ist

ziemlich oft muss oder müssen im Text

Um unsere Forschungsfrage zu operationalisieren...

Wie schön, dass ihr unseren Sammelband zitiert 😍

Damit eine solche Messung möglich ist...

Ihr nutzt oft Gedankenstriche, vielleicht weniger nutzen

verlinkt glaube auf das Falsche? Jedenfalls nicht auf das Kapitel davor.

Die Forschungsfrage aus dem vorherigen Abschnitt

La ciencia de la información, a grandes rasgos, es una disciplina que se encarga de gestionar datos, documentos e información para que no se pierdan y puedan cumplir una función útil, más allá de simplemente almacenarlos, se trata de otorgarles valor y establecer un ciclo que permita organizarlos, analizarlos y utilizarlos con un propósito definido, de este modo, la información deja de verse como algo sin importancia y se transforma en un recurso con poder, que requiere procesos propios para su adecuada gestión y aprovechamiento.

Considero que un gran campo de la Ciencia de la Información esta movido por los avances tecnológicos y como los profesionales en esta área nos adaptamos. esta tecnología. A las técnicos computacionales, la ciencia de datos y la analítica. Eso mezclado con una interacción entre personas y organizaciones las cuales ya tienen un sistema de información.

Para mi la Ciencia de la Información es un campo interdisciplinario que se encarga de analizar cómo se genera, recolecta, organiza, almacena, recupera y transmite la información.

En lugar de centrarse solo en los cables o el código , se enfoca en el vínculo entre las personas y los datos como tal objetivo principal es asegurar que la información sea accesible y útil para quien la necesite.

Opino que es importante tener en que es una ciencia interdisciplinaria interdisciplinaria porque integra conocimientos y métodos de diversas áreas para comprender, organizar y facilitar el acceso a la información, como las disciplinas que aquí se mencionan

Vorschlag für Formulierung: „Dieses JupyterBook umfasst mehrere Kapitel, die jeweils als eigenständige Open Educational Resources (OER) konzipiert sind. Sie sind durch eine gemeinsame Forschungsfrage thematisch miteinander verbunden, können jedoch auch unabhängig voneinander bearbeitet werden

Hier würde ich eine Verlinkung setzen

wo kann ich die denn jetzt auswähle?

verlinken?

Das Raketen Symbol fehlt vor den Kapiteln die interaktive sind

Hier würde ich auch externe Ressourcen verlinken, eine Person ohne R Kenntnisse brauch wahrscheinliche eine ausführlichere Erklärung. Die Erklärungen hier sind sehr kurz

ich verstehe das nicht so ganz, ist das immer noch eine beschreibende Hinleitung, damit ich dann endlich irgendwann ins Notebook komme,n kann und was lerne oder lerne ich hier schon was?

eine info mit der ich ohne kenntnis des dashboards nichts anfangen kann. vielleicht schon mal ein screenshot?

vorgestellt.

in der Visualisierung? der Analyse?

die Kapitel xyz der Fallstudie setzen sich daher mit der Übersetzung theoretischer Konzepte in messbare Variablen auseinander.

yes! wobei: aus der datenquelle werden keine Indikatoren entwickelt, sondern generiert, oder? Aber die Exploration der Datenquelle, also ihrer Inhalte und Struktur hilft der Entwicklung (bzw. Auswahl)

warum befinden wir uns immer noch in einer Meta-Kommunikation? Solltes dies Teil der Präambel sein? Sollte es hier nicht um die Bedeutung der Fragestellung und ihrer Beantwortung gehen? Eine solche Erläuterung eröffnet gedanklichen Vektoren für die Übertragung. (also implizit die Frage beantworten, zu welcher Art von Forschung dies hier ein exemplarisches Vorgehen sein kann)

dieser exemplarischen Fallstudie

Wir gendern mit : nicht mit * -> Auf Vorschriften achten

Leerzeichen zu viel

Leerzeichen zu viel

sollen die Sätze als Bullet formatiert sein? Falls ja, dann ist der ganze Absatz falsch Formatiert.

Leerzeichen zu viel

ein Leerzeichen zu viel

Gendern vergessen

mit "Gesellschaft für Informatik"

Ich finde durch die Boxen ist es sehr viel Text und viel Klicken zu den Lernzielen. Vielleicht besser ohne Boxen?

Unverständlich, würde ich löschen. Es wirkt wie eingeschoben.

löschen

Ich würde diesen Teilsatz löschen, weil ich ihn überflüssig finde, denn es werden ja bereits grundlegende statistische Konzepte genannt. Außerdem macht er den Satz unnötig lang.

in diesem Abschnitt wird niemand direkt angesprochen. im nächsten Abschnitt 1.2 werden dann die Leserinnen mit SIE angesprochen

brauche ich die jetzt oder nicht?

