring EFRs, SAM stimuli were presented at 85 dB SPL for humans vs. 30 dB above detection threshold (inferred from ABRs) for gerbils - I do not think the results strongly depend on this choice, but it would be good to comment on why you did not choose also to present stimuli 30 dB above thresholds in humans.
Simulations of EFRs using functional models could have been used to understand (at least in humans) how the differences in EFRs obtained between the two groups are quantitatively compatible with the differences in % of remaining synaptic connections known from histopathological studies for their age range (see the approach in Märcher-Rørsted et al., Hear. Res., 2022)
(3) Synergetic effects of CND and listening effort Could you test whether there is an interaction between CNR and listening effort? (e.g. one could hypothesize that MA subjects with largest CND have also the higher listening effort)
Comments on revised version:
The authors did well to address all the points raised in my review. This paper will make an important contribution to our assessment of the sources of age-related auditory processing deficits beyond the cochlea that impair speech intelligibility.
Synthèse des Priorités et Défis de la Haute-Commissaire à l'Enfance
La Haute-Commissaire insiste sur l'importance de l'interministérialité et de la pluridisciplinarité professionnelle comme leviers pour répondre aux défis complexes liés à l'enfance.
1. Mission et Définition de l'Enfance
L'objectif est de "penser autrement ces politiques publiques et donc de coordonner en étant d'une certaine manière le garant que l'enfant était au cœur et du coup il était au centre de ses réflexions".
Il réunit tous les acteurs (associatifs, administrations, éducatifs).
2. Priorités Thématiques et Actions Engagées
La Haute-Commissaire aborde plusieurs chantiers prioritaires, souvent interdépendants :
Service Public de la Petite Enfance (0-3 ans) :
Actions : Travail sur l'attractivité des métiers (VAE inversée, accompagnement des formations), aides aux communes pour la montée en charge des compétences, collaboration avec les fédérations pour la prochaine COG (Convention d'Objectifs et de Gestion), expérimentation de "solutions hybrides" comme les crèches familiales ou scolaires.
Écrans en petite enfance : Publication d'un "référentiel qualité" interdisant les écrans dans les lieux d'accueil des 0-3 ans, posant des règles claires et accompagnant les PMI.
Soutien à la Parentalité :
Adoption et Accès aux Origines : * Adoption : Améliorer les pratiques professionnelles pour accélérer les procédures et la mise en œuvre du "fichier national des familles adoptantes". Diffuser les outils législatifs existants (Loi Limon sur l'adoption simple). * Accueil durable bénévole/Tiers digne de confiance : Étude des différentes hypothèses, en soulignant l'importance de l'accompagnement spécialisé pour les familles adoptantes si elles envisagent un accueil durable, et la nécessité de "remuscler toutes les possibilités" d'accueil.
Recherche des "familles de cœur" pour apporter stabilité et favoriser la désinstitutionnalisation.
Reposer la question compte tenu des nouvelles réalités et de l'évolution législative en Europe.
Lutte contre les Violences Faites aux Enfants :
Poursuite des travaux de prévention, détection des "signaux faibles", et capacité à "mieux accueillir la parole des enfants".
Prise en charge : Déploiement des "Unités d'accueil spécialisées pour les enfants" (UEJ) dans toutes les juridictions, coordination du soin médical, psychologique, social et judiciaire. Renforcement du 119 (campagne d'information, amélioration des canaux dont le chat, traitement des rappels et priorisation).
Loi Votrein : Projet de loi en préparation par la Ministre Votrein proposant des mesures concrètes : autorisation du cumul d'activité pour l'accueil familial, droit au répit, réinterrogation des modalités d'indemnisation (y compris pour l'accueil durable bénévole), reconnaissance du tiers digne de confiance.
Enjeux Numériques et Écrans :
Cela a conduit au départ de certaines plateformes pornographiques ne souhaitant pas utiliser ces outils. Projet de "mini wallet européen".
Suivi des travaux de la Convention Citoyenne sur les temps de l'enfant avec la participation d'enfants.
3. Défis et Critiques Adressées à la Haute-Commissaire
Plusieurs députés expriment des préoccupations majeures, remettant en question l'action et les moyens du Haut-Commissariat :
Elle questionne l'investissement total de la Haute-Commissaire, engagée dans la campagne des municipales.
La Haute-Commissaire évoque le déploiement de "Santé Protégée Péas" et le rôle des coordinateurs.
Elle cite le rapport accablant de la commission d'enquête sur les "manques de pilotage national, rupture de parcours, recours abusif au placement à l'hôtel".
Elle demande des leviers pour garantir la scolarisation effective et la volonté d'intervenir à Mayotte. La Haute-Commissaire prend note des alertes et évoque un travail avec le Ministre des Outre-Mers pour renforcer les équipes et la priorité donnée à ces territoires.
4. Réponse de la Haute-Commissaire aux Critiques
La Haute-Commissaire se défend des accusations de manque d'investissement ou d'action, affirmant être pleinement engagée dans sa mission de coordination. Elle insiste sur :
En conclusion, la Haute-Commissaire à l'Enfance se positionne comme une figure de coordination interministérielle, cherchant à décloisonner les politiques publiques pour une approche centrée sur l'enfant.
Elle met en avant des chantiers concrets sur la petite enfance, la parentalité, l'adoption, la lutte contre les violences et le numérique.
Néanmoins, elle fait face à des critiques virulentes de députés qui soulignent l'urgence d'une crise de l'enfance, le manque de moyens concrets et des défaillances institutionnelles persistantes, notamment dans la protection de l'enfance, remettant en cause l'effectivité de son action.
arready carrt - ja carregar
fueron
La ec 2 no vale para rectificador de media onda. En los rectificadores de media onda la frecuencia es la misma que la frecuencia de la señal, ergo el factor dos de la ec 2, es la unidad.
Tanto para el diodo LED 2 como para el diodo Zener 3 los valores obtenidos del ajuste se asemejan a losvalores esperados de cada uno
Falto aclarar cual es la tensión umbral de los diodos. De hecho en, la figura 3, veo que al de silicio llegan a aplicarle un máximo de ~0.5 cuando es sabido que se abre totalmente a la conducción por arriba de los 0.6 V. Tenían que ir a valores más altos de tensión para ver la curva completa.
eq(3)
La ec 3 es una ecuacion del apendice
Figura 4:
En la leyenda, en vez de mediciones mejor datos. En el ajuste, se ve muy escalonado, lo mejor en este caso es definir un array nuevo para el eje x con una mayor densidad de puntos, para que al graficar la función esta se vea más suave
This method merges an embedding based protein homolog search with a genomic context similarity. This needs a multi-modal LM including aa and DNA seqs. - Genomic context examples: CRISPR/defense islands.
Modalities of protein homolog (sequence similarity) search
1. amino acid sequence based: BLAST, HMMER
2. Embedding based search: using ESM2 embeddings
- Structural search, using AlphaFold structures
- But all of these lack the extra boost provided by adding in genomic context which currently is only done manually!
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
Wang et al. investigated how sexual failure influences sweet taste perception in male Drosophila. The study revealed that courtship failure leads to decreased sweet sensitivity and feeding behavior via dopaminergic signaling. Specifically, the authors identified a group of dopaminergic neurons projecting to the suboesophageal zone that interacts with sweet-sensing Gr5a+ neurons. These dopaminergic neurons positively regulate the sweet sensitivity of Gr5a+ neurons via DopR1 and Dop2R receptors. Sexual failure diminishes the activity of these dopaminergic neurons, leading to reduced sweet-taste sensitivity and sugar-feeding behavior in male flies. These findings highlight the role of dopaminergic neurons in integrating reproductive experiences to modulate appetitive sensory responses.
Previous studies have explored the dopaminergic-to-Gr5a+ neuronal pathways in regulating sugar feeding under hunger conditions. Starvation has been shown to increase dopamine release from a subset of TH-GAL4 labeled neurons, known as TH-VUM, in the suboesophageal zone. This enhanced dopamine release activates dopamine receptors in Gr5a+ neurons, heightening their sensitivity to sugar and promoting sucrose acceptance in flies. Since the function of the dopaminergic-to-Gr5a+ circuit motif has been well established, the primary contribution of Wang et al. is to show that mating failure in male flies can also engage this circuit to modulate sugar-feeding behavior. This contribution is valuable because it highlights the role of dopaminergic neurons in integrating diverse internal state signals to inform behavioral decisions.
An intriguing discrepancy between Wang et al. and earlier studies lies in the involvement of dopamine receptors in Gr5a+ neurons. Prior research has shown that Dop2R and DopEcR, but not DopR1, mediate starvation-induced enhancement of sugar sensitivity in Gr5a+ neurons. In contrast, Wang et al. found that DopR1 and Dop2R, but not DopEcR, are involved in the sexual failure-induced decrease in sugar sensitivity in these neurons. I wish the authors had further explored or discussed this discrepancy, as it is unclear how dopamine release selectively engages different receptors to modulate neuronal sensitivity in a context-dependent manner.
Our immunostaining experiments showed that three dopamine receptors, Dop1R1, Dop2R, and DopEcR were expressed in Gr5a<sup>+</sup> neurons in the proboscis, which was consistent with previous findings by using RT-PCR (Inagaki et al 2012). As the reviewer pointed out, we found that Dop1R1 and Dop2R were required for courtship failure-induced suppression of sugar sensitivity, whereas Marella et al 2012 and Inagaki et al 2012 found that Dop2R and DopEcR were required for starvation-induced enhancement of sugar sensitivity. These results may suggest that different internal states (courtship failure vs. starvation) modulate the peripheral sensory system via different signaling pathways (e.g. different subsets of dopaminergic neurons; different dopamine release mechanisms; and different dopamine receptors). We have discussed these possibilities in the revised manuscript.
The data presented by Wang et al. are solid and effectively support their conclusions. However, certain aspects of their experimental design, data analysis, and interpretation warrant further review, as outlined below.
(1) The authors did not explicitly indicate the feeding status of the flies, but it appears they were not starved. However, the naive and satisfied flies in this study displayed high feeding and PER baselines, similar to those observed in starved flies in other studies. This raises the concern that sexually failed flies may have consumed additional food during the 4.5-hour conditioning period, potentially lowering their baseline hunger levels and subsequently reducing PER responses. This alternative explanation is worth considering, as an earlier study demonstrated that sexually deprived males consumed more alcohol, and both alcohol and food are known rewards for flies. To address this concern, the authors could remove food during the conditioning phase to rule out its influence on the results.
This is an important consideration. To rule out potential confound from food intake during courtship conditioning, we have now also conducted courtship conditioning in vials absent of food. In the absence of any feeding opportunity over the 4.5-hour courtship conditioning period, sexually rejected males still exhibited a robust decrease in sweet taste sensitivity compared with Naïve and Satisfied controls (Figure 1-supplement 1C). These data confirm that the suppression of PER is driven by courtship failure per se, rather than by differences in feeding during the conditioning phase.
(2) Figure 1B reveals that approximately half of the males in the Failed group did not consume sucrose yet Figure 1-S1A suggests that the total volume consumed remained unchanged. Were the flies that did not consume sucrose omitted from the dataset presented in Figure 1-S1A? If so, does this imply that only half of the male flies experience sexual failure, or that sexual failure affects only half of males while the others remain unaffected? The authors should clarify this point.
Our initial description of the experimental setup might be a bit confusing. Here is a brief clarification of our experimental design and we have further clarified the details in the revised manuscript, which should resolve the reviewer’s concerns:
After the behavioral conditioning, male flies were divided for two assays. On the one hand, we quantified PER responses of individual flies. As shown in Figure 1C, Failed males exhibited decreased sweet sensitivity (as demonstrated by the right shift of the dose-response curve). On the other hand, we sought to quantify food consumption of individual flies by using the MAFE assay (Qi et al 2005).
In the initial submission, we used 400 mM sucrose for the MAFE assay. When presented with 400 mM sucrose, approximately 100% of the flies in the Naïve and Satisfied groups, and 50% of the flies in the Failed group, extended their proboscis and started feeding, as a natural consequence of decreased sugar sensitivity (Figure 1B). We were able to quantify the actual volume of food consumed of these flies showing PER responses towards 400 mM sucrose and observed no change (Figure 1-supplement 1A, left). To avoid potential confusion, we have now repeated the MAFE assay with 800 mM sucrose, which elicited feeding in ~100% of flies among all three groups, as shown in Figure 1C. Again, we observed no change in food intake (Figure 1-supplement 1A, right).
These experiments in combination suggest that sexual failure suppresses sweet sensitivity of the Failed males. Meanwhile, as long as they still responded to a certain food stimulus and initiated feeding, the volume of food consumption remained unchanged. These results led us to focus on the modulatory effect of sexual failure on the sensory system, the main topic of this present study.
(3) The evidence linking TH-GAL4 labeled dopaminergic neurons to reduced sugar sensitivity in Gr5a+ neurons in sexually failed males could be further strengthened. Ideally, the authors would have activated TH-GAL4 neurons and observed whether this restored GCaMP responses in Gr5a+ neurons in sexually failed males. Instead, the authors performed a less direct experiment, shown in Figures 3-S1C and D. The manuscript does not describe the condition of the flies used in this experiment, but it appears that they were not sexually conditioned. I have two concerns with this experiment. First, no statistical analysis was provided to support the enhancement of sucrose responses following activation of TH-GAL4 neurons. Second, without performing this experiment in sexually failed males, the authors lack direct evidence to confirm that the dampened response of Gr5a+ neurons to sucrose results from decreased activity in TH-GAL4 neurons.
We have now quantified the effect of TH<sup>+</sup> neuron activation on Gr5a<sup>+</sup> neuron calcium responses. in Naïve males, dTRPA1-mediated activation of TH<sup>+</sup> cells significantly enhanced sucrose-induced calcium responses (Figure 3-supplement 1C); while in Failed males, the baseline activity of Gr5a<sup>+</sup> neurons was lower (Figure 3C), the same activation also produced significant (even slightly larger) effect on the calcium responses of Gr5a<sup>+</sup> neurons (Figure 3-supplement 1D).
Taken together, we would argue that these experiments using both Naïve and Failed males were adequate to show a functional link between TH<sup>+</sup> neurons and Gr5a<sup>+</sup> neurons. Combining with the results that these neurons form active synapses (Figure 3-supplement 1B) and that the activity of TH<sup>+</sup> neurons was dampened in sexually failed males (Figure 3G-I), our data support the notion that sexual failure suppresses sweet sensitivity via TH-Gr5a circuitry.
(4) The statistical methods used in this study are poorly described, making it unclear which method was used for each experiment. I suggest that the authors include a clear description of the statistical methods used for each experiment in the figure legends. Furthermore, as I have pointed out, there is a lack of statistical comparisons in Figures 3-S1C and D, a similar problem exists for Figures 6E and F.
We have added detailed information of statistical analysis in each figure legend.
(5) The experiments in Figure 5 lack specificity. The target neurons in this study are Gr5a+ neurons, which are directly involved in sugar sensing. However, the authors used the less specific Dop1R1- and Dop2R-GAL4 lines for their manipulations. Using Gr5a-GAL4 to specifically target Gr5a+ neurons would provide greater precision and ensure that the observed effects are directly attributable to the modulation of Gr5a+ neurons, rather than being influenced by potential off-target effects from other neuronal populations expressing these dopamine receptors.
We agree with the reviewer that manipulating Dop1R1 and Dop2R genes (Figure 4) and the neurons expressing them (Figure 5) might have broader impacts. For specificity, we have also tested the role of Dop1R1 and Dop2R in Gr5a<sup>+</sup> neurons by RNAi experiments (Figure 6). As shown by both behavioral and calcium imaging experiments, knocking down Dop1R1 and Dop2R in Gr5a<sup>+</sup> neurons both eliminated the effect of sexual failure to dampen sweet sensitivity, further confirming the role of these two receptors in Gr5a<sup>+</sup> neurons.
(6) I found the results presented in Fig. 6F puzzling. The knockdown of Dop2R in Gr5a+ neurons would be expected to decrease sucrose responses in naive and satisfied flies, given the role of Dop2R in enhancing sweet sensitivity. However, the figure shows an apparent increase in responses across all three groups, which contradicts this expectation. The authors may want to provide an explanation for this unexpected result.
We agree that there might be some potential discrepancies. We have now addressed the issues by re-conducting these calcium imaging experiments again with a head-to-head comparison with the controls (Gr5a-GCaMP, +/- Dop1R1 and Dop2R RNAi).
In these new experiments, Dop1R1 or Dop2R knockdown completely prevented the suppression of Gr5a<sup>+</sup> neuron responsiveness by courtship failure (Figure 6E), whereas the activities of Gr5a<sup>+</sup> neurons in Naïve/Satisfied groups were not altered. These results demonstrate that Dop1R1 and Dop2R are specifically required to mediate the decrease in sweet sensitivity following courtship failure.
(7) In several instances in the manuscript, the authors described the effects of silencing dopamine signaling pathways or knocking down dopamine receptors in Gr5a neurons with phrases such as 'no longer exhibited reduced sweet sensitivity' (e.g., L269 and L288), 'prevent the reduction of sweet sensitivity' (e.g., L292), or 'this suppression was reversed' (e.g. L299). I found these descriptions misleading, as they suggest that sweet sensitivity in naive and satisfied groups remains normal while the reduction in failed flies is specifically prevented or reversed. However, this is not the case. The data indicate that these manipulations result in an overall decrease in sweet sensitivity across all groups, such that a further reduction in failed flies is not observed. I recommend revising these descriptions to accurately reflect the observed phenotypes and avoid any confusion regarding the effects of these manipulations.
We have changed the wording in the revised manuscript. In brief, we think that these manipulations have two consequences: suppressing the overall sweet sensitivity, and eliminating the effect of sexual failure on sweet sensitivity.
Reviewer #2 (Public review):
Summary:
The authors exposed naïve male flies to different groups of females, either mated or virgin. Male flies can successfully copulate with virgin females; however, they are rejected by mated females. This rejection reduces sugar preference and sensitivity in males. Investigating the underlying neural circuits, the authors show that dopamine signaling onto GR5a sensory neurons is required for reduced sugar preference. GR5a sensory neurons respond less to sugar exposure when they lack dopamine receptors.
Strengths:
The findings add another strong phenotype to the existing dataset about brain-wide neuromodulatory effects of mating. The authors use several state-of-the-art methods, such as activity-dependent GRASP to decipher the underlying neural circuitry. They further perform rigorous behavioral tests and provide convincing evidence for the local labellar circuit.
Weaknesses:
The authors focus on the circuit connection between dopamine and gustatory sensory neurons in the male SEZ. Therefore, it is still unknown how mating modulates dopamine signaling and what possible implications on other behaviors might result from a reduced sugar preference.
We agree with the reviewer that in the current study, we did not examine the exact mechanism of how mating experience suppressed the activity of dopaminergic neurons in the SEZ. The current study mainly focused on the behavioral characterization (sexual failure suppresses sweet sensitivity) and the downstream mechanism (TH-Gr5a pathway). We think that examining the upstream modulatory mechanism may be more suitable for a separate future study.
We believe that a sustained reduction in sweet sensitivity (not limited to sucrose but extend to other sweet compounds Figure 1-supplement 1D-E) upon courtship failure suggests a generalized and sustained consequence on reward-related behaviors. Sexual failure may thus resemble a state of “primitive emotion” in fruit flies. We have further discussed this possibility in the revised manuscript.
Reviewer #3 (Public review):
Summary
In this work, the authors asked how mating experience impacts reward perception and processing. For this, they employ fruit flies as a model, with a combination of behavioral, immunostaining, and live calcium imaging approaches.
Their study allowed them to demonstrate that courtship failure decreases the fraction of flies motivated to eat sweet compounds, revealing a link between reproductive stress and reward-related behaviors. This effect is mediated by a small group of dopaminergic neurons projecting to the SEZ. After courtship failure, these dopaminergic neurons exhibit reduced activity, leading to decreased Gr5a+ neuron activity via Dop1R1 and Dop2R signaling, and leading to reduced sweet sensitivity. The authors therefore showed how mating failure influences broader behavioral outputs through suppression of the dopamine-mediated reward system and underscores the interactions between reproductive and reward pathways.
Concern
My main concern regarding this study lies in the way the authors chose to present their results. If I understood correctly, they provided evidence that mating failure induces a decrease in the fraction of flies exhibiting PER. However, they also showed that food consumption was not affected (Fig. 1, supplement), suggesting that individuals who did eat consumed more. This raises questions about the analysis and interpretation of the results. Should we consider the group as a whole, with a reduced sensitivity to sweetness, or should we focus on individuals, with each one eating more? I am also concerned about how this could influence the results obtained using live imaging approaches, as the flies being imaged might or might not have been motivated to eat during the feeding assays. I would like the authors to clarify their choice of analysis and discuss this critical point, as the interpretation of the results could potentially be the opposite of what is presented in the manuscript.
Please refer to our responses to the Public Review (Reviewer 1, Point 2) for details.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
(1) The label for the y-axis in Figure 1B should be "fraction", not "percentage".
We have revised the figure as suggested.
(2) I suggest that the authors indicate the ROIs they used to quantify the signal intensity in Figure 3E and G.
We have revised the figures as suggested.
(3) There is a typo in Figure 4A: it should be "Wilde type", not "Wide type".
We have revised the figure as suggested.
(4) The elav-GAL4/+ data in Figure 4-S1B, C, and D appears to be reused across these panels. However, the number of asterisks indicating significance in the MAT plots differs between them (three in panels B and C, and four in panel D). Is this a typo?