Satz wirkt irgendwie abschreckend. Was sind städtische Strukturdaten?

nur entworfen?

würde ich löschen

hier stimmt die Formatierung nicht.

für wen ist dies geschrieben? Für den User, den Lehrenden oder für den Projektträger?

dies ist kein Konzept (dabei wurde die Vertraut mit Konzepten angeteasert)

für wen ist dies geschrieben, für Lehrende, für Lernende (aus welchen Disziplinen?). Ich habe den Eindruck, dass ich die Frage für mich gar nicht beantworten kann, da ich den Bedeutungsumfang von 'städtischen Strukturdaten' nicht kenne (also ich kann nicht sagen, ob das, was mich interessiert, hier drin ist)

Steht im Kontrast zu der ersten Seite, auf der steht, dass Lernende Vorkenntnisse in R brauchen. Dieser Satz gibt einem die Schlussfolgerung, dass man keine fortgeschrittene Person sein muss. Wohin gegen das aber im Vorhinein geschrieben stand.

stellen wir uns die Frage

in Form von

meint ihr stinkender? kenne "stickend" nicht

ist das notwendig zu erwähnen? oder ist es nicht selbstverständlich?

nicht mit in den bibliographischen Eintrag übernommen

Gibt es eine Variante, wie beide in einer Klammer stehen?

dieser ganze Abschnitt ist redundant zu dem darunter

WIR UNS oder SIE als ansprache oder die Anwesenden ohnr direkte Ansprache.. das variiiert

???

ganz schön langer Satz, am besten Zwei draus machen

erster Schritt und zunächst -> das doppelt sich

Sehr wissenschaftlich geschrieben, vielleicht vereinfachen? OERs sollen wissen leicht verständlich rüberbringen

Zuerst erklären wir Ihnen in dem Kapitel....

Link öffnet im selben Fenster und führt von der Fallstudie weg, evtl. im neuen Fenster/Tab öffnen

Ich würde den Satz hier nicht unterbrechen mit einen Code snippet sondern zu Ende führen und dann den Code snippet einfügen und mit den Text danach weiter machen.

vielleicht noch 1-2 Sätze zum Projekt

23 See: Wikidata:Data round-tripping, https://www.wikidata.org/w/index.php?title=Wikidata:Data_round-tripping&oldid=2440906511

wird unten nicht aufgeführt und verlinkt auf spätere Seite

In einem Satz erklären, was NLPs sind

"Entwickeln Sie" oder "entwickeln wir"

Irgendwo erklären, was quantiative Methoden leisten, wie sie die Literaturwissenschaft voran bringen können

Erklären, was ein Korpus ist

Siehe mein Review von der Text2 Fallstudie zu Layout (Marthe). Die Abbildung kann ablenken

Es sind noch keine Links drin

Ihr habt hier noch nicht erklärt, was QUADRIGA ist. Den Block zu QUADRIGA am besten direkt am Anfang der Präambel #PR

yeah! 🥳

Sehr schön formuliert. Immer die Personen direkt ansprechen und somit aktivieren

Es setzt den neuen Spiegelstrich nicht um

fehlt hier ein Verb ?

Vielleicht kurz ein paar Beispiele für Internet-Browser nennen

Hier habt ihr das "wir" drin -> überlegen wir die Nutzenden am besten angesprochen werden sollen

Der Code ist in Jupyter Notebooks integriert und beschrieben (?)

Für die Fallstudie sind für Sie ... aufbereitet oder für die Fallstudie haben wir für Sie ... aufbereitet

Wort überflüssig; außerdem inkonsistent, denn vorher wird die lesende Person auch nicht direkt angesprochen

... und Tabellen die Hauptdatenform in der Verwaltung sind?

Vorschlag für Formulierung: Im 1. Schritt wird das notwendige Hintergrundwissen vermittelt.

statt zeigen: Wo lassen sich ... feststellen?

lies sich seltsam. "Hintergrundwissen .. vermitteln". Eher: Relevante Fach- und Kontextwissen aufzuarbeiten.

fortgeschrittene in was?

vielleicht nicht ihre wichtigkeit, sondern ihre Wirkung/Funktionalität benennen. also: Visualisierungen sind ein bewährtes Mittel der ..., um zu ..

unklar, warum es hier Überschriften gibt. Darüber steht bereits ".. Leitfragen:" Lösung: Unsichtbar schalten, falls dies für Anker-Link-Funktion bestehen bleiben muss

Anstelle

Dies irritiert, weil der Grund nicht klar ist. Mein Vorschlag: "Weil wir die Forschungsschritte exemplarisch darlegen wollen, skizzieren wir hier ein Szenario, in dem die hier adressierten Themen relevant sind".