It is indeed a typo, and we have revised the figure accordingly.
Reviewer #2 (Recommendations for the authors):
Additional comments:
The authors should add this missing literature about dopamine and neuromodulation in courtship:
Boehm et al., 2022 (eLife) - this study shows that mating affects olfactory behavior in females.
Cazalé-Debat et al., 2024 (Nature) - Mating proximity blinds threat perception.
Gautham et al., 2024 (Nature) - A dopamine-gated learning circuit underpins reproductive state-dependent odor preference in Drosophila females.
We have added these references in the introduction section.
Has the mating behavior been quantified? How often did males copulate with mated and virgin females?
We tried to examine the copulation behavior based on our video recordings. In the “Failed” group (males paired with mated females), we observed virtually no successful copulation events at all, confirming that nearly 100% of those males experienced sexual failure. In contrast, males in the “Satisfied” group (paired with virgin females) mated on average 2-3 times during the 4.5-hour conditioning period. We have added some explanations in the manuscript.
Do the rejected males live shorter? Is the effect also visible when they are fed with normal fly food, or is it only working with sugar?
We did not directly measure the lifespan of these males. But we conducted a relevant assay (starvation resistance), in which “Failed” males died significantly faster than both Naïve and Satisfied controls, indicating a clear reduction in their ability to endure food deprivation (Figure 1-supplement 1B). Since sweet taste is a primary cue for food detection in Drosophila, and sugar makes up a large portion of their standard diet, the drop in sugar sensitivity we observed in Failed males could likewise impair their perception and consumption of regular fly food, hence their resistance to starvation.
Also, the authors mention that the reward pathway is affected, this is probably the case as sugar sensation is impaired. One interesting experiment would be (and maybe has been done?) to test rejected males in normal odor-fructose conditioning. The data would suggest that they would do worse.
We have already measured how courtship failure affected fructose sensitivity (Figure 1 supplement 1D), and we found that the reduction in fructose perception was even more profound than for sucrose. We have not yet tested whether Failed males showed deficits in odor-fructose associative conditioning. That was indeed a very interesting direction to explore. But olfactory reward learning relies on molecular and circuit mechanisms distinct from those governing taste. We therefore argue such experiments would be more suitable in a separate, follow up study.
The authors could have added another group where males are exposed to other males. It would be interesting if this is also a "stressful" context and if it would also reduce sugar preference - probably beyond the scope of this paper.
In our experiments, all flies, including those in the Naïve, Failed, and Satisfied groups, were housed in groups of 25 males per vial before the conditioning period (and the Naïve group remained in the same group housing until PER testing). This means every cohort experienced the same level of “social stress” from male-male interactions. While it would indeed be interesting to compare that to solitary housing or other male-only exposures, isolation itself imposes a different kind of stress, and disentangling these effects on sugar preference would require a separate, dedicated study beyond the scope of the present work.
Would the behavior effect also show up with experienced males? Maybe this has been tested before. Does mating rejection in formerly successful males have the same impact?
As suggested by the reviewer, we performed an additional experiment in which males that had previously mated successfully were subsequently subjected to courtship rejection. As shown in Figure 1 supplement 1F, prior successful mating did not prevent the decline in sweet sensitivity induced by subsequent mating failure, indicating that even experienced males exhibit the reduction in sugar sensitivity after rejection.
Is the same circuit present and functioning in females? Does manipulating dopamine receptors in GR5a neurons in females lead to the same phenotype? This would suggest that different internal states in males and females could lead to the same phenotype and circuit modulations.
This is indeed a very interesting suggestion. In male flies, Gr5a-specific knockdown of dopamine receptors did not alter baseline sweet sensitivity, but it selectively prevented the reduction in sugar perception that followed mating failure (Figure 6C-D), indicating that this dopaminergic pathway is engaged only in the context of courtship rejection. By extension, knocking down the same receptors in female GR5a neurons would likewise be expected to leave their basal sugar sensitivity unchanged. Moreover, because there is currently no established paradigm for inducing mating failure in female flies, we cannot yet test whether sexual rejection similarly modulates sweet taste in females, or whether it operates via the same circuit.
Reviewer #3 (Recommendations for the authors):
Suggestions to the authors:
Introduction, line 61. I suggest the authors add references in fruit flies concerning the rewarding nature of mating. For example, the paper from Zhang et al, 2016 "Dopaminergic Circuitry Underlying Mating Drive" demonstrates the role of the dopamine rewarding system in mating drive. There is a large body of literature showing the link between dopamine and mating.
We have added this literature in the introduction section.
Figure 1B and Figure Supplement 1: If I understood correctly, Figure Supplement 1A shows that the total food consumption across all tested flies remains unchanged. However, fewer flies that failed to mate consumed sucrose. I would be curious to see the results for sucrose consumption per individual fly that did eat. According to their results, individual flies that failed to mate should consume more sucrose. This would change the conclusion. The authors currently show that a group of flies that failed to mate consumed less sucrose overall, but since fewer males actually ate, those that failed to mate and did eat consumed more sucrose. The authors should distinguish between failed and satisfied flies in two groups: those that ate and those that did not.
Please see our responses to the Public Review for details (Reviewer 1, Point 2).
Figure 1C, right: For a better understanding of all the "MAT" figures, I suggest the authors start the Y axis with the unit 25 and increase it to 400. This would match better the text (line 114) saying that it was significantly elevated in the failed group. As it is, we have the impression of a decrease in the graph.
We have revised the figures accordingly.
Line 103: When suggesting a reduced likelihood of meal initiation of these males, do these males take longer to eat when they did it? In other words, is the latency to eat increased in failed males? That would be a good measure of motivational state.
We tried to analyze feeding latency in the MAFE assay by measuring the time from sucrose presentation to the first proboscis extension, but it was too short to be accurately accounted. Nevertheless, when conducting the experiments, we did not feel/observe any significant difference in the feeding latency between Failed males and Naïve or Satisfied controls.
Line 117. I don't understand which results the authors refer to when writing "an overall elevation in the threshold to initiate feeding upon appetitive cues". Please specify.
This phrase refers to the fact that for every sweet tastant we tested, including sucrose (Figure 1C), fructose and glucose (Figure 1 supplement 1D-E), the concentration-response curve in Failed males shifted to the right, and the Mean Acceptance Threshold (MAT) was significantly higher. In other words, for these different appetitive cues, mating failure raised the concentration of sugar required to trigger a proboscis extension, indicating a general elevation in the threshold to initiate feeding upon an appetitive cue.
Figure 1D. Please specify the time for the satisfied group.
For clarity, the Naïve and Satisfied groups in Figure 1D each represent pooled data from 0 to 72 hours post-treatment, as their sweet sensitivity remained stable throughout this period. Only the Failed group was shown with time-resolved data, since it was the only group exhibiting a dynamic change in sugar sensitivity over time. We have now specified this in the figure legend.
Figure 1F. The phenotype was not totally reversed in failed-re-copulated males. Could it be due to the timing between failure and re-copulation? I suggest the authors mention in the figure or in the text, the time interval between failure and re-copulation.
We’d like to clarify that the interval between the initial treatment (“Failed”) and the opportunity for re copulation was within 30 minutes. The incomplete reversal in the Failed-re-copulated group indeed raised interesting questions. One possible explanation is that mating failure reduces synaptic transmissions between the SEZ dopaminergic neurons and Gr5a<sup>+</sup> sweet sensory neurons (Figure 3), and the regeneration of these transmissions takes a longer time. We have added this information to the figure legend and the Method section.
Line 227-228 and Figure 3E. The authors showed that the synaptic connections between dopaminergic neurons and Gr5a+ GRNs were significantly weakened. I am wondering about the delay between mating failure and the GFP observation. It would be informative to know this timing to interpret this decrease in synaptic connections. If the timing is relatively long, it is possible that we can observe a neuronal plasticity. However, if this timing is very short, I would not expect such synaptic plasticity.
The interval between the behavioral treatment and the GRASP-GFP experiment was approximately 20 hours. We chose this time window because it was sufficient for both GFP expression and accumulation. Therefore, the observed reduction in synaptic connections between dopaminergic neurons and Gr5a<sup>+</sup> GRNs likely reflects a genuine, experience-induced structural and functional change rather than an immediate, transient effect. We have added this information to the revised manuscript for clarity in the Method section.
Line 240-243: The authors demonstrated that there is a reduction of CaLexA-mediated GFP signals in dopaminergic neurons in the SEZ after mating failure, but not a reduction in Gr5a+ GRNs. I suggest replacing "indicate" with "suggest' in line 240.
We have made the change accordingly. Meanwhile, we would like to clarify that while we observed a reduction of NFAT signal in SEZ dopaminergic neurons (Figure 3G), we did not directly test NFAT signal in Gr5a<sup>+</sup> neurons. Notably, the results that the synaptic transmissions from SEZ dopaminergic neurons to Gr5a<sup>+</sup> neurons were weakened (Figure 3E-F), and the reduction of NFAT signal in SEZ dopaminergic neurons (Figure 3G-I), were in line with a reduction in sweet sensitivity of Gr5a<sup>+</sup> neurons upon courtship failure (Figure 3B-D).
Line 243: replace "consecutive" with "constitutive".
We have revised it accordingly.
Figure 5: I have trouble understanding the results obtained in Figure 5. Both constitutive activation and inhibition of Dop1R1 and Dop2R neurons lead to the same results, knowing that males who failed mating no longer exhibit decreased sweet sensitivity. I would have expected contrary results for both experimental conditions. I suggest the author to discuss their results.
Both activation and inhibition of Dop1R1 and Dop2R neurons eliminated the effect of courtship failure on sweet sensitivity (Figure 5). These results are in line with our hypothesis that courtship failure leads to changes in dopamine signaling and hence sweet sensitivity. If dopamine signaling via Dop1R1 and Dop2R was locked, either to a silenced or a constitutively activated state, the effect of courtship failure on sweet sensitivity was eliminated.
Nevertheless, as the reviewer pointed out, constitutive activation/inhibition should in principle lead to the opposite effect on Naïve flies. In fact, when Dop1R1<sup>+</sup>/Dop2R<sup>+</sup> neurons were silenced in Naïve flies, PER to sucrose was significantly reduced (Figure 5C-D), confirming that these neurons normally facilitate sweet sensation. Meanwhile, while neuronal activation by NaChBac did show a trend towards enhanced PER compared to the GAL4/+ controls, it did not exhibit a difference compared to +>UAS-NaChBac controls that showed a high PER level, likely due to a potential ceiling effect. We have added the discussions to the manuscript.
Figure 7: I suggest the authors modify their figure a bit. It is not clear why in failed mating, the red arrow in "behavioral modulation" goes to the fly. The authors should find another way to show that mating failure decreased the percentage of flies that are motivated to eat sugar.
We have modified the figure as suggested.
Overall, I would suggest the authors be precautious with their conclusion. For example, line 337= "sexual failure suppressed feeding behavior". This is not what is shown by this study. Here, the study shows that mating failure decreases the fraction of flies to eat sucrose. Unless the authors demonstrate that this decrease is generalizable to other metabolites, I suggest the authors modify their conclusion.
While we primarily used sucrose as the stimulant in our experiments, we also tested responses to two other sugars: fructose and glucose (Figure 1 supplement 1D-E). In all three cases, mating failure led to a significant reduction in sweet perception, suggesting that the effect of courtship failure is not limited to a single metabolite but rather reflects a general decrease in sweet sensitivity. Meanwhile, reduced sweet sensitivity indeed led to a reduction of feeding initiation (Figure 1).
Compte rendu détaillé : Rapport sur l'accompagnement à la parentalité
Ce document présente les principales thématiques, idées clés et faits importants tirés des extraits de la présentation du rapport sur l'accompagnement à la parentalité.
Thèmes principaux
Idées et faits importants
Elle est elle en est même le principal facteur explicatif bien devant les discriminations." * 90% des inégalités de revenus entre femmes et hommes sont liées à la parentalité. * Les femmes voient leurs revenus baisser de 38% au cours des 10 premières années de l'enfant. * "Plus de 6 femmes sur 10 estiment qu'être mère est un frein à la carrière alors qu'être père ne l'est pas." * Bérangère Couillard, Présidente du HCE, a qualifié la parentalité d'"éléphant dans la pièce" concernant l'égalité professionnelle. * Impact sur la santé des femmes : "Vous évoquez à juste titre les dépressions post-partum, l'isolement, l'épuisement parental, ce sont des réalités que nous ne pouvons plus ignorer." * La dépression post-partum touche 10 à 20% des mères et est la première cause de mortalité maternelle dans l'année suivant la naissance. * La "double injonction permanente": "Travailler comme si nous n'avions pas d'enfants, élever nos enfants comme si nous ne travaillions pas" est intenable et mène au burnout parental, qui touche davantage les femmes.
Répartition inégale des tâches :
Le congé paternité comme levier clé : * La durée actuelle du congé maternité est de 16 semaines, contre 28 jours pour le congé paternité, ce qui "peut consolider la dynamique d'une mère parent principal et d'un père auxiliaire". * Le rapport propose de porter progressivement le congé paternité à 16 semaines, à égalité avec le congé maternité, en s'inspirant du modèle espagnol. * En Espagne, le congé paternité a été porté à 16 semaines entre 2019 et 2021, avec un succès unanime et une augmentation systématique du taux de recours à chaque allongement. Là-bas, on parle de "congé de naissance et de soins de l'enfant". * La proposition inclut 8 semaines obligatoires dès le début (4 semaines à la naissance avec la mère, 4 semaines obligatoires après le congé maternité pour que le second parent puisse passer du temps seul avec l'enfant) et 8 semaines facultatives progressives. * Le coût de porter le congé paternité à 8 semaines obligatoires serait d'environ 820 millions d'euros (correction de la coquille "820 milliards"). Le coût maximum pour 16 semaines est estimé à 2,3 milliards. * L'allongement du congé paternité est vu comme un levier pour "lutter contre les stéréotypes de genre" et "inverser la dynamique parent principal-parent auxiliaire".
Propositions clés pour une parentalité égalitaire :
Accorder aux parents des autorisations d'absence (4 demi-journées/an) pour participer aux moments clés de la scolarité de leurs enfants (ex: réunions parents-professeurs). Réfléchir à l'organisation du travail (horaires atypiques, réunions avant 9h ou après 18h). * Accompagnement des pères : Associer les pères au projet parental dès le désir d'enfant, consacrer une séance de préparation à la naissance au projet parental en présence du père, permettre aux pères d'assister à tous les rendez-vous médicaux si la mère le souhaite. * Interruption de grossesse : En cas d'IVG, IMG ou fausse couche avant 22 semaines d'aménorrhée, la femme devrait bénéficier d'un arrêt maladie et le conjoint pourrait demander jusqu'à 3 jours d'autorisation d'absence. * Familles monoparentales (mères solos) : Défiscaliser la pension alimentaire. Étudier la création d'un statut des familles monoparentales avec des droits associés. Ouvrir les allocations logement (APL) aux deux parents. Possibilité offerte aux mères isolées de transférer une partie de leur "congé de paternité ou d'accueil et de soins de l'enfant" à une personne de confiance de leur choix. * Accompagnement des parents d'adolescents : Élargir les lieux d'accueil parents-enfants aux adolescents, renforcer l'offre en pédopsychiatrie, travailler avec la médecine scolaire, lancer une campagne nationale sur la santé mentale des adolescents. * Coût et bénéfices : La mesure d'allongement du congé paternité, bien que coûteuse (estimée à 820 millions d'euros pour 8 semaines obligatoires et jusqu'à 2,3 milliards d'euros pour 16 semaines), est jugée "pleine de bon sens" et susceptible de générer de "nombreuses externalités positives" (meilleure participation des femmes au marché du travail, baisse du temps partiel subi, augmentation des cotisations sociales, baisse du recours au congé parental, encouragement à la natalité).
Réflexions sur le congé maternité et le temps de travail :
La difficulté de ces familles à voir leur rôle de parent reconnu avant l'adoption ou la naissance a été soulignée, d'où l'insistance sur la reconnaissance de la parentalité "dès le projet parental".
La restricción del ejercicio físico vigoroso protege de la disección aórtica.
NO EJERCICIO
Los antagonistas de los conductos de calcio, una vez utilizados como sustituto del β-bloqueador, son perjudiciales para la aorta.
NO DAR ANTAGONISTAS DE CALCIO
Los β bloqueadores adrenérgicos administrados de forma crónico, ajustados a la tolerancia individual pero lo suficiente para producir un efecto inotrópico negativo (atenolol, 1 a 2 mg/kg por VO diarios)
Tratamiento.
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Nassim - 24/06 - Il est pas intéréssé pour le moment, peut l'être dans un an
Nassim - 24/06 - Peut être intéréssé, il m'a demandé de voir ça par mail
Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
Summary:
In their comprehensive analysis Diallo et al. deorphanise the first olfactory receptor of a nonhymenopteran eusocial insect - a termite and identified the well-established trail pheromone neocembrene as the receptor's best ligand. By using a large set of odorants the authors convincingly show that, as expected for a pheromone receptor, PsimOR14 is very narrowly tuned. While the authors first make use of an ectopic expression system, the empty neuron of Drosophila melanogaster, to characterise the receptor's responses, they next perform single sensillum recordings with different sensilla types on the termite antenna. By that, they are able to identify a sensillum that houses three neurons, of which the B neuron exhibits the narrow responses described for PsimOR14. Hence the authors do not only identify the first pheromone receptor in a termite but can even localize its expression on the antenna. The authors in addition perform a structural analysis to explain the binding properties of the receptor and its major and minor ligands (as this is beyond my expertise, I cannot judge this part of the manuscript). Finally, they compare expression patterns of ORs in different castes and find that PsimOR14 is more strongly expressed in workers than in soldier termites, which corresponds well with stronger antennal responses in the worker caste.
Strengths:
The manuscript is well-written and a pleasure to read. The figures are beautiful and clear. I actually had a hard time coming up with suggestions.
We thank the reviewer for the positive comments.
Weaknesses:
Whenever it comes to the deorphanization of a receptor and its potential role in behaviour (in the case of the manuscript it would be trail-following of the termite) one thinks immediately of knocking out the receptor to check whether it is necessary for the behaviour. However, I definitely do not want to ask for this (especially as the establishment of CRISPR Cas-9 in eusocial insects usually turns out to be a nightmare). I also do not know either, whether knockdowns via RNAi have been established in termites, but maybe the authors could consider some speculation on this in the discussion.
We agree that a functional proof of the PsimOR14 function using reverse genetics would be a valuable addition to the study to firmly establish its role in trail pheromone sensing. Nevertheless, such a functional proof is difficult to obtain. Due to the very slow ontogenetic development inherent to termites (several months from an egg to the worker stage) the CRISPR Cas-9 is not a useful technique for this taxon. By contrast, termites are quite responsive to RNAimediated silencing and RNAi has previously been used for the silencing of the ORCo co-receptor in termites resulting in impairment of the trail-following behavior (DOI: 10.1093/jee/toaa248). Likewise, our previous experiments showed a decreased ORCo transcript abundance, lower sensitivity to neocembrene and reduced neocembrene trail following upon dsPsimORCo administration to P. simplex workers, while we did not succeed in reducing the transcript abundance of PsimOR14 upon dsPsimOR14 injection. We do not report these negative results in the present manuscript so as not to dilute the main message. In parallel, we are currently developing an alternative way of dsRNA delivery using nanoparticle coating, which may improve the RNAi experiments with ORs in termites.
Reviewer #2 (Public review):
Summary:
In this manuscript, the authors performed the functional analysis of odorant receptors (ORs) of the termite Prorhinotermes simplex to identify the receptor of trail-following pheromone. The authors performed single-sensillum recording (SSR) using the transgenic Drosophila flies expressing a candidate of the pheromone receptor and revealed that PsimOR14 strongly responds to neocembrene, the major component of the pheromone. Also, the authors found that one sensillum type (S I) detects neocembrene and also performed SSR for S I in wild termite workers. Furthermore, the authors revealed the gene, transcript, and protein structures of PsimOR14, predicted the 3D model and ligand docking of PsimOR14, and demonstrated that PsimOR14 is higher expressed in workers than soldiers using RNA-seq for heads of workers and soldiers of P. simplex and that EAG response to neocembrene is higher in workers than soldiers. I consider that this study will contribute to further understanding of the molecular and evolutionary mechanisms of the chemoreception system in termites.
Strength:
The manuscript is well written. As far as I know, this study is the first study that identified a pheromone receptor in termites. The authors not only present a methodology for analyzing the function of termite pheromone receptors but also provide important insights in terms of the evolution of ligand selectivity of termite pheromone receptors.
We thank the reviewer for the overall positive evaluation of the manuscript.
Weakness:
As you can see in the "Recommendations to the Authors" section below, there are several things in this paper that are not fully explained about experimental methods. Except for this point, this paper appears to me to have no major weaknesses.
We address point by point the specific comments listed in the Recommendation to the authors chapter below.
Reviewer #3 (Public review):
Summary:
Chemical communication is essential for the organization of eusocial insect societies. It is used in various important contexts, such as foraging and recruiting colony members to food sources. While such pheromones have been chemically identified and their function demonstrated in bioassays, little is known about their perception. Excellent candidates are the odorant receptors that have been shown to be involved in pheromone perception in other insects including ants and bees but not termites. The authors investigated the function of the odorant receptor PsimOR14, which was one of four target odorant receptors based on gene sequences and phylogenetic analyses. They used the Drosophila empty neuron system to demonstrate that the receptor was narrowly tuned to the trail pheromone neocembrene. Similar responses to the odor panel and neocembrene in antennal recordings suggested that one specific antennal sensillum expresses PsimOR14. Additional protein modeling approaches characterized the properties of the ligand binding pocket in the receptor. Finally, PsimOR14 transcripts were found to be significantly higher in worker antennae compared to soldier antennae, which corresponds to the worker's higher sensitivity to neocembrene.