Vorschlag zur Formulierung: Anhand einer modellhaften Forschungsfrage werden die Möglichkeiten der Datenvisualisierung analysiert. In den einzelnen Kapiteln werden dabei die Themen Datenvisualisierung, Dashboards, die Manipulierbarkeit von Visualisierungen sowie die Kommunikation von Forschungsergebnissen systematisch behandelt

wieso ist dies kursiv?

Ausführlicher, Kompetenzen die allgemein empfohlen sind fürs das OER, nicht nur für das Dashboard bauen. Das wird zwar schon bei 1.1 zu den Kapitel gemacht, sollte aber finde ich hier nochmal gebündelt stehen

andere Formulierungsvorschläge: Konkret auf den Nutzen bezogen:

"Erkenntnisgewinn für die Verwaltungswissenschaft" "Implikationen für die verwaltungswissenschaftliche Forschung" "Relevanz für Theorie und Praxis der Verwaltungswissenschaft"

Knapper:

"Verwaltungswissenschaftliche Einordnung" "Forschungsbeitrag" "Wissenschaftstheoretische Verortung"

Diese Frage verstehe ich nicht. Meint ihr: Wie unterscheidet sich das Engagement der Bürgerinnen z.B.mit Blick auf den Bezirk, auf die dort vorhandene Baumdichte, auf das Alter der Bäume und auf die jeweilige Jahreszeit?

eben heißt es noch szenario .. vielleicht gleich lassen.

sollte ausgeschrieben werden.

explorativ?

Baumwässerungsverhalten der Bürgerinnnen oder wer vehält sich da?

gestalten anstatt gestaltet

Baumarten die Wo existieren? Was ist der Umfang? Geht es nur um Deutschland? Das sollte noch beigefügt werden.

Hierfür oder Hierzu

weil der lesende ja gar nicht weiß, dass diese alles eine Fallstudie ist, würde ich eher so beginnen: "Auf diesen Seiten bilden wir einen Forschungsverlauf einer Fallstudie in der Verwaltungswissenschaft in einem JupyterBook nach.

hier sollte es eine kurze Erklärung geben, was genau Manipulierbarkeit im Kontext Visualisierung bedeutet.

... wann gehts endlich los ..

Durch die Verlinkung auf die entsprechenden Kapitel öffnen sich die Kapitel teils unbeabsichtigt, weil man das Lernziel nur aufklappen möchte und direkt auf den Text drückt. Könnte man übergehen durch "(zum Kapitel)" mit Verlinkung?

einer Fallstudie in den digitalen Geisteswissenschaften Digitale Geisteswissenschaften schreiben oder Digital Humanities

Wieder sehr oft das Wort "kann"

Das klingt alles ein bisschen negativ. Pädagogisch sinnvoller, wäre zu schreiben: Sie lernen die Grundkonzepte des NLP....

Das methodische Vorgehen... kann erläutert werden

Habt ihr Metadaten schon erklärt? Vielleicht darauf verweisen, dass Metadaten noch erklärt werden

Was ist basal?

Zwei mal "können"

Genauer, den Zusammenhang erklären. Wie hängen die beiden Punkte mit der Überschrift zusammen

Musculation Scolaire et Déterminisme Décisionnel : Analyse des Stéréotypes de Genre

Résumé Exécutif

Ce document synthétise les travaux de Matthieu Lorieux (en collaboration avec Dorian Deemer) concernant l'influence des normes de genre sur les choix des élèves en musculation scolaire. L'étude révèle que, malgré les objectifs éducatifs de santé et d'autonomie, la pratique de la musculation en milieu scolaire reste massivement déterminée par des stéréotypes corporels sexués. Les garçons privilégient le développement du haut du corps et la puissance (virilité agissante), tandis que les filles se concentrent sur le bas du corps et la silhouette (esthétique de la minceur). L'analyse souligne un paradoxe : l'institution scolaire, en autorisant une approche analytique centrée sur les zones musculaires, risque d'institutionnaliser des déviances narcissiques et individualistes issues de la sphère sociale et des réseaux sociaux, plutôt que de favoriser un véritable esprit critique.

Contexte et État des Lieux de la Pratique

Une expansion sociale et scolaire massive

La musculation a connu une croissance exponentielle, quadruplant son nombre de pratiquants entre 2000 et 2020. Elle est aujourd'hui la deuxième activité physique la plus pratiquée en France, juste derrière la marche.