Strengths:
The study presents an excellent characterization of a trail pheromone receptor in a termite species. The integration of receptor phylogeny, receptor functional characterization, antennal sensilla responses, receptor structure modeling, and transcriptomic analysis is especially powerful. All parts build on each other and are well supported with a good sample size.
We thank the reviewer for these positive comments.
Weaknesses:
The manuscript would benefit from a more detailed explanation of the research advances this work provides. Stating that this is the first deorphanization of an odorant receptor in a clade is insufficient. The introduction primarily reviews termite chemical communication and deorphanization of olfactory receptors previously performed. Although this is essential background, it lacks a good integration into explaining what problem the current study solves.
We understand the comment about the lack of an intelligible cue to highlight the motivation and importance of the present study. In the current version of the manuscript the introduction has been reworked. As suggested by Reviewer 3 in the Recommendations section below, the introduction now integrates some parts of the original discussion, especially the part discussing the OR evolution and emergence of eusociality in hymenopteran social insects and in termites, while underscoring the need of data from termites to compare the commonalities and idiosyncrasies in neurophysiological (pre)adaptations potentially linked with the independent eusociality evolution in the two main social insect clades.
Selecting target ORs for deorphanization is an essential step in the approach. Unfortunately, the process of choosing these ORs has not been described. Were the authors just lucky that they found the correct OR out of the 50, or was there a specific selection process that increased the probability of success?
Indeed, we were extremely lucky. Our strategy was to first select a modest set of ORs to confirm the feasibility of the Empty Neuron Drosophila system and newly established SSR setup, while taking advantage of having a set of termite pheromones, including those previously identified in the P. simplex model, some of them de novo synthesized for this project. The selection criteria for the first set of four receptors were (i) to have full-length ORF and at least 6 unambiguously predicted transmembrane regions, and (ii) to be represented on different branches (subbranches) of the phylogenetic tree. Then it was a matter of a good luck to hit the PsimOR14 selectively responding to the genuine P. simplex trail-following pheromone main component. In the revised version, we state these selection criteria in the results section (Phylogenetic reconstruction and candidate OR selection).
The deorphanization attempts of additional P. simplex ORs are currently running.
The authors assigned antennal sensilla into five categories. Unfortunately, they did not support their categories well. It is not clear how they were able to differentiate SI and SII in their antennal recordings.
We agree that the classification of multiporous sensilla into five categories lacks robust discrimination cues. The identification of the neocembrene-responding sensillum was initially carried out by SSR measurements on individual olfactory sensilla of P. simplex workers one-by-one and the topology of each tested sensillum was recorded on optical microscope photographs taken during the SSR experiment. Subsequently, the SEM and HR-SEM were performed in which we localized the neocembrene sensillum and tried to find distinguishing characters. We admit that these are not robust. Therefore, in the revised version of the manuscript we decided to abandon the attempt of sensilla classification and only report the observations about the specific sensillum in which we consistently recorded the response to neocembrene (and geranylgeraniol). The modifications affect Fig. 4, its legend and the corresponding part of the results section (Identification of P. simplex olfactory sensillum responding to neocembrene).
The authors used a large odorant panel to determine receptor tuning. The panel included volatile polar compounds and non-volatile non-polar hydrocarbons. Usually, some heat is applied to such non-volatile odorants to increase volatility for receptor testing. It is unclear how it is possible that these non-volatile compounds can reach the tested sensilla without heat application.
The reviewer points at an important methodological error we made while designing the experiments. Indeed, the inclusion of long-chain hydrocarbons into Panel 1 without additional heat applied to the odor cartridges was inappropriate, even though the experiments were performed at 25–26 °C. We carefully considered the best solution to correct the mistake and finally decided to remove all tested ligands beyond C22 from Panel 1, i.e. altogether five compounds. These changes did not affect the remaining Panels 2-4 (containing compounds with sufficient volatility), nor did they affect the message of the manuscript on highly selective response of PsimOR14 to neocembrene (and geranylgeryniol). In consequence, Figures 2, 3 and 5 were updated, along with the supplementary tables containing the raw data on SSR measurements. In addition, the tuning curve for PsimOR14 was re-built and receptor lifetime sparseness value re-calculated (without any important change). We also exchanged squalene for limonene in the docking and molecular dynamics analysis and made new calculations.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
(1) L 208: "than" instead of "that"
Corrected.
(2) L 527+527 strange squares (•) before dimensions
Apparently an error upon file conversion, corrected.
(3) L553 "reconstructing" instead of "reconstruct"
Corrected.
(4) Two references (Chahda et al. and Chang et al. appear too late in the alphabet.
Corrected. Thank you for spotting this mistake. Due to our mistake the author list was ordered according to the alphabet in Czech language, which ranks CH after H.
Reviewer #2 (Recommendations for the authors):
(1) L148: Why did the authors select only four ORs (PsimOR9, 14, 30, and 31) though there are 50 ORs in P. simplex? I would like you to explain why you chose them.
Our strategy was to first select a modest set of ORs to confirm the feasibility of the Empty Neuron Drosophila system and newly established SSR setup, while taking advantage of having a set of termite pheromones, including those previously identified in the P. simplex model, some of them de novo synthesized for this project. Then, it was a matter of a good luck to hit the PsimOR14 selectively responding to the genuine P. simplex trail-following pheromone main component, while the deorphanization attempts of a set of additional P. simplex ORs is currently running. In the revised version of the manuscript, we state the selection criteria for the four ORs studied in the Results section (Phylogenetic reconstruction and candidate OR selection).
(2) L149: Where is Figure 1A? Does this mean Figure 1?
Thank you for spotting this mistake. Fig. 1 is now properly labelled as Fig. 1A and 1B in the figure itself and in the legend. Also the text now either refers to either 1A or 1B.
(3) Figure 1: The authors also showed the transcription abundance of all 50 ORs of P. simplex in the right bottom of Figure 1, but there is no explanation about it in the main text.
The heatmap reporting the transcript abundances is now labelled as Fig. 1B and is referred to in the discussion section (in the original manuscript it was referred to on the same place as Fig. 1).
(4) L260-265: The authors confirmed higher expression of PsimOR14 in workers than soldiers by using RNA-seq data and stronger EAG responses of PsimOR14 to neocembrene in workers than soldiers, but I think that confirming the expression levels of PsimOR14 in workers and soldiers by RT-qPCR would strengthen the authors' argument (it is optional).
qPCR validation is a suitable complement to read count comparison of RNA Seq data, especially when the data comes from one-sample transcriptomes and/or low coverage sequencing. Yet, our RNA Seq analysis is based on sequencing of three independent biological replicates per phenotype (worker heads vs. soldier heads) with ~20 millions of reads per sample. Thus, the resulting differential gene expression analysis is a sufficient and powerful technique in terms of detection limit and dynamic range.
We admit that the replicate numbers and origin of the RNA seq data should be better specified since the Methods section only referred to the GenBank accession numbers in the original manuscript. Therefore, we added more information in the Methods section (Bioinformatics) and make clear in the Methods that this data comes from our previous research and related bioproject.
(5) L491: I think that "The synthetic processes of these fatty alcohols are ..." is better.
We replaced the sentence with “The de novo organic synthesis of these fatty alcohols is described …”
(6) L525 and 527: There are white squares between the number and the unit. Perhaps some characters have been garbled.
Apparently an error upon file conversion, corrected.
(7) L795: ORCo?
Corrected.
(8) L829-830 & Figure 4: Where is Figure 4D?
Thank you for spotting this mistake from the older version of Figure 4. The SSR traces referred to in the legend are in fact a part of Figure 5. Moreover, Figure 4 is now reworked based on the comments by Reviewer 3.
(9) L860-864: Why did the authors select the result of edgeR for the volcano plot in Figure 7 although the authors use both DESeq2 and edgeR? An explanation would be needed.
Both algorithms, DESeq2 and EdgeR, are routinely used for differential gene expression analysis. Since they differ in read count normalization method and statistical testing we decided to use both of them independently in order to reduce false positives. Because the resulting fold changes were practically identical in both algorithms (results for both analyses are listed in Supplementary table S15), we only reported in Fig. 7 the outputs for edgeR to avoid redundancies. We added in the Results section the information that both techniques listed PsimOR14 among the most upregulated in workers.
Reviewer #3 (Recommendations for the authors):
The discussion contains many descriptions that would fit better into the introduction, where they could be used to hint at the study's importance (e.g., 292-311, 381-412). The remaining parts often lack a detailed discussion of the results that integrates details from other insect studies. Although references were provided, no details were usually outlined. It would be helpful to see a stronger emphasis on what we learn from this study.
Along with rewriting the introduction, we also modified the discussion. As suggested, the lines 292-311 were rewritten and placed in the introduction. By contrast, we preferred to keep the two paragraphs 381-412 in the discussion, since both of them outline the potential future interesting targets of research on termite ORs.
As suggested, the discussion has been enriched and now includes comparative examples and relevant references about the broad/narrow selectivity of insect ORs, about the expected breadth of tuning of pheromone receptors vs. ORs detecting environmental cues, about the potential role of additional neurons housed in the neocembrene-detecting sensillum of P. simplex workers, etc. From both introduction and discussion the redundant details on the chemistry of termite communication have been removed.
This includes explanations of the advantages of the specific methodologies the authors used and how they helped solve the manuscript's problem. What does the phylogeny solve? Was it used to select the ORs tested? It would be helpful to discuss what the phylogeny shows in comparison to other well-studied OR phylogenies, like those from the social Hymenoptera.
We understand the comment. In fact, our motivation to include the phylogenetic tree of termite ORs was essentially to demonstrate (i) the orthologous nature of OR diversity with few expansions on low taxonomic levels, and (ii) to demonstrate graphically the relationship among the four selected sequences. We do not attempt here for a comprehensive phylogenetic analysis, because it would be redundant given that we recently published a large OR phylogeny which includes all sequences used in the present manuscript and analysed them in the proper context of related (cockroaches) and unrelated insect taxa (Johny et al., 2023). This paper also discusses the termite phylogenetic pattern with those observed in other Insecta. This paper is repeatedly cited on appropriate places of the present manuscript and its main observations are provided in the Introduction section. Therefore, we feel that thorough discussion on termite phylogeny would be redundant in the present paper.
The authors categorized the sensilla types. Potential problems in the categorization aside, it would be helpful to know if it is expected that you have sensilla specialized in perceiving one specific pheromone. What is known about sensilla in other insects?
We understand. In the discussion of the revised version, we develop more about the features typical/expected for a pheromone receptor and the sensillum housing this receptor together with two other olfactory sensory neurons, including examples from other insects.
As the manuscript currently stands, specialist readers with their respective background knowledge would find this study very interesting. In contrast, the general reader would probably fail to appreciate the importance of the results.
We hope that the re-organized and simplified introduction may now be more intelligible even for non-specialist readers.
(1) L35: Should "workers" be replaced with "worker antennae"?
Corrected.
(2) L62: Should "conservativeness" be replaced by "conservation"?
Replaced with “parsimony”.
(3) L129: How and why did the authors choose four candidate ORs? I could not find any information about this in the manuscript. I wondered why they did not pick the more highly expressed PsimOr20 and 26 (Figure 7).
As already replied above in the Weaknesses section, we selected for the first deorphanization attempts only a modest set of four ORs, while an additional set is currently being tested. We also explained above the inclusion criteria, i.e. (i) full-length ORF and at least 6 unambiguously predicted transmembrane regions, and (ii) presence on different branches (subbranches) of the OR phylogeny. For these reasons, we did not primarily consider the expression patterns of different ORs. As for Fig. 7, it shows differential expression between soldiers and workers, which was not the primary guideline either and the data was obtained only after having the ORs tested by SSR. Yet, even though we had data on P. simplex ORs expression (Fig. 1B), we did not presume that pheromone receptors should be among the most expressed ORs, given the richness of chemical cues detected by worker termites and unlike, e.g., male moths, where ORs for sex pheromones are intuitively highly expressed.
The strategy of OR selection is specified in the results section of the revised manuscript under “Phylogenetic reconstruction and candidate OR selection”.
(4) 198 to 200: SI, II, and III look very similar. Additional measurements rather than qualitative descriptions are required to consider them distinct sensilla. The bending of SIII could be an artifact of preparation. I do not see how the authors could distinguish between SI and SII under the optical microscope for recordings. A detailed explanation is required.
As we responded above in “Weaknesses” chapter, we admit that the sensilla classification is not intelligible. Therefore, we decided in the revised version to abandon the classification of sensilla types and only focus on the observations made on the neocembreneresponding sensillum. To recognize the specific sensillum, we used its topology on the last antennal segment. Because termite antennae are not densely populated with sensilla, it is relatively easy to distinguish individual sensilla based on their topology on the antenna, both in optical microscope and SEM photographs. The modifications affect Fig. 4, its legend and the corresponding part of the results section (Identification of P. simplex olfactory sensillum responding to neocembrene).
(5) 208: "Than" instead of "that"
Corrected.
(6) 280: I suggest replacing "demand" with "capabilities"
Corrected.
(7) 312: Why "nevertheless? It sounds as if the authors suggest that there is evidence that ORs are not important for communication. This should be reworded.
We removed “Nevertheless” from the beginning of the sentence.
(8) 321 to 323: This sentence sounds as if something is missing. I suggest rewriting it.
This sentence simply says that empty neuron Drosophila is a good tool for termite OR deorphanization and that termite ORs work well Drosophila ORCo. We reworded the sentence.
(9) 323: I suggest starting a new paragraph.
Corrected.
(10) 421: How many colonies were used for each of the analyses?
The data for this manuscript were collected from three different colonies collected in Cuba. We now describe in the Materials and Methods section which analyses were conducted with each of the colonies.
(11) 430: Did the termites originate from one or multiple colonies and did the authors sample from the Florida and Cuba population?
The data for this manuscript were collected from three different colonies collected in Cuba. We now describe in the Materials and Methods section which analyses were conducted with each of the colonies.
(12) 501: How was the termite antenna fixated? The authors refer to the Drosophila methods, but given the large antennal differences between these species, more specific information would be helpful.
Understood. We added the following information into the Methods section under “Electrophysiology”: “The grounding electrode was carefully inserted into the clypeus and the antenna was fixed on a microscope slide using a glass electrode. To avoid the antennal movement, the microscope slide was covered with double-sided tape and the three distal antennal segments were attached to the slide.”
(13)509: I want to confirm that the authors indicate that the outlet of the glass tube with the airstream and odorant is 4 cm away from the Drosophila or termite antenna. The distance seems to be very large.
Thank you for spotting this obvious mistake. The 4 cm distance applies for the distance between the opening for Pasteur pipette insertion into the delivery tube, the outlet itself is situated approx. 1 cm from the antenna. This information is now corrected.
(14) 510/527: It looks like all odor panels were equally applied onto the filter paper despite the difference in solvent (hexane and paraffin oil). How was the solvent difference addressed?
In our study we combine two types of odorant panels. First, we test on all four studied receptors a panel containing several compounds relevant for termite chemical communication including the C12 unsaturated alcohols, the diterpene neocembrene, the sesquiterpene (3R,6E)-nerolidol and other compounds. These compounds are stored in the laboratory as hexane solutions to prevent the oxidation/polymerization and it is not advisable to transfer them to another solvent. In the second step we used three additional panels of frequently occurring insect semiochemicals, which are stored as paraffin oil solutions, so as to address the breadth of PsimOR14 tuning. We are aware that the evaporation dynamics differ between the two solvents but we did not have any suitable option how to solve this problem. We believe that the use of the two solvents does not compromise the general message on the receptor specificity. For each panel, the corresponding solvent is used as a control. Similarly, the use of two different solvents for SSR can be encountered in other studies, e.g. 10.1016/j.celrep.2015.07.031.
(15) 518: delta spikes/sec works for all tables except for the wild type in Table S5. I could not figure out how the authors get to delta spikes/sec in that table.
Thank you for your sharp eye. Due to our mistake, the values of Δ spikes per second reported in Table S5 for W1118 were erroneously calculated using the formula for 0.5 sec stimulation instead of 1 sec. We corrected this mistake which does not impact the results interpretation in Table S5 and Fig. 2.
522: Did the workers and soldiers originate from different colonies or different populations?
We now clearly describe in the Material and Methods section the origin of termites for different experiments. EAG measurements were made using individuals (workers, soldiers) from one Cuban colony.
(16) Figure 6C/D: I suggest matching colors between the two figures. For example, instead of using an orange circle in C and a green coloration of the intracellular flap in D, I recommend using blue, which is not used for something else. In addition, the binding pocket could be separated better from anything else in a different color.
We agree that the color match for the intracellular flap was missing. This figure is now reworked and the colors should have a better match and the binding region is better delineated.
(17) Figure 7/Table S15: It is unclear where the transcriptome data originate and what they are based on. Are these antennal transcriptomes or head transcriptomes? Do these data come from previous data sets or data generated in this study? Figure 7 refers to heads, Table S15 to workers and soldiers, and the methods only refer to antennal extractions. This should be clarified in the text, the figure, and the table.
We admit that the replicate numbers and origin of the RNA seq data should be better specified and that the information that the RNASeq originated from samples of heads+antennae of workers and soldiers should be provided at appropriate places. Therefore, we added more information on replicates and origin of the data in the Methods section (Bioinformatics) and make clear that this data comes from our previous research and refer to the corresponding bioproject. Likewise, the Figure 7 legend and Table S15 heading have been updated.
Compte rendu détaillé : La justice face aux violences sexuelles, entre tradition punitive et voie restaurative
La discussion se focalise sur les limites de la justice punitive traditionnelle face aux crimes de violences sexuelles, en particulier l'inceste, et propose des alternatives telles que la justice restaurative et des évolutions législatives.
1. La nature et l'ampleur des crimes sexuels, en particulier l'inceste
Antoine Garapon mentionne le chiffre de "160 000 enfants subissent des violences sexuelles chaque année" en France, une statistique qu'il met en perspective avec les 1600 homicides annuels, soulignant que les violences sexuelles sont "10 000 fois plus" fréquentes.
Ces crimes sont caractérisés par :
L'identité de l'agresseur : Majoritairement des hommes, souvent majeurs. Les pères (27%), les frères (19%) et les oncles (13%) sont fréquemment cités comme agresseurs.
Leur nature "fondatrice" et paradoxale : Antoine Garapon les décrit comme des crimes "réputés les plus graves, les plus fondateurs", mais paradoxalement "les moins condamnés, étaient même les moins dénoncés".
L'exemple des crimes sexuels commis par des prêtres est particulièrement mis en avant, car une institution qui doit annoncer le salut "sème la mort", ce qui est une contradiction totale.
Un "système du silence" prévalait, souvent lié à un "conflit de loyauté", où la loyauté envers l'institution (comme l'Église) ou la famille était "supérieure à au crédit porté à un enfant".
L'affaire de l'Abbé Pierre est citée comme un exemple criant où "tout le monde savait" mais les autorités n'ont pas agi, abordant le crime uniquement par rapport à la loi morale, "pas un mot pour les victimes".
La thèse audacieuse de Dorothée Dussy, partagée par Garapon, suggère que les enfants victimes étaient en quelque sorte "le prix de l'ordre familial, de l'ordre ecclésial", participant par leur silence à l'ordre social général.
2. L'évolution de la "conscience commune" et le rôle du mouvement #MeToo
La perception de ces crimes a radicalement évolué. Reprenant la définition de Durkheim, qui définit le crime comme "ce qui choque la conscience commune", Antoine Garapon affirme qu'aujourd'hui, "ces crimes sont considérés comme étant les plus choquants dans la conscience générale. Peut-être même plus que les homicides".
La société ne supporte plus que des dominés (enfants, femmes) soient l'objet de violences impunies, d'autant plus que le viol est quasi équivalent au crime en termes de répression pénale.
3. Les limites de la justice pénale traditionnelle et les souffrances des victimes
La justice pénale traditionnelle, bien qu'essentielle, montre ses limites :
Cependant, Antoine Garapon soutient que "s'intéresser au rétablissement des personnes à commencer par celui de la victime, c'est de la justice".
L'"amnésie traumatique" peut durer des années, empêchant même la conscience des faits.
Les aveux de l'auteur restent souvent la preuve maîtresse.
Plus le nombre de traumatismes est élevé, plus les conséquences à l'âge adulte sont graves.
Une personne ayant subi deux traumatismes majeurs dans l'enfance a "20 ans d'espérance de vie de moins que la population générale".
Plus de la moitié des victimes d'inceste font ou ont fait une tentative de suicide.
4. La justice restaurative : une alternative centrée sur la victime
Antoine Garapon promeut la justice restaurative comme une "alternative" ou un complément à la justice pénale :
5. Les évolutions législatives et les défis de la prescription
Les intervenants abordent les débats actuels autour de la prescription des crimes sexuels :
Elle ajoute que "le sentiment d'intranquillité qui habite la victime lui est à vie" et qu'il devrait "venir hanter l'agresseur".
6. Le rôle des associations et les besoins des victimes
L'association "Face à l'Inceste", créée il y a 25 ans par une victime, Isabelle Aubry, joue un rôle crucial :
7. Vers une "autre justice" et la "politisation de l'intime"
Antoine Garapon plaide pour une "autre justice", plus "accomplie", qui intègre différentes facettes :
En conclusion, la discussion met en lumière la nécessité d'une approche plus globale et empathique face aux violences sexuelles, qui ne se limite pas à la seule punition de l'agresseur mais qui inclut une reconnaissance profonde de la souffrance des victimes, un soutien adapté, et des mécanismes de réparation qui favorisent leur reconstruction et leur capacité à vivre.
Reviewer #1 (Public review):
The authors examine how probabilistic reversal learning is affected by dopamine by studying the effects of methamphetamine (MA) administration. Based on prior evidence that the effects of pharmacological manipulation depend on baseline neurotransmitter levels, they hypothesized that MA would improve learning in people with low baseline performance. They found this effect, and specifically found that MA administration improved learning in noisy blocks, by reducing learning from misleading performance, in participants with lower baseline performance. The authors then fit participants' behavior to a computational learning model and found that an eta parameter, responsible for scaling learning rate based on previously surprising outcomes, differed in participants with low baseline performance on and off MA.
Questions:
(1) It would be helpful to confirm that the observed effect of MA on the eta parameter is responsible for better performance in low baseline performers. If performance on the task is simulated for parameters estimated for high and low baseline performers on and off MA, does the simulated behavior capture the main behavioral differences shown in Figure 3?
(2) In Figure 4C, it appears that the main parameter difference between low and high baseline performance is inverse temperature, not eta. If MA is effective in people with lower baseline DA, why is the effect of MA on eta and not IT?
Also, this parameter is noted as temperature but appears to be inverse temperature as higher values are related to better performance. The exact model for the choice function is not described in the methods.
Comments on revisions:
Thanks to the authors for their thorough responses and revisions. One typo to note: in the Methods, the "drug effects" paragraph is repeated.
Reviewer #2 (Public review):
Summary:
Kirschner and colleagues test whether methamphetamine (MA) alters learning rate dynamics in a validated reversal learning task. They find evidence that MA can enhance performance for low-performers, and that the enhancement reflects a reduction in the degree to which these low-performers dynamically up-regulate their learning rates when they encounter unexpected outcomes. The net effect is that poor performers show more volatile learning rates (e.g. jumping up when they receive misleading feedback), when the environment is actually stable, undermining their performance over trials.
Strengths:
The study has multiple strengths, including a large sample size, placebo control, double-blind randomized design, and rigorous computational modeling of a validated task. Additionally, the analytic methods are rigorous and offer new types of analyses for people interested in exploring learning as a function of dynamically changing volatility.
Weaknesses:
The limitations, which are acknowledged, include that the drug they use, methamphetamine, can influence multiple neuromodulatory systems including catecholamines and acetylcholine, all of which have been implicated in learning rate dynamics. They also do not have any independent measures of any of these systems, so it is impossible to know which is having an effect.
Another limitation which they should acknowledge is that the fact that participants were aware of having different experiences in the drug sessions means that their blinding was effectively single-blind (to the experimenters) and not double-blind. That said, the authors do provide some evidence that subjective effects of drugs (e.g. arousal, mood, etc.) did not drive differences in performance.
Comments on revisions:
The authors have done an outstanding job responding to, and allaying my prior concerns about their analyses.
Synthèse : Le Consentement au Cœur du Débat en France
Il met en lumière la complexité de cette notion, les défis liés à sa compréhension et son application, ainsi que les efforts déployés pour l'intégrer pleinement dans la loi et les mentalités.
1. Le Consentement : Une Notion Émergente et Centralisée
Le procès des viols de Mazan, avec la condamnation de Dominique Pélico pour avoir drogué et violé sa femme pendant dix ans, a été un catalyseur majeur.
Une des personnes interrogées souligne la simplicité apparente mais la profondeur de la notion :
"Quand une fille dit non, j'ai l'impression quand même que souvent ça sous-entend que c'est c'est non. Non.
D'accord. Ah ouais. Ah faut bien c'est pas si simple. Faut bien choper le truc hein. Oui ou non ? Deux petits mots de trois lettres. Mais qui change absolument tout."
Cette prise de conscience a conduit à des appels à inscrire le consentement dans la loi, exigeant que les agresseurs présumés prouvent avoir obtenu un accord explicite avant tout acte sexuel.
Le slogan "Jamais sans mon consentement" est devenu un cri de ralliement dans les cortèges féministes.
2. La Compréhension du Consentement : Défis et Manques
Pauline, victime de viol à 14 ans par son premier petit ami, témoigne de la difficulté à identifier le viol et à en parler, d'autant plus en l'absence d'éducation sexuelle adéquate : "Je savais pas ce que c'était les rapports.
Donc pour moi c'était un peu la norme entre guillemets... je savais pas trop comment en parler et après j'ai mis du temps avant de d'accepter aussi le terme viol parce que c'est un mot quand même très fort."
Elle évoque aussi l'influence de la pornographie, qui "ne parle pas du tout" du consentement à cet âge.
Les témoignages révèlent que le "non" n'est pas toujours respecté, et que la peur peut paralyser les victimes, comme Elodie qui a été agressée sexuellement à 17 ans : "J'étais tellement peur que c'est comme si j'étais paralysée. J'arrivais pas à crier. J'étais vraiment tétanisée."
3. L'Éducation et la Prévention : Des Outils Essentiels
Face à ces lacunes, des interventions en milieu scolaire se multiplient. Une gendarme intervient dans un collège pour expliquer le consentement aux élèves de 3ème.
Elle définit l'agression sexuelle comme "le fait de toucher les parties intimes sans consentement, sans son autorisation."
Elle insiste sur la clarté du "oui" ou du "non", verbal ou par des gestes, et surtout, sur le fait qu'en l'absence de réponse, il faut considérer que c'est un "non".
L'importance de parler "sans cacher les mots" est soulignée par la gendarme, car "on a beau dire non du plus plus fort qu'on peut, si l'autre en face n'entend pas, il fera quand même ce qu'il a envie de faire qui est illégal."
Ces interventions sont jugées cruciales, car la discussion sur le consentement est "très peu abordée aussi bien par les parents à la maison qui peuvent être embarrassés... et même les établissements scolaires sont parfois dépourvus de moyens."
4. La Réalité des Violences Sexuelles : Souvent le Fait de Proches
Un point crucial est la démystification de l'image de l'agresseur. Contrairement à l'imaginaire collectif, un violeur n'est pas toujours un inconnu armé : "Dans 90 % des cas, l'agresseur connaît sa victime.
Dans la moitié des cas, c'est son partenaire ou un ex amoureux." De plus, les femmes sont majoritairement les victimes, avec 91% des auteurs de violences sexuelles étant des hommes.
5. La Complexité Juridique et la Subjectivité du Consentement
Un accusé, qui nie les viols dont il est accusé malgré la condamnation à 7 ans de prison, exprime cette ambiguïté : "La notion de consentement est pour moi acquise...
À aucun moment, ell m'ont elles m'ont dit non clairement." Il ajoute avoir dit à une victime "C'est comme un viol, ce n'en est pas un," illustrant la "limite très fine" de la compréhension.
Tout ça est adossé à la culture du viol, au patriarcat, à la domination des femmes et c'est ça qui change tout."
6. L'Inscription Légale du Consentement : L'Exemple International
Le débat sur l'inscription du consentement dans la loi française s'inspire de législations étrangères :
7. Changer l'Imaginaire Collectif pour une Culture du Consentement
Les films et les médias sont critiqués pour leurs représentations stéréotypées où l'insistance masculine est glorifiée et le "non" féminin est souvent interprété comme un "oui" latent.
Des initiatives, comme l'association Sex et Consentement, proposent des supports (cartes postales, préservatifs) avec des messages explicites pour normaliser la demande de consentement.
Les jeunes interrogés y voient un moyen de "nous forcer à réfléchir et à demander à l'autre aussi si elle est d'accord oui ou non."
En conclusion, l'émission souligne une transformation profonde des mentalités et du cadre légal autour du consentement en France, tirant des leçons des expériences individuelles et des législations internationales pour mieux protéger les victimes et éduquer les nouvelles générations.
Compte Rendu Détaillé : L'école et les enfants d'immigrés en France
Les discussions, menées par Louis Touret, impliquent le sociologue Stéphane Beau, auteur de "La France des Béloui, portrait de famille 1977-207", et Véronique Desquer, enseignante en éducation prioritaire depuis 30 ans.
Magid Cherfi, chanteur du groupe Zebda et écrivain, et Dominique Garcia, archéologue et président de l'INRAP, partagent également leurs expériences personnelles.
L'émission s'articule autour de l'impact de l'école française sur les enfants d'immigrés, l'évolution du rôle des enseignants et de la politique en la matière, les défis de la ségrégation sociale et spatiale, et le rôle crucial de l'école dans l'intégration sociale.
I. L'École comme Porte d'Entrée et Facteur de Réussite Sociale
L'école est présentée comme un lieu d'espoir et de possibilités, particulièrement pour les enfants d'immigrés. Stéphane Beau souligne l'importance du diplôme dans la société française et le rôle majeur de l'école dans la transmission des savoirs en milieu populaire.
L'instituteur y incarne un principe d'égalité, contrastant avec les discriminations vécues dans la rue où ils sont traités comme des "bougoules" ou des "indigènes". Ce sentiment d'égalité est "presque martien" pour lui. * Le parcours exemplaire de Samira Beloui : Stéphane Beau raconte l'histoire de Samira, l'aînée d'une famille de huit enfants immigrés d'Algérie. Arrivée en France à 7 ans sans maîtriser le français, elle est "éblouie, entre guillemets, sauvée par un système scolaire français". Ses institutrices sont décrites comme des figures dévouées : "Moi, j'ai une une affection sans borne pour mes institutrices. Elles m'ont aidé, elles m'ont sauvé". L'une d'elles restait même "une heure avec cette jeune fille Samira lui apprenant le français, ne comptant pas son temps." Samira obtient un bac et devient infirmière, une "réussite éclatante" malgré ses responsabilités familiales. * La stratégie parentale et la mixité sociale : Le père des Beloui, bien qu'analphabète, a "choisi un HLM où il y a moins d'étrangers que dans les tours et les barres du même quartier", favorisant une école primaire "un peu plus mixte avec des enfants de classe moyenne pavillonnaires". Ce choix, combiné à l'engagement des instituteurs des années 70, a contribué à la réussite scolaire des aînés. * L'école, dernier service public : Véronique Desquer réfute l'idée que l'école aurait "démissionné" en soulignant qu'elle est souvent "le dernier service public qui est encore ouvert dans le quartier".
II. L'Évolution du Rôle des Enseignants et les Conditions d'Enseignement
L'émission met en lumière un changement significatif dans le profil et les conditions de travail des enseignants, particulièrement dans les quartiers populaires.
Ces enseignants étaient souvent issus de milieux populaires, des "miraculés scolaires" qui avaient "à cœur de transmettre ce savoir et notamment aux enfants de milieu populaire comme eux". L'esprit de corps et l'engagement pour la République étaient forts.
L'école normale leur offrait des "études supérieures pour des gens qui n'ont pas les moyens d'aller à l'université". Aujourd'hui, beaucoup d'enseignants vivent leur métier comme un "déclassement" en raison des salaires faibles et des difficultés à trouver des postes près de chez eux.
III. Les Inégalités et la Ségrégation Sociale et Spatiale
L'émission aborde la permanence et l'aggravation des inégalités scolaires, étroitement liées aux changements socio-spatiaux.
L'échec de l'école française pour les enfants de catégories populaires : Une étude du KNESCO est citée, indiquant que l'école française est "pas très forte pour faire réussir les enfants d'immigrés" car elle n'est "pas très performante à faire réussir les enfants de catégorie populaire où on trouve les enfants d'immigrés".
La précarisation des familles : Véronique Desquer et Stéphane Beau mettent en avant la "précarité" et "l'instabilité" croissante des familles populaires. Les parents, "tellement envahis par leurs propres problèmes", sont moins "disponibles psychiquement pour leurs enfants".
Cette "accumulation de d'instabilité" affecte directement la scolarité des enfants.
Stéphane Beau fait le parallèle avec la famille Beloui, qui, bien que "pauvre", a vécu une enfance "pas malheureuse" grâce à une "sécurisation affective" et un "avenir qui était ouvert devant eux".
Auparavant, ces quartiers étaient vus comme des lieux où habitaient des professionnels de toutes sortes, y compris des footballeurs célèbres.
Véronique Desquer ajoute que le mot "cité" désignait les "cités de transit" entre les bidonvilles et les ZUP, qui étaient alors "enviées par tout le monde".
IV. La Différence Filles/Garçons et son Explication Sociologique
Une différence "majeure et cruciale" est observée dans les parcours scolaires au sein de la famille Beloui : "cinq filles Bachelière (...) et des garçons beaucoup moins diplômés".
Elles savent que "ce n'est que par l'école qu'elles pourront réussir une vie professionnelle et surtout échapper pour cette génération des aînés au mariage qui les attend".
Cette menace du mariage est une "formidable incitation à réussir à l'école". Les filles font preuve de "bonne volonté scolaire, une disposition scolaire, un suivi d'elle-même, un travail régulier".
C'est à ce moment que "va jouer à fond le rôle des pères [pairs], des amis, des copains et cetera et des sorties". Les filles ayant "beaucoup moins le droit de sortie", cette dynamique affecte davantage les garçons qui "vont progressivement dérailler".
Les garçons ont tendance à "reprocher au système scolaire, aux enseignants" leur échec, mais certains finiront par admettre : "C'est vrai, j'ai déconné à l'école".
Les filles, moins sujettes aux sorties, ont "plus envie sans doute de progresser et d'apprendre pour s'en sortir parce que il y a un enfermement".
Les sœurs aînées jouent un rôle de "protection" pour leurs frères, "elles vont suivre elles vont (...) surveiller les bulletins, qui vont rencontrer les enseignants".
V. Le Rapport à la Politique et à la Gauche
L'émission explore également l'interaction entre les familles immigrées et le monde politique, en particulier la gauche.
Elles s'intéressent à la politique parce que "la politique va s'intéresser à eux". La fréquentation des clubs de sport et des associations militantes locales (souvent communistes ou de gauche) joue un rôle crucial.
Leila, la deuxième sœur, est "très marquée par justement ce qu'on fait pour nous malgré tout, les vacances pour nous et cetera sans distinction à égalité".
Elle estime que la mairie PC de Bobigny a perdu la ville car elle n'a pas su faire de place aux habitants issus de l'immigration. Stéphane Beau cite le livre "La gauche et les cités enquête sur un rendez-vous manqué" de Lilier Mascle, qui explique comment "le ratage la succession des générations ne s'est pas faite".
Les trois sœurs aînées se disent "Charlie" et manifestent, tandis que les autres, notamment les garçons moins diplômés, sont plus "réactifs" et "dans la théorie du complot", manifestant une "logique de provocation". Stéphane Beau insiste sur la complexité de ces positions, ni "Charlie" ni "anti-Charlie".
VI. Le Récit du Réel et la Mobilité Sociale
Les intervenants soulignent l'importance de raconter la réalité des familles populaires et immigrées pour contrer les discours sensationnalistes.
Il s'oppose aux récits sensationnalistes centrés sur la "tentation radicale", affirmant que "la France des Benoui, c'est la France majoritaire de ces familles immigrées, mais celle dont on parle jamais parce que tranquillement leurs enfants essayent de faire leur place dans la société française".
Il y voit une "utilité publique" pour "donner une autre image de la société".
Elle déplore les "affirmations d'autant plus péremptoires qu'elles ne sont étayées par rien" de la part de "spécialistes de la banlieue auto-proclamés".
Elle conclut sur une note positive, soulignant que les familles d'origine maghrébine "sont en train de quitter progressivement les HLM pour aller vivre ailleurs", ce qui est "la preuve d'une vraie mobilité sociale" et démontre que "l'école n'a rien réussi [mais] la mobilité sociale qui est celle de ces familles issues de l'immigration fera preuve puisque ils n'ont hérité de rien hein les béomis à part de l'école publique."
VII. L'Archéologie de l'Enfance et la Découverte du Monde
Le témoignage de Dominique Garcia offre une perspective différente sur l'impact de l'école.
Un club d'archéologie au collège, animé par un professeur de lettres classiques, lui a ouvert le monde en lui montrant que "les Romains sont venus dans la région pour exploiter du cuivre c'est eux qui introduit la vigne".
Cette "géographie était mêlée d'histoire", l'incitant à "élargir son champ de vision" et à voyager pour fouiller des sites archéologiques.
Elle souligne le rôle crucial de l'école comme vecteur de mobilité sociale, tout en pointant du doigt les transformations qui ont rendu son action plus difficile, notamment la précarisation des familles et la ségrégation sociale.
Le rôle des enseignants, l'évolution de la politique éducative et la nécessité de récits authentiques pour comprendre ces réalités sont également des fils conducteurs majeurs.
Compte-rendu détaillé : Éducation Populaire et Liens avec l'École
Source : Extraits de "France culture être et savoir Tuerie dans un lycée de Nantes L'éducation populaire, quelles relations avec l'école 11192-28.04.2025-ITEMA_24116950-2025C14993S0118-NET_MFC_1282E914-3484-4849-8E09-1FE806076BE5-21.mp3"
Introduction : Un événement tragique comme point de départ et la nécessité de l'éducation populaire
L'émission s'ouvre sur le rappel d'une attaque au couteau survenue le 24 avril dans un lycée privé de Nantes, Notre Dame de toutes aide, où un élève de seconde a poignardé quatre camarades, causant la mort d'une lycéenne.
L'agresseur, Justin P., 16 ans, était inconnu des services de police et ses camarades le décrivent comme "un jeune homme perturbé".
Suite à cet événement, la sociologue Nathalie Paton, spécialiste des school shootings aux États-Unis, intervient pour commenter les réactions politiques, notamment la proposition du Premier ministre d'installer des portiques de sécurité.
Elle juge cette mesure "démesurée et presque légèrement délirante" dans le contexte français, soulignant que les school shootings sont un phénomène isolé en France, contrairement aux États-Unis où ils sont quotidiens et où de telles mesures n'ont pas prouvé leur efficacité, pouvant même générer un sentiment d'insécurité.
L'analyse des motivations de l'agresseur tend vers une "belle psychose" et un "délire", comme en témoigne un manifeste mêlant des références disparates (Hitler, Écoid).
Nathalie Paton souligne l'importance d'une approche psychiatrique pour comprendre cet acte, soulignant que le jeune homme était "clairement très mal, très délirant" et que son acte a été un "passage à l'acte" débordant d'une "grande angoisse".
Thème central : Le sous-financement de la pédopsychiatrie et de la médecine scolaire
Le cas de Nantes met en lumière les graves lacunes de la prise en charge de la santé mentale des jeunes en France. Nathalie Paton insiste sur l'état "extrêmement préoccupant" de la pédopsychiatrie française et le "délaiement" de la médecine scolaire.
Elle s'interroge sur l'absence de repérage et de prise en charge préalable de l'agresseur : "Qu'est-ce qui fait que il n'avait pas été pris en charge avant ?
Ça ça paraît difficile de penser que ça allait déborder pour la première fois ce jour-là."
Elle dénonce le manque de psychologues scolaires et le fait que la psychiatrie soit considérée comme une "médecine pauvre" par les politiques publiques, manquant cruellement de "politiques et d'investissements".
Cette première partie de l'émission sert de tremplin pour aborder le rôle crucial de l'éducation populaire dans la construction du lien social et la prévention, en complément de l'école.
L'Éducation Populaire : Histoire, Valeurs et Fonctions L'émission explore ensuite en détail le monde de l'éducation populaire, souvent invisible mais pourtant essentiel pour deux tiers des enfants et adolescents français (périscolaire, centres de loisirs, colonies de vacances, activités sportives et artistiques).
1. Fondements et mission historique : Former le citoyen éclairé
Il s'agit de faire en sorte que le vote populaire soit pas ne soit pas détourné parce que parce que ce vote populaire ne serait pas éclairé."
L'objectif est de "favoriser la création d'une école d'une école publique laïque" et de "former les citoyens pour que la démocratie s'exerce vraiment dans la République."
La Ligue agit "un mouvement complémentaire de l'école publique et elle agit y compris au sein de l'école publique."
2. Une éducation "au côté ou à côté de l'école" : Complémentarité et différences
L'éducation populaire se positionne en complément de l'école, mais avec des approches différentes. Wahid Ben Hamed, directeur du centre de formation des CEMÉA Île-de-France, insiste sur la nature des métiers de l'éducation populaire : "C'est des métiers du lien social.
C'est des métiers de la cohésion sociale." Il met en avant la dimension collective de l'apprentissage : "On apprend ensemble on apprend lorsqu'on se met autour d'objets communs."
Distinction fondamentale : L'absence de jugement et de compétition
Une différence majeure avec l'école est l'absence de jugement et d'évaluation. Laurent Bess, maître de conférence en histoire contemporaine, explique que "les animateurs par principe refusent de juger que ce soit les pratiques ou les réalisations des enfants alors que bah l'enseignant, il dit ce qui est vrai, ce qui est faux, ce qui est juste, ce qui est bon."
Cette approche favorise une "volonté de conserver la cohérence du groupe" en "abolissant ce jugement qui crée effectivement des différences entre les enfants."
Wahid Ben Hamed renchérit en affirmant : "C'est pas un concours, c'est jamais c'est ce qui différencie par exemple de la profession d'enseignant."
Pour lui, l'enjeu est de "réinterroger les représentations du groupe" pour "favoriser l'émancipation".
Il cite l'exemple du sport où l'on peut "imaginer autre chose" que le simple fait de gagner ou de perdre.
3. L'évolution de l'éducation populaire : Des cours du soir aux loisirs émancipateurs
Laurent Bess retrace l'histoire de l'éducation populaire, situant son "âge d'or" entre l'entre-deux-guerres et les années 1970.
Si au 19ème siècle, elle était davantage centrée sur des modèles scolaires (cours du soir), elle se transforme dans l'entre-deux-guerres autour de la "démocratisation des loisirs", visant à permettre aux enfants des milieux populaires d'accéder à de nouvelles pratiques (artistiques, sportives, plein air).
Des instituteurs ont d'abord encadré ces activités via les "œuvres laïques", avant d'être progressivement remplacés par des professionnels, les "animateurs socioculturels".
Aujourd'hui, l'accent est mis sur "l'aspect non scolaire de l'éducation populaire sur la reconnaissance des individus l'accent mis sur des relations qui se veulent horizontales des pratiques qui se veulent ludiques qui visent à former toujours."
Bien que l'ambition de former le citoyen demeure, le contenu politique est "moins mise en avant".
4. Le rôle crucial du "vivre ensemble" et de la "transformation sociale"
Patricia Ménard, directrice du périscolaire pour l'école du Four au sein de la Fondation Léo Lagrange (fondée en 1936), insiste sur les valeurs de son institution : "le vivre ensemble, la découverte et l'épanouissement de l'enfant et la mixité culturelle."
Elle définit le "vivre ensemble" comme "partager, c'est être ensemble, essayer de comprendre les autres, c'est vivre ensemble en tant que citoyen aussi sur un dans le loisir au sein de l'école, d'avoir les mêmes règles de l'école et du loisir, c'est être un enfant parmi toute une collectivité et être à plusieurs pour être bien en fait."
Il souligne que l'éducation populaire vise la "transformation sociale", en "essayant d'ouvrir une porte et de s'approprier en fait ce qu'on lui propose."
Défis et Perspectives de l'Éducation Populaire
1. La précarité des financements et ses conséquences
La question du financement est jugée "cruciale" par Hélène Lacassagne. Mohamed Magassa explique que son association dépend "systématiquement" de "subventions" et "d'appels à projet".
Hélène Lacassagne déplore que les appels à projet et les marchés publics se soient "substitués à la subvention", ce qui pose un "une vraie difficulté parce que le diagnostic n'est plus porté par l'association."
Elle regrette que cela mette en danger la "capacité d'innovation" des associations, autrefois moteurs de dispositifs comme les bibliobus.
Ce modèle, qui exige du temps, est menacé par des politiques publiques qui ne "rencontrent pas les personnes pour lesquelles elle a été inventée", car le "dernier kilomètre, c'est le premier" pour les acteurs de terrain.
2. Le défi de l'attractivité des métiers et de l'innovation pédagogique
Les métiers de l'éducation populaire sont "pas très bien payés".
La motivation des professionnels comme Cyriel, une animatrice Léo Lagrange qui a créé l'atelier "raconte-toi", réside dans le sens de leur travail : "Je n'ai pas l'impression d'aller au travail.
En fait tous les jours, on a une situation différente et moi je trouve que c'est une chance de pouvoir leur transmettre des valeurs et les écouter."
Wahid Ben Hamed insiste sur "l'innovation pédagogique" au sein des centres de formation des CEMÉA, qui accueillent de nombreux jeunes ayant "une méfiance et une réticence au fait d'apprendre" suite à un "échec" ressenti vis-à-vis de l'Éducation Nationale.
L'approche des CEMÉA est non-verticale : "on part du principe que les gens qui sont ici et les apprenants ont des choses à nous apprendre nous à formateur en tant que formateur. Ils ont des des choses à apprendre au groupe qui est là."
L'exemple de la "Newton Room" au collège Jean-Mermoz d'Angers, un atelier scientifique scandinave, illustre cette volonté d'innover pour rendre les mathématiques "concrètes" et offrir des outils de qualité.
Ce type de partenariat vise à valoriser l'école publique et à lui donner une "étiquette" pour "exister sur des des établissements qui ont pignon sur rue" (privés).
3. Accueillir tous les publics et déconstruire les sujets sensibles
Hélène Lacassagne souligne la nécessité d'une approche qui ne soit pas seulement "prestataire" mais qui permette de "recréer une relation, de remettre les de faire vraiment éducation populaire, c'est-à-dire de mettre les personnes en situation, de porter l'action, d'être non pas dans une relation de de consommation d'une action, mais d'être associé au diagnostic, au faire et à l'évaluation de la chose de façon à ce que les personnes se sentent reconnu en égale dignité avec les autres citoyennes, les autres citoyens."
Mohamed Magassa explique comment son association Reconnectus aborde les "questions vives" avec les jeunes.
Ces derniers "ramènent en fait les sujets qu'ils avaient entendu à l'école pour les déconstruire avec nous", abordant par exemple la discrimination avant la laïcité.
Leur propre expérience de la discrimination leur permet de mieux accompagner les jeunes : "le sujet de la laïcité s'impose à travers la discrimination."
Conclusion
Face à cela, l'éducation populaire apparaît comme un pilier essentiel, bien que souvent sous-estimé et sous-financé.
Son rôle complémentaire de l'école, axé sur le lien social, l'émancipation individuelle et collective, et l'absence de jugement, en fait un acteur clé pour répondre aux besoins des jeunes et des familles.
Cependant, la pérennité et la capacité d'innovation de l'éducation populaire sont menacées par les modes de financement actuels, qui entravent la co-construction de projets adaptés aux réalités du terrain et au "premier kilomètre" des citoyens.
Le plaidoyer des intervenants est clair : reconnaître et soutenir davantage ce secteur pour qu'il puisse continuer à former des citoyens éclairés et à renforcer le tissu social.
Author response:
The following is the authors’ response to the original reviews.
Reviewer 1:
Comment 1: Indirect Estimates of White Matter Connections: While dMRI is a valuable tool, it inherently provides indirect and inferred information about neural pathways. The accuracy and specificity of tractography can be influenced by various factors, including fiber crossing, partial volume effects, and algorithmic assumptions. A potential limitation in the accuracy of indirect estimates might affect the precision of spatial extent measurements, introducing uncertainty in the interpretation of cortico-thalamic connectivity patterns. Addressing the methodological limitations associated with indirect estimates and considering complementary approaches could strengthen the overall robustness of the findings.
We appreciate the reviewer’s comment and agree tractography is an indirect estimate and subject to limitations. Regarding this manuscript, the key question is not whether the anatomical tracts are without false positives or negatives, and in fact we argue that this question is outside the scope of this manuscript and has been addressed in several previous studies (e.g. Thomas et al. 2015, Schilling et al., 2020, Grisot et al. 2021, and many others). Instead, the key question for this manuscript is whether the focality of termination patterns within the thalamus is systematically biased in a way that the observation of a hierarchy effect is artifactual. The many supplementary analyses in this manuscript do help address this question and increase our confidence that the indirect nature of tractography does not systematically bias the EDpc1 measure such that association areas only appear to have more diffuse connectivity patterns relative to sensorimotor areas.
Comment 2: An over-arching theme of my review is that, each time I found myself wondering about a detail, a null, or a reference, I had only to read the next sentence or paragraph to find my concern handled in a clear and concise fashion. This is, in my opinion, the mark of work of the highest order. I congratulate the authors on their excellent work, which I believe will be impactful and well-received.
I have no notes that I feel can help improve what is already an impeccable piece of work.
We thank the reviewer for the kind comment.
Reviewer #2:
Comment 1: Structural thalamocortical connectivity was estimated from diffusion imaging data obtained from the HCP dataset. Consequently, the robustness and accuracy of the results depend on the suitability of this data for such a purpose. Conducting tractography on the cortical-thalamic system is recognized as a challenging endeavor for several reasons. First, diffusion directions lose their clearly defined principal orientations once they reach the deep thalamic nuclei, rendering the tracking of structures on the medial side, such as the medial dorsal (MD) and pulvinar nuclei difficult. Somewhat concerning is those are regions that authors found to show diffuse connectivity patterns. Second, the thalamic radiata diverge into several directions, and routes to the lateral surface often lack the clarity necessary for successful tracking. It is unclear if all cortical regions have similar levels of accuracy, and some of the lateral associative regions might have less accurate tracking, making them appear to be more diffuse, biasing the results.
As mentioned in the weakness section, it is crucial to address the need for better validation or the inclusion of control analyses to ensure that the results are not systematically biased due to known issues, such as the difficulty in tracking the medial thalamus and the potential for higher false positives when tracking the lateral frontal cortex.
We thank that reviewer for bringing up an important point. To determine if some areas of the thalamus were more difficult to track and, in turn, biased the EDpc1 measure we added an additional supplemental figure (S31). In this figure, shown below, we calculate the total SC of all ipsilateral cortical areas to each thalamic voxel. We show that, indeed, medial thalamic voxels have a lower total streamline count to ipsilateral cortex, and we see reduced total streamline counts to lateral thalamic areas and the very posterior end of the thalamus. We determined if some cortical areas preferentially projected to parts of the thalamus with lower ipsilateral total SC (i.e. by calculating the overlap between SC and total cortical SC for each thalamic voxel) and found only a weak relationship with our measure. Furthermore, we regressed each voxel’s mean ipsilateral cortical SC from streamline count matrix. We found that the EDpc1 measure didn’t significantly change after the regression.
Additionally, we note that this analysis assumes that all thalamic voxels should have equal strength of connectivity (i.e., total SC) to the ipsilateral cortex and that such a measure is a proxy for “accuracy.” While both of these assumptions may not be entirely valid, this figure does demonstrate that potential reductions in tracking from the medial thalamus does not significantly affect the EDpc1 measure.
Comment 2: While the methodology employed by the authors appears to be state-of-the-art, there exists uncertainty regarding its appropriateness for validation, given the well-documented issues of false positives and false negatives in probabilistic diffusion tractography, as discussed by Thomas et al. 2014 PNAS. Although replicating the results in both humans and non-human primates strengthens the study, a more compelling validation approach would involve demonstrating the method's ability to accurately trace known tracts from established tracing studies or, even better, employing phantom track data. Many of the control analyses the authors presented, such as track density, do not speak to accuracy.
In addition to or response to Reviewer 1 Comment 1, we would like to add the following:
We agree with the reviewer that tractography methods have known limitations. We would also like to point out that several studies have already performed the studies suggested by the reviewer. Many studies have compared tracts reconstructed from diffusion data using tractography methods to tracer-derived connections (eg. Thomas et al., 2014, as mentioned by the reviewer; Donahue et al., 2016, J Neurosci; Dauguet et al., 2007 NeuroImage; Gao et al., 2013 PloS One; van den Heuvel et al., 2015, Hum Brain Map; Azadbakht et al., 2015 Cereb Cortex; Ambrosen et al., 2020 NeuroIamge). Notably, studies comparing tractography and tracer-derived white matter tracts in the same animal (e.g. Grisot et al., 2021; Gao et al., 2013 PloS One) have demonstrated that tractography errors may be inflated in studies comparing tractography and tracer-derived connections in different animals.
Additionally, others have employed phantoms to assess the validity of tractography methods (e.g. Drobnjak et al., 2021). For the purposes of this manuscript, phantom data would not be an adequate control because phantom data would likely not capture the biological complexities of tracking subcortical white matter tracts and identifying projections within subcortical grey matter.
While a comparison of our tractography-derived ED measure to ED calculated on terminations from tracer studies within the thalamus from several somatomotor and associative regions in macaques would provide additional confidence for our results, such a control is certainly outside the scope of this study. Additionally, such a study would not provide a ground truth comparison for the human data. Even if this hypothetical experiment was performed, a negative finding would not refute our results, as any differences could be attributed to evolutionary differences. Unfortunately, there exists no ground truth to compare human white matter connectivity patterns to, which is why we stress-tested our results in as many ways as possible. These stress tests revealed that our main findings are very robust.
Specifically, as the key validity question of our study was whether there was a confound that systematically biased the ED measure as to make the hierarchy effect artifactual, the control analyses we performed to determine if track density, cortical geometry, bundle integrity, etc in fact do speak the robustness of the results. Regarding the track density analyses we argue that these control analyses do speaks to accuracy. The reviewer mentioned above that some cortical areas may be biased because their anatomical tracts may be more difficult to reconstruct using tractography. The mean streamline count is meant to reflect the density of a fiber bundle, but corticothalamic tracts that are more difficult to track will, by nature, have fewer streamline counts. So, the mean streamline not only reflects the density of a fiber bundle but also how easily that tract is to reconstruct. Therefore, if it was the case that cortical areas with more difficult to reconstruct white matter tracts to the thalamus are also more diffuse, then we should observe a strong positive correlation between the ED measure and the mean streamline count, which we tested directly and found only a weak correlation (Fig. S11). This is true for tracking to the entire thalamus, and the additional supplemental Figure S31 shows that reduced tracking to specific parts of the thalamus (e.g. the medial portion) also does not strongly relate to the ED measure. So, tracts that are more difficult to reconstruct may also be more diffuse, but this seems to add only a little noise and does not account for the strong relationship between the ED measure and T1w/T2w and RSFCpc1 measures the reflect the cortical hierarchy.
Comment 3: If tracking the medial thalamus is indeed less accurate, characterized by higher false positives and false negatives, it could potentially lead to increased variability among individual subjects. In cases where results are averaged across subjects, as the authors have apparently done, this could inadvertently contribute to the emergence of the "diffuse" motif, as described in the context of the associative cortex. This presents a critical issue that requires a more thorough control analysis and validation process to ensure that the main results are not artifacts resulting from limitations in tractography.
Additionally, conducting a control analysis to demonstrate that individual variability in tracking endpoints within the thalamus, when averaged across subjects, does not artificially generate a more diffuse connectivity pattern, is essential.
We thank the reviewer for bringing up this point, and the reviewer is correct that a simple group average of streamline counts across that thalamus could make some thalamic patterns appear more diffuse if those patterns vary slightly in location across people. The simplest way to address this concern is to show that diffuse patterns are present in individual subjects. Fig. 2 panels B, C, H, and I are all subject-level figures, which show that we can replicate the group level findings in Fig. 2 panels F, G. Specifically, Fig 2. Panels H and I show that the effect of association areas exhibiting more diffuse connectivity patterns within the thalamus relative to sensorimotor areas is generalizable across subjects.
To the reviewer’s point, the other way that averaged streamline counts could make focal connections seem diffuse is by averaging within cortical areas (e.g. to test the possibility that association areas may have highly variability focal patterns, and when averaged within the cortical area it makes these focal patterns appear more diffuse). To test this, we show that we can replicate the hierarchy effect at the vertex level, by calculating the extent of connectivity patterns for every cortical vertex and correlated vertex-level EDpc1 values to vertex-level T1w/T2w and RSFC_pc1 values (Fig S20).
Hopefully the data shown in Fig. 2 (replication at the individual level) and Fig. S20 (replication at the vertex level) ameliorate the reviewer’s concerns that averaging highly variable focal connectivity patterns within the thalamus (either across people or across vertices) does not artifactually produce diffuse thalamic connectivity patterns for associative cortical areas.
Comment 4: Because the authors included data from all thresholds, it seems likely that false positive tracks were included in the results. The methodology described seems to unavoidably include anatomically implausible pathways in the spatial extent analyses.
The thresholding approach taken in the manuscript aimed to control for inter-areal differences in anatomical connection strength that could confound the ED estimates. Here I am not quite clear why inter-areal differences in anatomical connection strength have to be controlled. A global threshold applied on all thalamic voxels might kill some connections that are weak but do exist. Those weak pathways are less likely to survive at high thresholds. In the meantime, the mean ED is weighted, with more conservative thresholds having higher weights. That being said, isn't it possible that more robust pathways might contribute more to the mean ED than weaker pathways?
This is a good point from the reviewer, and we appreciate them bringing up these points about our thresholding rationale. We would like to clarify two points: why it was appropriate for our question to threshold thalamic voxels for each cortical area separately and why we iteratively thresholded thalamic voxels.
Regarding thalamic connectivity differences between cortical areas: a global threshold would indeed exclude weak, but potentially true, connections. This was part of our rationale for thresholding thalamic voxels for each cortical area separately. Too conservative of a global threshold would exclude all thalamic voxels for some cortical areas and too liberal of a threshold would include many potentially false positive connections for other cortical areas. Our method of thresholding each cortical area’s thalamic voxels separately ensured that we were sampling thalamic voxels in an equitable manner across cortical areas. We updated the text to clarify this:
Methods section, pg. 11, section Framework to quantify the extent of thalamic connectivity patterns via Euclidean distance (ED)
“We used Euclidean distance (ED) to quantify the extent of each cortical area's thalamic connectivity patters. Probabilistic tractography data require thresholding before the ED calculation. To avoid the selection of an arbitrary threshold (Sotiropoulos et al., 2019, Zhang et al., 2022), we calculated ED for a range of thresholds (Figure 1a). Our thresholding framework uses a tractography-derived connectivity matrix as input. We iteratively excluded voxels with lower streamline counts for each cortical parcel such that the same number of voxels was included at each threshold. At each threshold, ED was calculated between the top x\% of thalamic voxels with the highest streamline counts. This produced a matrix of ED values (360 cortical parcels by 100 thresholds). This matrix was used as input into a PCA to derive a single loading for each cortical parcel. While alternative thresholding approaches have been proposed, this framework optimizes the examination of spatial patterns by proportionally thresholding the data, enabling equitable sampling of each cortical parcel's streamline counts within the thalamus.
This approach controlled for inter-areal differences in anatomical connection strength that could confound the ED estimates. In contrast, a global threshold, which is applied to all cortical areas, may exclude all thalamic streamline counts for some cortical areas that are more difficult to reconstruct, thus making it impossible to calculate ED for that cortical area, as there are no surviving thalamic voxels from which to calculate ED. This would be especially problematic for white matter tracts are more difficult to reconstruct (e.g. the auditory radiation), and cortical areas connected to the thalamus by those white matter tracts would have a disproportionate number of thalamic voxels excluded when using a global threshold.”
Regarding thalamic connectivity differences across the thalamus for a given cortical area, the thresholding method we use does include anatomically implausible connections in the ED calculation because we sample voxels iteratively, and as more and more thalamic voxels are included in the ED analysis the likelihood that they reflect spurious connections increases. This approach made the most sense to us, because there is no way to identify a threshold that only includes true positive connections. And since this method does not exist, we sampled all thresholds and leveraged the behavior of the ED metric across thresholds to quantify the spread of a connectivity pattern. As the reviewer points out, since the measure is effectively “weighted,” more “robust” or anatomically plausible pathways should contribute more to the EDpc1 rather than weaker pathways. This is exactly the balanced approach we aimed for: a measure that is driven by connections that have the highest likelihood of being a true positive but does not rely on an arbitrary threshold.
We did also replicate our main findings after thresholding and binarizing the data for separate thresholds, which show that our main effect was strongest only when thalamic voxels with the highest streamline counts (which are assumed to have a lower chance of being false positives) are included in the ED calculation (Fig. S5). This more traditional method of thresholding also supported our results, and increases our overall confidence that associative cortical areas have more diffuse connectivity patterns within the thalamus relative to somatomotor areas.
Comment 5: In the introduction, there is a bit of ambiguity that needs clarification. The overall goal of the study appears to be the examination of anatomical connectivity from the cortex to the thalamus, specifically whether a cortical region projects to a single thalamic subregion or multiple thalamic subregions. However, certain parts of the introduction also suggest an exploration of the concept of thalamic integration, which typically means a single thalamic region integrating input from multiple cortical regions (converging input). These two patterns, many cortical regions to one thalamic region versus one cortical region to many different thalamic regions, represent distinct and fundamentally different concepts that should be clarified in the manuscript.
We thank the reviewer for pointing out this ambiguity and have edited the introduction to clarify this point:
Our argument for a potential mechanism for integration is the following: because corticothalamic connectivity is topographically organized, if a cortical area has a more diffuse anatomical projection across the thalamus that means its connections overlap with more cortical areas. To the reviewer’s point, our argument is simply that one cortical area targeting multiple thalamic nuclei inherently suggests that such a cortical area has overlapping connectivity patterns with many other cortical areas in the same thalamic subregion. We have updated the introduction to clarify this further.
Intro, pg 1.
“Studies of cortical-thalamic connectivity date back to the early 19th century, yet we still lack a comprehensive understanding of how these connections are organized (see 13 and 14 for review). The traditional view of the thalamus is based on its histologically-defined nuclear structure (6). This view was originally supported by evidence that cortical areas project to individual thalamic nuclei, suggesting that the thalamus primarily relays information (15). However, several studies have demonstrated that cortical connectivity within the thalamus is topographically organized and follows a smooth gradient across the thalamus (16–21). Additionally, some cortical areas exhibit extensive connections within the thalamus, which target multiple thalamic nuclei (22? ). These extensive connections may enable information integration within the thalamus through overlapping termination patterns from different cortical areas, a key mechanism for higher-order associative thalamic computations (23– 25). However, our knowledge of how thalamic connectivity patterns vary across cortical areas, especially in humans, remains incomplete. Characterizing cortical variation in thalamic connectivity patterns may offer insights into the functional roles of distinct cortico-thalamic loops (6, 7).”
Discussion, pg 9. Section: The spatial properties of thalamic connectivity pat- terns provide insight into the role of the thalamus in shaping brain-wide information flow.
“In this study, we demonstrate that association cortical areas exhibit diffuse anatomical connections within the thalamus. This may enable these cortical areas to integrate information from distributed areas across the cortex, a critical mechanism supporting higher-order neural computations. Specifically, because thalamocortical connectivity is organized topographically, a cortical area that projects to a larger set of thalamic subregions has the potential to communicate with many other cortical areas. We observed that anterior cingulate cortical areas had some of the most diffuse thalamic connections. This observation aligns with findings from Phillips et al. that area 24 exhibited the most diffuse anatomical terminations across the mediodorsal nucleus of the thalamus relative to other prefrontal cortical area…”
Reviewer 3:
Comment 1: Potential weaknesses of the study are that it seems to largely integrate aspects of the thalamus that have been already described before. The differentiation between sensory and association systems across thalamic subregions is something that has been described before (see: Oldham and Ball, 2023; Zheng et al., 2023; Yang et al., 2020 Mueller, 2020; Behrens, 2003).
It is true that previous studies have shown that corticothalamic systems vary between sensory and associative cortical areas. Furthermore, there is much evidence that indicates that the sensory-association hierarchy is a major principle of brain organization in general. However, how and why these circuits are different is still not fully known, both across the whole brain and in corticothalamic circuits specifically.
Our study is the first to compare patterns of anatomical connectivity within the thalamus and determine if cortical areas vary in the extent of those patterns. So our main finding isn't that sensory and association cortical areas show differences in thalamic connectivity, it is that they specifically show differences in their pattern of connectivity within the thalamus. This provides a unique insight into how sensory and associative systems differ in their thalamic connectivity in primates.
Additionally, we show evidence that provides some insight into why these differences may exist. Although we cannot provide causal evidence, our data suggest that differences in patterns of anatomical connectivity within the thalamus were related to how different cortical areas process information via the thalamus, which aligns with speculations from Phillips et al 2021.
So our main finding isn't that sensory and association cortical areas show differences in thalamic connectivity, is it that they specifically show differences in their pattern of connectivity within the thalamus and these differences may help us understand how these cortical areas process information and, in turn, how they may support different types of computations, both of which are major goals in neuroscience. To better clarify this in the manuscript, we made the following changes:
Discussion, Paragraph 1, pg 8:
“This study contributes to the rich body of literature investigating the organization of cortico-thalamic systems in human and non-human primates. Prior research has shown that features of thalamocortical connectivity differ between sensory and association systems, and our work advances this understanding by demonstrating that these systems also differ in the pattern and spatial extent of their anatomical connections within the thalamus. Using dMRI-derived tractography across species, we show that these connectivity patterns vary systematically along the cortical hierarchy in both humans and macaques. These findings are critical for establishing the anatomical architecture of how information flows within distinct cortico-thalamic systems. Specifically, we identify reproducible tractography motifs that correspond to sensorimotor and association circuits, which were consistent across individuals and generalize across species. Collectively, this study offers convergent evidence that the spatial pattern of anatomical connections within the thalamus differs between sensory and association cortical areas, which may support distinct computations across cortico-thalamic systems.”
Comment 2: (1) Why not formally test the association between humans and macaques by bringing the brains to the same space?
We thank the reviewer for this query. We were primarily interested in using the macaque data as a validation of the human data, because it was acquired at a much higher resolution, there are no motion confounds, and it provides a bridge with the tract tracing literature in macaques. We are currently studying interspecies differences in patterns of thalamic connectivity, as well as extensions of our approach into structure-function coupling, and we believe these topics warrant their own paper.
Comment 3: (2) Possibly flesh out the differences between this study and other studies with related approaches a bit further.
We updated the discussion section to better clarify the differences in this study from previous research. See response to Reviewer 3 Comment 1 for text changes.
Comment 4: (3) The current title entails 'cortical hierarchy' but would 'differentiation between sensory and association regions' not be more correct? Or at least a reflection on how cortical hierarchy can be perceived?
We treat these phrases as synonymous terms. Our definition of cortical hierarchy is a smooth transition in features between sensory and motor areas to higher-order associative areas. The use of cortical hierarchy is meant to reflect that our measure continuously varies across the cortex. We updated the manuscript to make this clearer:
Abstract, pg 1.
“Additionally, we leveraged resting-state functional MRI, cortical myelin, and human neural gene expression data to test if the extent of anatomical connections within the thalamus varied along the cortical hierarchy, from sensory and motor to multimodal associative cortical areas.”
Comment 5: (4) For the core-matrix map, there is a marked left-right differences and also there are only two donors in the right hemisphere, possibly note this as a limitation?
We thank the reviewer for this observation. We updated Fig. S28 Panel D to show that the correspondence between EDpc1 and the Core-Matrix (CPc) cortical maps holds when the correlation was done for left and right cortex, separately.
RRID:AB_2313606
DOI: 10.1016/j.stem.2025.04.010
Resource: (Vector Laboratories Cat# BA-1000, RRID:AB_2313606)
Curator: @scibot
SciCrunch record: RRID:AB_2313606
RRID:AB_10807410
DOI: 10.1016/j.celrep.2025.115899
Resource: (Millipore Cat# MABN50, RRID:AB_10807410)
Curator: @scibot
SciCrunch record: RRID:AB_10807410
Document d'information détaillé sur les études de genre :
Ce document d'information examine les principales thématiques et les idées ou faits les plus importants concernant les études de genre, en s'appuyant sur les extraits de l'émission "France Culture Questions du soir : le débat Études de genre : pourquoi tant de polémiques".
1. La nature controversée des études de genre :
Certains les perçoivent comme une "remise en cause des repères", tandis que d'autres les considèrent comme un "outil utile pour penser les inégalités". * Controverses politiques et médiatiques : Aux États-Unis, des recherches ont été "freinées voire arrêtées sous l'administration Trump".
En France, des "polémiques régulières alimentent la méfiance, même dans les sphères ministérielles".
Le collectif "La Manif pour tous" s'oppose à l'intrusion du "gender à l'école", affirmant que cela "favoriserait l'indifférenciation entre les sexes et la théorie du genre", et que l'idéologie du genre à l'école "signifie propager l'idée aux enfants qu'ils peuvent changer d'identité sexuelle".
2. Qu'est-ce que les études de genre ?
Ça va des sciences sociales, à la philosophie, mais aussi à la biologie ou à toutes sortes de disciplines."
Pluralité des théories : Il n'existe pas une "théorie du genre" monolithique, mais "des théories qui peuvent s'opposer". Sylviane Agacinski, philosophe, confirme qu'il s'agit d'une "caricature", d'une "simplification" de parler d'une idéologie monolithique, car "il y a plusieurs théories, c'est-à-dire il y a aussi plusieurs usages du mot genre."
Un concept central : le "genre" comme "sexe social" : Le concept de genre a été "approprié par le féminisme à partir des années 70" et s'est transformé. Il signifiait initialement le "sexe social", comme l'a utilisé Ann Oakley.
Cette notion est cruciale pour comprendre que "quand on parle des femmes, on parle toujours à mon avis simultanément des femmes telles qu'elles sont dans telle ou telle société, dans telle ou telle culture.
C'est-à-dire que en tant que sexe [...] elles sont toujours socialisées, de même que le masculin est toujours socialisé."
3. Le débat sur la biologie et le sexe :
Critique du "biologisme" : Le reproche courant est que les études de genre nieraient l'importance de la biologie. Cependant, Éric Fassin explique que ce qui est critiqué n'est pas la biologie en tant que fait, mais le "biologisme", c'est-à-dire "l'idée que nous serions tout entier posé par cette définition."
La perspective d'Anne Fausto-Sterling : Cette biologiste féministe utilise le concept de genre pour "déconstruire l'idée même de notre rapport à la biologie".
Elle remet en question la dualité homme/femme, soulignant une "variété bien plus grande que le simple sexe mâle et femelle" et la possibilité de penser le sexe à "différents niveaux : chromosomal, hormonal, formation des organes génitaux, gonades, et développement humain".
Elle propose que la discipline biologique propose "des manières d'organiser le réel" mais que cela "ne veut pas dire que c'est le réel".
Catégorisation et hiérarchie : Éric Fassin insiste sur le fait que "catégoriser, c'est-à-dire organiser le monde selon des catégories, c'est pas simplement décrire de manière neutre, c'est toujours déjà organiser des hiérarchies."
Le point de vue de Sylviane Agacinski sur la reproduction et le sexe : Agacinski rejette l'approche de Fausto-Sterling comme un "biologisme réductionnisme". Pour elle, "la définition du sexe se donne par la fécondité, par la reproduction".
Elle considère que la distinction mâle/femelle est "universelle" et que les personnes intersexes, bien qu'humaines, sont des "exceptions" qui "confirment la règle".
Il utilise l'exemple de Donald Trump qui veut "restaurer le sexe biologique", montrant que "c'est un fantasme la biologie" dans ce cas.
La controverse sur "l'homme enceinte" découle de l'abandon de la stérilisation pour le changement de sexe, montrant que "c'est l'État, c'est la politique qui détermine le sexe." Sylviane Agacinski conteste l'idée que l'on puisse "changer de sexe" facilement, affirmant que les réalités physiologiques persistent.
4. Les études de genre face à l'individualisme et aux normes sociales :
Les violences sexuelles en sont un exemple, où il y a eu "une prise de conscience que il y a des normes démocratiques, c'est-à-dire de respecter la liberté, c'est-à-dire la capacité de consentir et l'égalité".
5. Pourquoi les études de genre cristallisent-elles tant de polémiques ?
Touche à l'intimité et aux peurs : Éric Fassin explique que la controverse vient du fait que "ça touche à notre intimité et mobiliser l'intimité, les peurs sur l'intimité et sur les changements de l'ordre amoureux, de l'ordre sexuel et bien c'est politiquement efficace".
Un langage politique pour les rapports de pouvoir : Il souligne une deuxième partie de la définition des études de genre, telle que donnée par Joan Scott : "une manière de signifier les rapports de pouvoir".
Cela signifie que le genre "ne parle pas seulement des hommes et des femmes", mais aussi d'"immigration, de laïcité, d'islam, d'identité nationale, etc."
C'est un "langage politique pour mobiliser des troupes" et jouer sur des "questions raciales, sur des questions économiques". * Instrumentalisation politique : Sylviane Agacinski reconnaît une "instrumentalisation" et une "utilisation politique". Elle évoque des "violences activistes" qui peuvent se mêler à la "réflexion et la théorie", ce qu'elle déplore.
Il alerte sur le fait que des leaders comme Trump, Milei, Orban et Poutine "défendent l'idée que l'ordre sexuel et bien ça ne doit pas bouger", ce qui a des "effets sur des gens bien réels et pas simplement sur des minorités sexuelles mais aussi sur des femmes."
Elles sont l'objet de vifs débats, souvent politisés, concernant la nature du sexe, la relativité des normes sociales et leur rôle dans la compréhension et la contestation des rapports de pouvoir.
Document de Synthèse : Violence en Milieu Scolaire – Sécurité vs. Santé Mentale
Source : Extraits de "France Culture Questions du soir : Sécurité ou santé mentale, quelles solutions contre la violence à l'école ?". Émission du 16.06.2025.
Invités : * Sylvain Berrios (Député indépendant apparenté Horizon), * Catherine Nafbecti (Secrétaire générale de la CFDT Éducation Formation Recherche Publique), * Johanna Dagorne (Sociologue et chercheuse à l'Observatoire international de la violence à l'école).
Introduction
L'assassinat d'une surveillante de 31 ans à Nogent par un élève de 14 ans a ravivé le débat sur la recrudescence des violences en milieu scolaire.
L'émission explore deux approches principales pour lutter contre ce phénomène : une réponse sécuritaire (portiques, fouilles, sanctions pénales) et une approche axée sur la santé mentale des jeunes (formation du personnel, détection des signaux faibles, augmentation des professionnels de santé scolaire).
I. Constat et Nature de la Violence
Hausse des violences graves mais pas généralisée :
Cependant, elle précise : "Nous syndicalement, on n'a pas forcément un retour de nos équipes nous disant c'est une explosion des violences permanentes.
Mais depuis le Covid, beaucoup de collègues nous disent, quel que soit leur métier qu'ils perçoivent que des élèves en plus grand nombre qu'auparavant sont fragiles en terme de rapport aux autres, de rapport à la collectivité".
Elle se fonde sur des statistiques montrant une diminution de 4% des faits sur les mineurs l'année dernière, tout en reconnaissant une augmentation des violences "paroxistiques".
Origine de la violence : Société vs. Génération :
Il établit un lien entre cette exposition, la perte de repères d'autorité et les actes dramatiques, comme celui de Nogent.
Elle souligne plutôt "la question à la fois du sexe des auteurs, sur la question de la masculinité toxique, sur la question des violences retournées contre autrui."
Elle explique que les filles expriment davantage la violence par des automutilations, tandis que les garçons le font par l'agressivité.
Le cas de l'agresseur de Nogent :
Il est adepte de jeux vidéos violents sans pour autant être addicte". Plus important, il "apparaît en perte de repère quant à la valeur de la vie humaine à laquelle il ne semble pas attacher une importance particulière."
II. Solutions Proposées : Sécurité ou Prévention Humaine ?
L'approche sécuritaire : Limites et contre-productivité :
Cependant, il reconnaît les limites : "vous savez très bien que mois de septembre, il y aura pas des portiques partout.
Donc tout ça et je vous rappelle encore une fois que le drame qui a eu lieu a eu lieu un moment où on a fouillé des sacs, où il y a des gendarmes, où c'était un jeune qui était un bon élève, qui était référent harcèlement."
Catherine Nafbecti et Johanna Dagorne critiquent vivement ces mesures :
Le couteau de Nogent n'aurait pas été détecté.
Les fouilles par les forces de l'ordre sont logistiquement complexes, retardent le début des cours et créent des attroupements dangereux aux abords.
Johanna Dagorne estime que la prévention situationnelle (portiques, vidéosurveillance) "ne va faire qu'augmenter en effet les rencœurs, la colère, les injustices et donc par conséquent va engendrer un manque d'autorité institutionnelle évidemment, mais également des violences."
Elle soutient que la violence étant majoritairement endogène (perpétrée par les élèves eux-mêmes), la prévention doit être "humaine".
Justice pénale et centres éducatifs fermés : Sylvain Berrios préconise une réponse pénale précoce, y compris des détentions dans des centres éducatifs.
Johanna Dagorne conteste l'efficacité de l'enfermement : "la dernière enquête de Bran et de Choqué [sur les contrats éducatifs fermés] montre qu'il y a 70 % de réitération de récidive.
On sait très bien que la question de l'enfermement de la coercition et là je suis juste uniquement sur le volet pragmatique, ça ne marche pas, c'est contreproductif."
L'approche basée sur la santé mentale et l'humain :
Détection des signaux faibles et formation du personnel :
C'est la priorité de Johanna Dagorne et Catherine Nafbecti.
Un élève "en repli sur soi, lorsqu'il est en décrochage, qu'il a de l'anxiété, une irritabilité soudaine" sont des signes faibles de danger.
Les chiffres sont alarmants : "un infirmier pour 1600 élèves, un psychologue pour 1500, un assistant ou une assistante sociale pour 4000 élèves et un médecin scolaire pour 13000 élèves.
Chez les médecins scolaires, le nombre de postes vacants dépasse par ailleurs les 40 %".
Les médecins et infirmières scolaires, majoritairement des femmes, perdent le sens de leur travail et ne rencontrent plus les élèves.
Il n'y a pas de moyens budgétaires suffisants pour la revalorisation de ces métiers.
Donc augmenter les salaires, il y aura beaucoup plus de personnes qui se porteront volontaires. C'est systémique". Catherine Nafbecti partage cet avis, ajoutant la question des conditions de travail et du remboursement des frais de déplacement.
La question de l'autorité et de la justice scolaire :
Les recherches montrent un lien fort entre la justice scolaire perçue par les élèves et la baisse des violences. Des évaluations encourageantes sont préférables à la coercition.
Elle questionne si la justice scolaire, bien qu'importante pour éviter le sentiment d'injustice, est suffisante pour prévenir les violences extrêmes.
Elle souligne le manque de moyens pour que les enseignants puissent être attentifs au harcèlement et aux signes de dégradation de la santé mentale.
III. Le Rôle des Familles et la Co-éducation
Responsabilisation des familles :
Il déplore que l'école ait parfois pris le pas sur les parents dans l'éducation. Il observe que les familles aisées et instruites accompagnent mieux leurs enfants que les familles fragiles. * Il suggère une "fusion" ou une "accroche" entre la santé scolaire et la santé familiale.
Limites et culpabilisation :
Johanna Dagorne estime que demander un effort aux parents les plus éloignés du système scolaire est "au mieux illusoire au pire culpabilisant", dans un système éducatif de plus en plus compétitif et "en train de trier l'élite plutôt qu'à réduire les inégalités sociales et scolaires".
Catherine Nafbecti défend les parents, affirmant qu'ils ont "à cœur de bien éduquer leurs enfants" et qu'ils ne sont pas démissionnaires, même dans les cas dramatiques comme celui de Nogent.
Elle souligne aussi le manque de temps des parents qui travaillent pour participer aux réunions scolaires.
Conclusion
L'accent est mis sur l'urgence d'investir massivement dans la santé mentale des jeunes, via la revalorisation et l'augmentation des professionnels de santé scolaire, ainsi que sur une approche humaine de la prévention, fondée sur la détection des signaux faibles et une "justice scolaire" cohérente.
La co-éducation entre famille et école est jugée essentielle, mais doit se faire sans culpabilisation des parents les plus fragiles.
Note de synthèse : Les différences cognitives entre les sexes
Source : Extraits de la conférence "Les différences cognitives entre les sexes : lesquelles ? pourquoi ? comment ?" animée par Franck Ramus.
Cette conférence aborde la question complexe des différences cognitives entre les sexes, en s'appuyant sur des données scientifiques pour démystifier les idées reçues et explorer les diverses causes possibles (biologiques, environnementales, sociales).
Thèmes principaux et idées clés :
1. Existence de différences cognitives moyennes et leur nature :
quel rôle a joué notre évolution dans cette différenciation ? sont-elles construites par l'éducation ? sont-elles universelles ou liées à une époque et une culture et un environnement ?
et surtout que peut-on vraiment prouver aujourd'hui quand on interroge la réalité de ces écarts ?"
2. Le cas des performances en mathématiques chez les filles et les garçons :
Les rapports officiels (ADEP, inspection générale) montrent que les filles sont minoritaires dans les options scientifiques au lycée et dans les filières scientifiques de l'enseignement supérieur, une situation stable depuis 20 ans après une progression.
Des évaluations nationales récentes ont révélé que les garçons sont en moyenne plus performants en mathématiques dès le milieu du CP.
Cet écart est spécifique aux mathématiques, car globalement, les filles ont de meilleurs résultats scolaires dans d'autres matières (français, langues, etc.).
Ce phénomène n'est pas franco-français et s'observe dans la plupart des pays de l'OCDE (évaluations PISA), à quelques exceptions près comme la Finlande où il n'y a pas de différence ou elle est inversée.
Citation : "dès le dès le milieu du CP il se passe quelque chose au cours du CP qui fait que tout d'un coup les filles commencent à perdre du terrain sur les garçons dans l'apprentissage des mathématiques et c'est assez spécifique aux mathématiques parce que finalement c'est dans un contexte où globalement les filles ont des meilleurs résultats scolaires que les garçons".
3. Analyse des causes potentielles des différences : un modèle multifactoriel :
Franck Ramus propose un modèle causal hypothétique incluant :
4. Analyse critique des facteurs sociaux :
Les filles issues de familles plus stéréotypiques ont de moins bons scores.
5. La perspective évolutionnaire et les différences à travers les espèces :
6. Lien entre la cognition, l'évolution et les mathématiques :
Les mathématiques ne sont pas une capacité cognitive primaire sélectionnée, mais un objet culturel complexe.
Capacités visuo-spatiales (rotation mentale 3D) : Les hommes sont en moyenne meilleurs.
Des études chez les bébés (3-16 mois) et des "expériences de la nature" (filles avec hyperplasie congénitale des surrénales) suggèrent une origine biologique précoce, potentiellement liée à la testostérone prénatale/postnatale.
Citation : "c'est un indice possible que pour une capacité cognitive bien spécifique il pourrait y avoir un avantage au garçon qui soit observé dès la naissance et que peut-être il est là parce que il a été sélectionné".
Cependant, le lien entre ces capacités spécifiques et la performance globale en mathématiques est complexe et non linéaire, car les mathématiques sollicitent de nombreuses fonctions cognitives (mémoire, raisonnement, etc.) où les avantages sont répartis entre les sexes.
Les tests de compétences mathématiques précoces chez les bébés (dès 6 mois) ne montrent pas de différences entre garçons et filles.
7. Différences de préférences et de variabilité :
Cette plus grande variabilité masculine est expliquée par la plus grande variabilité reproductive des mâles (potentiel de descendants extrêmes), ce qui favorise la sélection de phénotypes extrêmes (très bons ou très mauvais).
Conséquence : Même avec une moyenne égale, une plus grande variabilité masculine signifie qu'il y aura plus d'hommes aux extrêmes (à la fois les meilleurs et les moins bons), ce qui impacte les filières sélectives (ingénierie, sciences).
Conclusion générale :
Concernant les différences en mathématiques :
En somme, l'explication des différences en mathématiques est multifactorielle, impliquant des interactions complexes entre facteurs biologiques, environnementaux et sociaux, sans qu'aucune cause unique ne puisse tout expliquer.
La focalisation sur les mathématiques est justifiée par leur lien avec les carrières rémunératrices, mais une attention similaire devrait être portée aux difficultés des garçons en français/langage.
Synthèse des Thèmes et Idées Principales : La Grande Solitude des Adolescents et le Manque de Liens Sociaux
L'émission "France culture être et savoir" aborde la problématique croissante de la solitude chez les adolescents, un phénomène qui dépasse même les questions de santé mentale, et ses conséquences sur la violence et le bien-être général des jeunes.
Les intervenantes, Louia Bris (coordinatrice jeunesse), Laurence Touroude (spécialiste des sciences de l'éducation), Sophie Vénétitay (secrétaire générale du SNES FSU) et Marie-Rose Morau (pédopsychiatre), explorent les causes de cette solitude et les pistes pour recréer du lien social.
1. La Solitude Existentielle et Banale des Adolescents
Le thème central de l'émission est la "solitude immense et banale de nos enfants" (Introduction de l'émission).
Marie-Rose Morau souligne que les adolescents d'aujourd'hui se sentent "isolé", ce qui est "la chose la plus grave et la plus importante qui arrive à nos adolescents aujourd'hui".
Cette solitude est si prégnante que même une jeune stagiaire de seconde, Agathe, témoigne de classes où "on est tous restés inconnus les uns des autres", où "on se parlait pas tant que ça" et où elle ne connaissait pas "les prénoms des gens de ma classe".
Ce manque de lien est perçu comme une "solitude existentielle" par les adolescents eux-mêmes, qui, une fois hospitalisés, apprécient les échanges mais craignent que "quand je vais sortir, ma solitude va recommencer."
2. Le Manque de Liens Sociaux et la Difficulté à "Vivre Ensemble"
Plusieurs facteurs sont identifiés comme contribuant à ce manque de lien social :
Cela conduit à une méfiance entre eux car "ils se connaissent pas bien qui justement qui a pas ces liens de générosité de et de fraternité". L'école, qui devrait être un lieu collectif, a selon elle "renoncé à être ce lieu collectif".
L'impact des écrans et des réseaux sociaux : Louia Bris observe que "de plus en plus, les parents vont plutôt nous dire en fait, il préfère rester à la maison, il préfère être devant ses écrans". Agathe, la stagiaire, l'explique directement : "Les jeunes passent de nos jours beaucoup plus de temps sur les réseaux sociaux qu'en personne avec les gens parce que bah ils peuvent communiquer, ils peuvent s'envoyer des messages, des vocaux comme si c'était la vie réelle." Ce comportement entraîne un repli sur soi où des élèves "s'enferment, ils ont leur casques, leur téléphone, ils parlent à personne".
La disparition des espaces collectifs : Les intervenantes soulignent le manque de lieux où les adolescents peuvent se rassembler et construire ensemble. Marie-Rose Morau déplore l'absence de "lieux où ces adolescents construisent finalement contre nous, j'allais dire contre la génération d'avant qui a encore le pouvoir, construisent ses propres valeurs". Sophie Vénétitay ajoute que "la démocratie lycéenne, la démocratie collégienne, ça fait partie de tous ces espaces qui aujourd'hui nous manquent pour créer du collectif et créer du vivre ensemble aujourd'hui." Elle cite également la disparition progressive des "clubs, les associations, la maison des lycéens".
L'incapacité à décoder les émotions et interagir : Louia Bris, travaillant en centre social, constate une "incapacité à des moments à créer du lien avec les autres" et une difficulté à comprendre "ce que l'autre ressent".
Ce constat est partagé par les écoles, centres de loisirs et clubs de sport locaux, tous d'accord sur le fait que les jeunes "ne savent vraiment plus communiquer".
3. Les Conséquences du Manque de Lien : Violence, Souffrance Psychologique et Isolement du Personnel
Le manque de liens a des répercussions graves :
4. Pistes et Solutions : Recréer du Lien et du Collectif Malgré la gravité de la situation, des solutions sont esquissées :
En conclusion, l'émission met en lumière une crise profonde du lien social chez les jeunes, exacerbée par les évolutions sociétales et éducatives.
Face à cette "angoisse de cette solitude" (Laurence Touroude), il est impératif de repenser collectivement les cadres et les espaces qui permettent la construction du "vivre ensemble" et le partage de la parole.
régime écosystémique
Pour penser les bibliothèques numériques dans toute leur complexité, il est nécessaire d’articuler les dimensions d’infrastructure, de plateforme et d’écosystème numérique. Il faudrait, selon moi, revenir là-dessus et reprendre l'articulation de ces dimensions. L’infrastructure constitue le socle invisible : elle rassemble les normes, les bases de données, les formats et les protocoles qui rendent possible la structuration et la circulation de l’information. La plateforme, quant à elle, donne forme à cette infrastructure en la rendant accessible à travers des interfaces, des outils de consultation et des dispositifs de médiation destinés aux usagers. Ces deux niveaux s’inscrivent dans un écosystème numérique plus large, où interviennent, comme vous le suggérez, des acteurs institutionnels, des politiques techniques et culturelles, des usages sociaux, et des logiques économiques. C’est dans l’interdépendance de ces trois dimensions que se joue la capacité d’un dispositif comme celui de la BnF à répondre, ou pas, aux enjeux actuels de diffusion, de mémoire et d'organisation/appropriation des contenus dans le (dés)ordre des savoirs. Ainsi, la re-médiation ne serait pas un simple transfert de support, mais un processus à plusieurs niveaux (technique, symbolique, institutionnel) qui mobilise infrastructure, plateforme et écosystème comme autant de conditions médiatrices (si l'on redéfinit cette notion ainsi), voir commentaire précédent aussi). Repenser la re-médiation à l’ère de la datafication impliquerait donc d’en reconnaître la dimension systémique, en l’inscrivant dans ces couches emboîtées du numérique. Je pense qu'il faut préciser ces aspects et ces pistes que vous ouvrez pour consolider votre argumentaire général.
plateforme
En outre, pourquoi ici limiter la discussion à la notion de plateforme ici ? Si le terme plateforme est couramment utilisé pour désigner la bibliothèque numérique de la BnF, il demeure partiel pour rendre compte de l’ensemble du dispositif à l’œuvre. Une plateforme renvoie principalement à l’interface visible offerte aux usagers : un environnement de consultation, de navigation et d’appropriation des contenus. En revanche, l’infrastructure désigne aussi l’ensemble des couches techniques, logicielles et normatives sous-jacentes – bases de données, formats de métadonnées, protocoles d’interopérabilité, dispositifs d’archivage – qui rendent possible cette expérience numérique. Autrement dit, la plateforme repose sur une infrastructure, mais ne l’épuise pas. Penser la bibliothèque numérique uniquement en termes de plateforme risque donc d’en réduire la portée : c’est aussi une infrastructure documentaire complexe, au croisement de logiques de conservation, de standardisation, de connectivité et de médiation. Une analyse complète de la re-médiation opérée par la BnF gagnerait ainsi à articuler ces deux niveaux.
re-médiation (numérique)
Il conviendrait de décider si l’on souhaite poursuivre avec le concept de re-médiation. La troisième phase décrite semble en relever, mais dans une forme particulière : alors même qu’on en a plutôt proposé une lecture en termes de « médiation des médiations », marquant une rupture ou un déplacement par rapport à la re-médiation. Ce retour à la notion de re-médiation impliquerait donc d’en adopter une acception élargie ou repensée, capable d’intégrer les logiques de modélisation, d’abstraction et de traitement algorithmique propres à la datafication.
Les vertus de la représentation cartographique se doublent donc de contraintes sémiotiques telles, notamment dans l’agencement en deux dimensions contiguës, qu’un impératif de cohérence peut émerger du medium lui-même et non de son objet, escamotant ici les pratiques extractivistes nécessaires à la fabrique de la liquidité des données : notamment les chaînes de production et de traitement des données, le travail amont sur celles-ci, l’infrastructure matérielle, énergivore et polluante, mais également la disparition de tout ce qui ne se transforme pas en numérique, qui ne se datafie pas.
Simplifier et clarifier le propos, la phrase est bien longue et chargée.
En ce sens, cette étape de “datafication” ne peut pas exactement être envisagée comme une re-médiation au sens propre du terme, s’agissant plutôt de la construction d’un tiers espace d’équivalence générale entre des unités minimales de construction de sens, circulables et manipulables en-deçà ou au-delà de tout cloisonnement historiquement hérité. Il s’agirait en quelque sorte de la médiation des médiations
Vous touchez ici un point conceptuel intéressant et stimulant : si la datafication produit un « tiers espace d’équivalence générale » entre unités signifiantes, peut-on encore parler de re-médiation au sens classique, par ex. Bolter & Grusin, 1999 (mais re-médiation, dans le cadre de ce texte n'a pas encore été définie, cela demeure un enjeu ici encore, comme tout au long de l'exercice) ? Ou alors s'agit-il d’un niveau méta, que l'on pourrait qualifier de « médiation des médiations » ? En supposant, la médiation (au sens classique), je comprends que, dans cette perspective, la datafication ne relève pas exactement de la re-médiation (au sens classique), qui suppose une transposition identifiable d’un média dans un autre. Il ne s’agit pas ici de reformuler un contenu dans un nouveau support (comme dans le passage de l’imprimé au numérique), mais de transformer des objets déjà médiatisés — œuvres numérisées, notices, corpus — en unités minimales d’information, interopérables, manipulables, et recombinables. Ce processus opère ainsi un changement de régime : il ne re-médie pas des contenus directement, mais restructure les médiations elles-mêmes en les inscrivant dans un espace d’équivalence générale régi par des normes de traitement des données. On peut ainsi parler, à juste titre, de médiation des médiations, pour souligner que la datafication agit à un niveau second, en réagencant les formes de circulation, de description et d’accès déjà instituées. Cela suggère, peut-être cependant, que la re-médiation doit être repensée ou élargie, de fait, pour intégrer ces formes de modélisation algorithmique et de recomposition documentaire qui excèdent la logique de représentation médiatique. Une autre catégorie de re-médiation peut-être ? C'est à discuter et à clarifier davantage, mais intéressant!
Comparé notamment à la fondation de Rome
Peut-être partager et mettre entre parenthèse la citation qui réfère à cette comparaison audacieuse.
l’étude des pratiques de navigation des usagers de Gallica
Pour faire un juste parallèle avec P.L.A.O, il faudrait encore s'interroger, plus largement, sur les pratiques de lecture (lecture distante) et pas seulement sur les pratiques de navigation, ou encore sur « la pluralisation des modalités d’accès au contenu en fonction de l’unité documentaire pertinente pour l’usage, ce qui se traduit par des régimes de navigation ou de consultation pluriels et une conséquente relinéarisation dans la production de sens par cheminement » qui est évoquée plus bas dans le paragraphe suivant.
la re-médiation de la BnF, la bibliothèque numérique se situe donc à l’intersection d’une collection numérisée et d’une plateforme de consultation
Dire que la bibliothèque numérique est à l’intersection d’une collection numérisée et d’une plateforme de consultation est une simplification utile, mais insuffisante. Il vaudrait mieux élargir cette description à au moins trois dimensions : 1) une infrastructure documentaire (bases de données, formats, standards, interopérabilité); 2) une médiation numérique (interface, outils de recherche, éditorialisation, parcours thématiques); 3) un espace de production et d’appropriation (usages savants, pédagogiques, citoyens; annotations, projets collaboratifs). Autrement dit, la re-médiation opérée par la BnF à travers sa bibliothèque numérique ne peut être réduite à l’articulation entre une collection numérisée et une simple plateforme de consultation. Elle se situe plutôt à l’intersection d’une infrastructure documentaire, d’une médiation éditoriale numérique, et d’un espace d’appropriation et d’usage — autant de dimensions qui transforment les modalités d’accès, d’interprétation/lecture et de valorisation de ses collections.
la définition de la médiation comme inscription d’une chose informe dans un régime signifiant, l’émergence du sens a lieu ici dans la pratique documentaire, c’est-à-dire dans la saisie par un·e lecteur·ice d’une unité documentaire à travers des media particuliers.
Proposer une source svp.
“pivot documentaire”
Que recouvrait cette notion de « pivot documentaire » au-delà de la centralisation des données, si c'est le cas?
n André Zysberg qui déclare publiquement que “la BNF n’est pas un laboratoire de recherche mais une bibliothèque” (
Je trouve également fort intéressante la question qui émerge au plan des orientations à savoir : Est-ce qu'une bibliothèque de recherche, comme une bibliothèque nationale, a un mandat de recherche ou simplement celui de servir les chercheurs ? Quel est son rapport à la recherche ? Je dirais que toutes les bibliothèques de recherche ont le mandat de soutenir la recherche. Certaines bibliothèques, notamment les bibliothèques nationales, ont aussi un mandat actif de production de recherche, explicite ou implicite, souvent en collaboration avec le milieu universitaire. Ce mandat de production semble croissant, notamment dans les domaines numériques, patrimoniaux et sociétaux. Library of Congress offre un exemple de cela; la BNF également en dépit des hésitations, apparemment, à certains moments de son évolution.
elon les travaux de Gaëlle Béquet, cette première étape de numérisation de la bibliothèque qui fait la part belle au “modèle de la lecture savante” se fait en opposition à “la démocratisation de l’accès au savoir et l’accueil de nouveaux lecteurs dans les salles de lecture” (Béquet 2014, p. 63). Pour Alain Giffard, alors directeur de l’informatique et des nouvelles technologies de l’EPBF, cela conduit à des frictions entre les “prérogatives patrimoniales” de l’historique Bibliothèque nationale et “l’effort technologique” de la nouvelle Bibliothèque de France (Giffard interrogé par Béquet, 2014, pp. 66-67). Et Gaëlle Béquet de noter les “groupes marginaux” de cette bibliothèque numérique projetée que fut le P.L.A.O. : outre les usagers non académiques, “les bibliothécaires, et particulièrement ceux de la Bibliothèque nationale (BN), les éditeurs et les auteurs” (Béquet 2014, p. 63sq).
Expliquer plus clairement les groupes en tension, les tensions, et les objectifs poursuivis par les uns et les autres. La cartographie de cette « controverse » et les enjeux pourrait être élaborer davantage et contextualisés dans une perspective bibliothéconomique. En cela, je crois que mon commentaire rejoint celui de E Bermes plus avant.
pan outillage du projet P.L.A.O.
Plutôt : le volet outillage du projet P.L.A.O. ou encore : la composante dédiée aux outils et aux dispositifs de soutien dans le cadre du projet P.L.A.O
Cette re-médiation à caractère anthropologique peut apparaître aussi quelque peu téléologique car orientée vers la concrétisation d’un dispositif logiciel modulaire.
Expliquer.
La numérisation de la bibliothèque est donc d’abord envisagée comme la numérisation, et l’outillage en conséquence, des pratiques de lecture et d’écriture des chercheur·euses.
Fort intéressant !
remédier
Le terme re-médiation est-il ici le plus pertinent ? Si tel est le cas — et en supposant qu’il ait été défini préalablement — il serait pertinent de développer/élaborer davantage l’idée selon laquelle la BnF et/ou Gallica (à préciser) ont fait l’objet de multiples formes de re-médiation, dont la numérisation ne constitue qu’une modalité parmi d’autres. Celle-ci ne saurait, à elle seule, épuiser la richesse des processus de transformation, de recontextualisation et de reconfiguration à l’œuvre dans l’environnement numérique. Par ailleurs — toujours dans l’hypothèse où re-médiation est bien le terme visé — il conviendrait de souligner que ce processus ne doit pas être envisagé uniquement du point de vue des œuvres, comme c’est souvent le cas, mais aussi à travers celui des pratiques, des gestes documentaires, ou des « opérations intellectuelles ». Cela dit, la définition attendue apportera peut-être les précisions nécessaires à cet égard.
un essai libre
d'une analyse et d'une réflexion ? La notion d' « essai libre » pourrait peut-être dévaloriser votre propos.
Reviewer #3 (Public review):
Summary:
This manuscript by Toth et al reveals a conserved phosphorylation site within the RIN4 (RPM1-interacting protein 4) R protein that is exclusive to two of the four nodulating clades, Fabales and Rosales. The authors present persuasive genetic and biochemical evidence that phosphorylation at the serine residue 143 of GmRIN4b, located within a 15-aa conserved motif with a core five amino acids 'GRDSP' region, by SymRK, is essential for optimal nodulation in soybean. The experimental design and results are robust, the manuscript's discussion has been satisfactorily updated. Results described here are important to understand how the symbiosis signaling pathway prioritizes associations with beneficial rhizobia, while repressing immunity-related signals.
Strengths:
The manuscript asks an important question in plant-microbe interaction studies with interesting findings.
Overall, the experiments are detailed, thorough and very well-designed. The findings appear to be robust.
The authors provide results that are not overinterpreted and are instead measured and logical.
Weaknesses:
No major weaknesses.
Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public Review):
The authors set out to illuminate how legumes promote symbiosis with beneficial nitrogen-fixing bacteria while maintaining a general defensive posture towards the plethora of potentially pathogenic bacteria in their environment. Intriguingly, a protein involved in plant defence signalling, RIN4, is implicated as a type of 'gatekeeper' for symbiosis, connecting symbiosis signalling with defence signalling. Although questions remain about how exactly RIN4 enables symbiosis, the work opens an important door to new discoveries in this area.
Strengths:
The study uses a multidisciplinary, state-of-the-art approach to implicate RIN4 in soybean nodulation and symbiosis development. The results support the authors' conclusions.
Weaknesses:
No serious weaknesses, although the manuscript could be improved slightly from technical and communication standpoints.
Reviewer #2 (Public Review):
Summary:
The study by Toth et al. investigates the role of RIN4, a key immune regulator, in the symbiotic nitrogen fixation process between soybean and rhizobium. The authors found that SymRK can interact with and phosphorylate GmRIN4. This phosphorylation occurs within a 15 amino acid motif that is highly conserved in Nfixation clades. Genetic studies indicate that GmRIN4a/b play a role in root nodule symbiosis. Based on their data, the authors suggest that RIN4 may function as a key regulator connecting symbiotic and immune signaling pathways.
Overall, the conclusions of this paper are well supported by the data, although there are a few areas that need clarification.
Strengths:
This study provides important insights by demonstrating that RIN4, a key immune regulator, is also required for symbiotic nitrogen fixation.
The findings suggest that GmRIN4a/b could mediate appropriate responses during infection, whether it is by friendly or hostile organisms.
Weaknesses:
The study did not explore the immune response in the rin4 mutant. Therefore, it remains unknown how GmRIN4a/b distinguishes between friend and foe.
Reviewer #3 (Public Review):
Summary:
This manuscript by Toth et al reveals a conserved phosphorylation site within the RIN4 (RPM1-interacting protein 4) R protein that is exclusive to two of the four nodulating clades, Fabales and Rosales. The authors present persuasive genetic and biochemical evidence that phosphorylation at the serine residue 143 of GmRIN4b, located within a 15-aa conserved motif with a core five amino acids 'GRDSP' region, by SymRK, is essential for optimal nodulation in soybean. While the experimental design and results are robust, the manuscript's discussion fails to clearly articulate the significance of these findings. Results described here are important to understand how the symbiosis signaling pathway prioritizes associations with beneficial rhizobia, while repressing immunity-related signals.
Strengths:
The manuscript asks an important question in plant-microbe interaction studies with interesting findings.
Overall, the experiments are detailed, thorough, and very well-designed. The findings appear to be robust.
The authors provide results that are not overinterpreted and are instead measured and logical.
Weaknesses:
No major weaknesses. However, a well-thought-out discussion integrating all the findings and interpreting them is lacking; in its current form, the discussion lacks 'boldness'. The primary question of the study - how plants differentiate between pathogens and symbionts - is not discussed in light of the findings. The concluding remark, "Taken together, our results indicate that successful development of the root nodule symbiosis requires cross-talk between NF-triggered symbiotic signaling and plant immune signaling mediated by RIN4," though accurate, fails to capture the novelty or significance of the findings, and left me wondering how this adds to what is already known. A clear conclusion, for eg, the phosphorylation of RIN4 isoforms by SYMRK at S143 modulates immune responses during symbiotic interactions with rhizobia, or similar, is needed.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
I have no major criticism of the work, although it could be improved by addressing the following minor points:
(1) Page 8, Figure 2 legend. Consider changing "proper symbiosis formation" to "normal nodulation" or something that better reflects control of nodule development/number.
We thank you for the suggestion, the legend was changed to “...required for normal nodule formation” (see Page 10, revised manuscript)
(2) Page 9. Cut "newly" from the first sentence of paragraph 2, as S143 phosphorylation was identified previously.
Thank you for the suggestion, we removed “newly” from the sentence.
(3) Page 10, Figure 3. Panels B showing green-fluorescent nodules are unnecessary given the quantitative data presented in the accompanying panel A. This goes for similar supplemental figures later.
We appreciate the comment; regarding Figure 3 (complementing rin4b mutant, we updated the figures according to the other reviewer’s comment) and Suppl Figure 6 (OE phenotype of phospho-mimic/negative mutants), we removed the panels showing the micrographs. At the same time, we did not modify Figure 2 (where micrographs showing transgenic roots carrying the silencing constructs) for the sake of figure completeness. (See Page 10, revised manuscript)
(4) Consider swapping Figure 3 for Supplemental Figure S7, which I think shows more clearly the importance of RIN4 phosphorylation in nodulation.
We appreciate the comment and have swapped the figures according to the reviewer’s suggestion. Legend, figure description, and manuscript text have been updated accordingly. (See page 12 and 38, revised manuscript)
(5) Page 10. Replace "it will be referred to S143..." with "we refer to S143 instead of ....".
We replaced it according to the comment.
(6) Page 11, delete "While" from "While no interactions could be observed...".
We deleted it according to the suggestion.
(7) Page 33, Fig S5. How many biological replicates were performed to produce the data presented in panel C and what do the error bar and asterisk indicate? Check that this information is provided in all figures that show errors and statistical significance.
Thank you for the remark. The experiment was repeated three times, and this note was added to the figure description. All the other figure legends with error bar(s) were checked whether replicates are indicated accordingly.
(8) Page 37, Fig S11, panel B. Are averages of data from the 2 biological and 3 technical replicates shown? Add error bars and tests of significant difference.
Averages of a total of 6 replicates (from 2 biological replicates, each run in triplicates) are shown. We thank the reviewer for pointing out the missing error bars and statistical test, we have updated the figure accordingly.
(9) Fig S12. Why are panels A, C, E, and G presented? The other panels seem to show the same data more clearly- showing the linear relationship between peak area ratio and protein concentration.
We have taken the reviewer’s comment into consideration and revised the figure, removing the calibration curves and showing only four panels. The figure legend has been corrected accordingly. (Please see page 43, revised masnuscript). The original figure (unlike other revised figures) had to be deleted from the revised manuscript,as it caused technical issues when converting the document into pdf.
Reviewer #2 (Recommendations For The Authors):
Some small suggestions:
(1) It's good to include a protein schematic for RIN4 in Figure 1.
We appreciate the reviewer’s suggestion and we have drawn a protein schematic and added it to Figure 1. The figure legend was updated accordingly.
(2) There appears to be incorrect labeling in Figure 2c; please double-check and make the necessary corrections.
With respect, we do not understand the comment about incorrect labeling. Would the reviewer please help us out and give more explanation? In Figure 2C, RIN4a and RIN4b expression was checked in transgenic roots expressing either EV (empty vector) or different silencing constructs targeting RIN4a/b.
Reviewer #3 (Recommendations For The Authors):
I enjoyed the level of detail and precision in experimental design.
A discussion point could be - What does it mean that nodule number but not fixation is affected? Is RIN4 only involved in the entry stage of infection but not in nodules during N-fixation?
Current/Our data suggest that RIN4 does indeed appear to be involved in infection. This hypothesis is supported by the findings that RIN4a/b was found phosphorylated in root hairs but not in root (or it was not detected in the root). The interaction with the early signaling RLKs also suggests that RIN4 is likely involved in the early stage of symbiosis formation.
How would the authors explain their observation "However, the motif is retained in non-nodulating Fabales (such as C. canadensis, N. schottii; SI Appendix, Figure S2) and Rosales species as well." What does this imply about the role in symbiosis that the authors propose?
We appreciate the reviewer’s question. The motif seems to be retained, however, it might be not only the motif but also the protein structure that in case of nodulating plants might be different. We have not investigated the structure of RIN4, how it would look based on certain features/upon interaction with another protein and/or post-translational modification(s). Griesman et al, (2018) showed the absence of certain genes within Fabales in non-nodulating species, we can speculate that these absent genes can’t interact with RIN4 in those species, therefore the lack of downstream signaling could be possible (in spite of the retained motif in non-nodulating species). At this point, there is not enough data or knowledge to further speculate.
qPCR analysis of symbiotic pathway genes showed that both NIN-dependent and NIN-independent branches of the symbiosis signaling pathway were negatively affected in the rin4b mutant. Please derive a conclusion from this.
We appreciate the comment, it also prompted us to correct the following sentence; original: “Since NIN is responsible for induction of NF-YA and ERN1 transcription factors, their reduced expression in rin4b plants was not unexpected (Fig. 5). “As ERN1 expression is independent of NIN (Kawaharada et al, 2017). The following sentences were also deleted as it represented a repetition of a statement above these sentences: “Soybean NF-YA1 homolog responded significantly to rhizobial treatment in rin4b plants, whereas NF-YA3 induction did not show significant induction (Fig. 5).“
We added the following conclusion/hypothesis: “Based on the results of the expression data presented above, it seems that both NIN-dependent and NINindependent branches of the symbiotic signaling pathways are affected in the rin4b mutant background. This indicates that the role of RIN4 protein in the symbiotic pathway can be placed upstream of CYCLOPS, as the CYCLOPS transcription activating complex is responsible (directly or indirectly) for the activation of all TFs tested in our expression analysis (Singh et al, 2014/47, 48).” (Please see Page 16, revised manuscript)
The authors are highly encouraged to write a thoughtful discussion that would accompany the detailed experimental work performed in this manuscript.
We appreciate the comment, and we did some work on the discussion part of the document. (Please see Pages 17-19, revised manuscript)
Some minor suggestions for overall readability are below.
What about immune signaling genes? Given that authors hypothesize that "Absence of AtRIN4 leads to increased PTI responses and, therefore, it might be that GmRIN4b absence also causes enhanced PTI which might have contributed to significantly fewer nodules." Could check marker immune signaling gene expression FLS2 and others.
We appreciate the reviewer’s comment, and while we believe those are very interesting questions/suggestions, answering them is out of the scope of the current manuscript. Partially because it has been shown that several defenseresponsive genes that were described in leaf immune responses could not be confirmed to respond in a similar manner in root (Chuberre et al., 2018). It was also shown that plant immune responses are compartmentalized and specialized in roots (Chuberre et al., 2018). If we were looking at immune-responsive genes, the signal might be diluted because of its specialized and compartmentalized nature. Another reason why these questions cannot be answered as a part of the current manuscript is because finding a suitable immune responsive gene would require rigorous experiments (not only in root, but also in root hair (over a timecourse) which would be a ground work for a separate study (root hair isolation is not a trivial experiment, it requires at least 250-300 seedlings per treatment/per time-point).
Regarding FLS2, it is known in Arabidopsis that its expression is tissue-specific within the root, and it seems that FLS2 expression is restricted to the root vasculature (Wyrsch et al, 2015). In our manuscript, we showed that RIN4a/b is highly expressed in root hairs, as well as RIN4 phosphorylation was detectable in root hair but not in the root; therefore, we do not see the reason to investigate FLS2 expression.
"in our hands only ERN1a could be amplified. One possible explanation for this observation is that primers were designed based on Williams 82 reference genome, while our rin4b mutant was generated in the Bert cultivar background." Is the sequence between the two cultivars and the primers that bind to ERN1b in both cultivars so different? If not, this explanation is not very convincing.
At the time of performing the experiment the genomic sequence of the Bert cultivar (used for generating rin4b edited lines) was not publicly available. In accordance with the reviewer’s comment, we removed the explanation, as it does not seem to be relevant. (See page 16, revised manuscript)
The figures are clear and there is a logical flow. The images of fluorescing nodules in Figure 2,3 panels with nodules are not informative or unbiased .
We appreciate the comment, as for Figure 3 (complementing rin4b mutant), we updated the figures according to the other reviewer’s comment and Suppl. Figure 6 (OE phenotype of phospho-mimic/negative mutants) we removed the panels showing the micrographs. At the same time, we did not modify Figure 2 (where micrographs showing transgenic roots carrying the silencing constructs) for the sake of figure completeness. (See pages 10, 12 and 38, revised manuscript)
What does the exercise in isolation of rin4 mutants in lotus tell us? Is it worth including?
Isolation of the Ljrin4 mutant suggests that RIN4 carries such an importance that the mutant version of it is lethal for the plant (as in Arabidospis, where most of the evidence regarding the role of RIN4 has been described), and an additional piece of evidence that RIN4 is similarly crucial across most land plant species.
Sentence ambiguous. "Co-expression of RIN4a and b with SymRKßΔMLD and NFR1α _resulted in YFP fluorescence detected by Confocal Laser Scanning Microscopy (SI Appendix, Figure S8) suggesting that RIN4a and b proteins closely associate with both RLKs." Were all 4 expressed together?
Thank you for the remark. Not all 4 proteins were co-expressed together. We adjusted the sentence as follows: “Co-expression of RIN4a/ and b with SymRKßΔMLD as well as and NFR1α resulted in YFP fluorescence…” I hope it is phrased in a clearer way. (See page 13, revised manuscript)
Minor spelling errors throughout.. Costume-made (custom made?)
Thank you for noticing. According to the Cambridge online dictionary, it is written with a hyphen, therefore, we added a hyphen and corrected the manuscript accordingly.
CRISPR-cas9 or CRISPR/Cas9? Keep it consistent throughout. CRISPR-cas9 is the latest consensus.
We corrected it to “CRISPR-Cas9” throughout the manuscript.
References are missing for several 'obvious statements' but please include them to reach a broader audience. For example the first 5 sentences of the introduction. Also, statements such as 'Root hairs are the primary entry point for rhizobial infection in most legumes.'.
Thank you for the comment. To make it clearer, we also added reference #1, after the third sentence of the introduction, as well as we added an additional review as reference. This additional review was also cited as the source for the sentence “Root hairs are the primary…” (Please see page 2, revised manuscript)
Can you provide a percent value? Silencing of RIN4a and RIN4b resulted in significantly reduced nodule numbers on soybean transgenic roots in comparison to transgenic roots carrying the empty vector control. Also, this wording suggests it was a double K.D. but from the images, it appears they were individually silenced.
We appreciate the reviewer's comment. We observed a 50-70% reduction in the number of nodules. We adjusted the text according to the reviewer's remark. (See page 9, revised manuscript)
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Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
Summary
This manuscript reports preliminary evidence of successful optogenetic activation of single retinal ganglion cells (RGCs) through the eye of a living monkey using adaptive optics (AO).
Strengths
The eventual goals of this line of research have enormous potential impact in that they will probe the perceptual impact of activating single RGCs. While I think more data should be included, the four examples shown look quite convincing. Weaknesses
While this is undoubtedly a technical achievement and an important step along this group's stated goal to measure the perceptual consequences of single-RGC activations, the presentation lacks the rigor that I would expect from what is really a methods paper. In my view, it is perfectly reasonable to publish the details of a method before it has yielded any new biological insights, but in those publications, there is a higher burden to report the methodological details, full data sets, calibrations, and limitations of the method. There is considerable room for improvement in reporting those aspects. Specifically, more raw data should be shown for activations of neighboring RGCs to pinpoint the actual resolution of the technique, and more than two cells (one from each field of view) should be tested.
We have expanded sections discussing both the methodology and limitations of this technique via a rewrite of the results and discussion section. The data used in the paper is available online via the link provided in the manuscript. We agree that a more detailed investigation of the strengths and limitations of the approach would have been a laudable goal. However, before returning to more detailed studies, we have shifted our effort to developing the monkey psychophysical performance we need to combine with the single cell stimulation approach described here. In addition, the optogenetic ChrimsonR used in this study is not the best choice for this experiment because of its poor sensitivity. We are currently exploring the use of ChRmine (as described in lines 93-97), which is roughly 2 orders of magnitude more sensitive. We have also been working on methods to improve probe stabilization to reduce tracking errors during eye movements. Once these improvements have been implemented, we will undertake the more detailed studies suggested here. Nonetheless, as a pragmatic matter, we submit that it is valuable to document proof-of-concept with this manuscript.
Some information about the density of labeled RGCs in these animals would also be helpful to provide context for how many well-isolated target cells exist per animal.
We agree. Getting reliable information about labeled cell density would be difficult without detailed histology of the retina, which we are reluctant to do because it would require sacrificing these precious and expensive monkeys from which we continue to get valuable information. We are actively exploring methods to reduce the cell density to make isolation easier including the use of the CAMKII promoter as well as the use of intracranial injections via AAV.retro that would allow calcium indicator expression in the peripheral retina where RGCs form a monolayer. It may be that the rarity of isolated RGCS will not be a fundamental limitation of the approach in the future.
Reviewer #2 (Public Review):
This proof-of-principle study lays important groundwork for future studies. Murphy et al. expressed ChrimsonR and GCaMP6s in retinal ganglion cells of a living macaque. They recorded calcium responses and stimulated individual cells, optically. Neurons targeted for stimulation were activated strongly whereas neighboring neurons were not.
The ability to record from neuronal populations while simultaneously stimulating a subset in a controlled way is a high priority for systems neuroscience, and this has been particularly challenging in primates. This study marks an important milestone in the journey towards this goal.
The ability to detect stimulation of single RGCs was presumably due to the smallness of the light spot and the sparsity of transduction. Can the authors comment on the importance of the latter factor for their results? Is it possible that the stimulation protocol activated neurons nearby the targeted neuron that did not express GCaMP? Is it possible that off-target neurons near the targeted neuron expressed GCaMP, and were activated, but too weakly to produce a detectable GCaMP signal? In general, simply knowing that off-target signals were undetectable is not enough; knowing something about the threshold for the detection of off-target signals under the conditions of this experiment is critical.
We agree with these points. We cannot rule out the possibility that some nearby cells were activated but we could not detect this because they did not express GCaMP. We also do not know whether cells responded but our recording methods were not sufficiently sensitive to detect them. A related limitation is that we do not know of course what the relationship is between the threshold for detection with calcium imaging and what the psychophysical detection threshold would have been an awake behaving monkey. Nonetheless, the data show that we can produce a much larger response in the target cell than in nearby cells whose response we can measure, and we suggest that that is a valuable contribution even if we can’t argue that the isolation is absolute. We’ve acknowledged these important limitations in the revised manuscript in lines 66-77.
Minor comments:
Did the lights used to stimulate and record from the retina excite RGCs via the normal lightsensing pathway? Were any such responses recorded? What was their magnitude?
The recording light does activate the normal light-sensing pathway to some extent, although it does not fall upon the RGC receptive fields directly. There was a 30 second adaptation period at the beginning of each trial to minimize the impact of this on the recording of optogeneticallymediated responses, as described in lines 222-224. The optogenetic probe does not appear to significantly excite the cone pathway, and we do not see the expected off-target excitations that would result from this.
The data presented attest to a lack of crosstalk between targeted and neighboring cells. It is therefore surprising that lines 69-72 are dedicated to methods for "reducing the crosstalk problem". More information should be provided regarding the magnitude of this problem under the current protocol/instrumentation and the techniques that were used to circumvent it to obtain the data presented.
The “crosstalk problem” referred to in this quote refers to crosstalk caused by targeting cells at higher eccentricities that are more densely packed, which are not represented in the data. The data presented is limited to the more isolated central RGCs.
Optical crosstalk could be spatial or spectral. Laying out this distinction plainly could help the reader understand the issues quickly. The Methods indicate that cells were chosen on the basis that they were > 20 µm from their nearest (well-labeled) neighbor to mitigate optical crosstalk, but the following sentence is about spectral overlap.
We have added a clearer explanation of what precisely we mean by crosstalk in lines 213-221.
Figure 2 legend: "...even the nearby cell somas do not show significantly elevated response (p >> 0.05, unpaired t-test) than other cells at more distant locations." This sentence does not indicate how some cells were classified as "nearby" whereas others were classified as being "at more distant locations". Perhaps a linear regression would be more appropriate than an unpaired t-test here.
The distinction here between “nearby” and “more distant” is 50 µm. We have clarified this in the figure caption. Performing a linear regression on cell response over distance shows a slight downward trend in two of the four cells shown here, but this trend does not reach the threshold of significance.
Line 56: "These recordings were... acquired earlier in the session where no stimulus was present." More information should be provided regarding the conditions under which this baseline was obtained. I assume that the ChrimsonR-activating light was off and the 488 nmGCaMP excitation light was on, but this was not stated explicitly. Were any other lights on (e.g. room lights or cone-imaging lights)? If there was no spatial component to the baseline measurement, "where" should be "when".
Your assumptions are correct. There was no spatial component to the baseline measurement, and these measurements are explained in more detail in lines 240-243.
Please add a scalebar to Figure 1a to facilitate comparison with Figure 2.
This has been done.
Lines 165-173: Was the 488 nm light static or 10 Hz-modulated? The text indicates that GCaMP was excited with a 488 nm light and data were acquired using a scanning light ophthalmoscope, but line 198 says that "the 488 nm imaging light provides a static stimulus".
The 488nm is effectively modulated at 25 Hz by the scanning action of the system. I believe the 10 Hz modulated you speak of is the closed-loop correction rate of the adaptive optics. The text has been updated in lines 217-219 to clarify this.
A potential application of this technology is for the study of visually guided behavior in awake macaques. This is an exciting prospect. With that in mind, a useful contribution of this report would be a frank discussion of the hurdles that remain for such application (in addition to eye movements, which are already discussed).
Lines 109-130 now offer an expanded discussion of this topic.
Reviewer #3 (Public Review):
This paper reports a considerable technical achievement: the optogenetic activation of single retinal ganglion cells in vivo in monkeys. As clearly specified in the paper, this is an important step towards causal tests of the role of specific ganglion cell types in visual perception. Yet this methodological advance is not described currently in sufficient detail to replicate or evaluate. The paper could be improved substantially by including additional methodological details. Some specific suggestions follow.
The start of the results needs a paragraph or more to outline how you got to Figure 1. Figure 1 itself lacks scale bars, and it is unclear, for example, that the ganglion cells targeted are in the foveal slope.
The results have been rewritten with additional explanation of methodology and the location of the RGCs has been clarified.
The text mentions the potential difficulties targeting ganglion cells at larger eccentricities where the soma density increases. If this is something that you have tried it would be nice to include some of that data (whether or not selective activation was possible). Related to this point, it would be helpful to include a summary of the ganglion cell density in monkey retina.
This is not something we tried, as we knew that the axial resolution allowed by the monkey’s eye would result in an axial PSF too large to only hit a single cell. The overall ganglion cell density is less relevant than the density of cells expressing ChrimsonR/GCaMP, which we only have limited info about without detailed histology.
Related to the point in the previous paragraph - do you have any experiments in which you systematically moved the stimulation spot away from the target ganglion cell to directly test the dependence of stimulation on distance? This would be a valuable addition to the paper.
We agree that this would have been a valuable addition to the paper, but we are reluctant to do them now. We are implementing an improved method to track the eye and a better optogenetic agent in an entirely new instrument, and we think that future experiments along these lines would be best done when those changes are completed.
The activity in Figure 1 recovers from activation very slowly - much more slowly than the light response of these cells, and much more slowly than the activity elicited in most optogenetic studies. Can you quantify this time course and comment on why it might be so slow?
We attribute the slow recovery to the calcium dynamics of the cell, and this slow recovery time is consistent with calcium responses seen in our lab elicited via the cone pathway. Similar time courses can be seen in Yin (2013) for RGCs excited via their cone inputs.
Traces from non-targeted cells should be shown in Figure 1 along with those of targeted cells.
We have added this as part of Figure 2.
analyse
Een analyse-eenheid is het object waar je uitspraken over doet in je onderzoek.
Simpel uitgelegd: Het is waar je iets over wil zeggen na je analyse.
Voorbeelden: Onderzoek je nieuwsartikelen? → Dan is het artikel de analyse-eenheid. Onderzoek je personen in interviews? → Dan is de persoon de analyse-eenheid. Onderzoek je tv-afleveringen? → Dan is de aflevering de analyse-eenheid.
Author response:
The following is the authors’ response to the previous reviews.
Recommendations for the authors:
Reviewer #1:
The authors addressed my previous concerns successfully. However, some critiques are addressed only in the response letter but not in the text (major comment 3, minor point 2). It will be great if they mention these in some parts of their manuscript.
Major 3: We now mention the effect of acs-2i on life span in the discussion, lines 475-480:
“Interestingly, acs-2 knockdown abolished glp-1 longevity (data not shown), consistent with previous work showing that NHR-49, a transcription factor that drives acs-2 expression, is required for glp-1 longevity (Ratnappan et al., 2014). Thus, inhibiting fatty acid β-oxidation promotes MML-1 nuclear localization under hxk-1i but abolishes lifespan extension, potentially due to epistatic effects on other transcription factors or processes.”
Minor 2: We now speculate on the differences concerning hxk-3 knockdown on MML-1 nuclear localization resulting from the low expression of hxk-3 in adults, lines 99-102:
“Among the three C. elegans hexokinase genes, hxk-1 and hxk-2 more strongly affected MML 1 nuclear localization in two independent MML-1::GFP reporter strains (Figure 1B, Supplementary Figure 1A), while hxk-3 had just a small effect on MML-1 nuclear localization, probably due to its low expression in adult worms (Hutter & Suh, 2016).”
Reviewer #2:
The authors have adequately addressed my previous concerns in their revised manuscript. However, I have one remaining minor concern regarding the link between lipid metabolism and MML-1 regulation. As proposed by the authors, HXKs modulate MML-1 localization between LD/mito and the nucleus. They have provided evidence supporting the roles of hxk-2 and the PPP in this regulatory process. Nonetheless, the involvement of hxk-1 and fatty acid oxidation (FAO) within this proposed framework remains unclear. Although FAO is generally believed to affect LD size, the potential effects of hxk-1 and FAO on LD should be investigated within the current study to further substantiate their model.
We thank the reviewer for this comment. We now examine how hxk-1 and acs-2 affect lipid droplet size. Interestingly, we found that knockdown of acs-2 and hxk-1 acs-2 double knockdown resulted in a mild but significant increase in LD size (Supplementary Figure 4I), supporting the notion that the two hexokinases regulate MML-1 via distinct mechanisms, reflected in the updated model (Figure 5E).