• En milieu scolaire : Elle est la deuxième activité la plus pratiquée au baccalauréat (toutes filières confondues) et la première en filières technologique et professionnelle.

• Enjeux de santé : Cette expansion s'inscrit dans un contexte de sédentarité accrue (la majorité des jeunes ne respectent pas les recommandations de l'OMS). Cependant, une confusion s'opère entre la santé (forme intrinsèque) et la beauté (forme extrinsèque), largement entretenue par l'industrie du fitness.

L'influence des réseaux sociaux

90 % des adolescents utilisent quotidiennement les réseaux sociaux. Ce canal diffuse des normes corporelles strictes via des cadrages spécifiques (plongée/contre-plongée) et des filtres, exacerbant la comparaison constante des corps et l'impératif de répondre à des standards plastiques.

La Dialectique des Corps : Féminité vs Masculinité

L'étude met en évidence une structuration binaire et opposée des aspirations corporelles selon le genre.

| Genre | Zones Valorisées | Qualités Physiques Associées | Idéal Social | | --- | --- | --- | --- | | Masculin | Haut du corps (bras, dos, pectoraux) | Puissance musculaire, volume | Activité, virilité, force "invisible" | | Féminin | Bas du corps (cuisses, fessiers) | Endurance, tonicité, silhouette | Passivité esthétique, minceur, galbe |

La construction de l'identité par l'opposition

Pour les adolescents, l'identité se construit par le rejet des attributs du genre opposé. L'apparence physique est le premier vecteur de rapports sociaux et de pressions psychologiques, pouvant mener au harcèlement scolaire en cas d'écart aux normes.

Analyse des Choix des Élèves en Contexte Scolaire

L'étude de Matthieu Lorieux a porté sur deux classes de terminale, analysant leurs questionnaires et carnets d'entraînement.

Préférences musculaires et rejet

Les résultats montrent un réinvestissement direct des normes sociales dans les choix d'exercices :

• Garçons : 74 % privilégient le triptyque bras-dos-pectoraux. 58 % rejettent explicitement le travail des cuisses et des fessiers.

• Filles : 54 % valorisent prioritairement les cuisses et les fessiers. 52 % rejettent le travail du haut du corps (bras-pectoraux).

Choix des thèmes d'entraînement

Les thèmes choisis reflètent les qualités idéales attribuées à chaque sexe :

• Le thème "Puissance" : Choisi par 58 % des garçons, visant l'expression de la virilité par la performance.

• Le thème "Volume" : Choisi par 67 % des filles. Bien que le nom suggère la masse, il est perçu comme le seul thème à visée esthétique disponible pour remodeler la silhouette.

• Le thème "Endurance" : Totalement absent chez les garçons, confirmant que la masculinité doit s'exprimer par des charges lourdes et non par un effort prolongé à faible intensité.

L'Évolution des Justifications : Entre Esthétique et Conformisme

L'analyse des carnets d'entraînement montre une évolution des justifications au fil de la séquence d'enseignement.

1. Début de séquence : Les justifications sont purement esthétiques ("avoir des biceps") ou liées à des pratiques sportives extrascolaires pour les garçons.

2. Fin de séquence : On observe une explosion des justifications basées sur les "ressentis" et les "sensations" (+58 points chez les garçons).

3. Le paradoxe du conformisme : Cette focalisation sur les sensations semble être une stratégie de conformisme scolaire. Les élèves adoptent le langage attendu par l'enseignant et l'institution (référentiel baccalauréat) pour rendre leurs choix acceptables, tout en conservant leurs motivations esthétiques profondes et stéréotypées.

◦ Exemple : Une élève (Agnès) justifie le travail du bas du corps par ses sensations, tout en avouant qu'elle cible cette zone car elle "complexe" sur ses fesses.

Conclusions et Perspectives Pédagogiques

Les risques d'une musculation analytique

L'étude suggère que permettre aux élèves de choisir leurs zones musculaires à travailler (musculation analytique) revient à institutionnaliser les déviances narcissiques de la société. L'école, au lieu de libérer des stéréotypes, pourrait paradoxalement offrir un espace pour les renforcer.

Vers des approches alternatives

Pour contrer ce déterminisme, plusieurs pistes sont proposées :

• Musculation fonctionnelle : Remplacer le ciblage par groupe musculaire par une approche par fonctions motrices (pousser, tirer, flexion/squat, soulever, balancer).

• Entrée par l'expérience : Privilégier des thèmes centrés sur des paramètres physiologiques neutres (comme la fréquence cardiaque) plutôt que sur des thèmes à connotation esthétique.

• Développement de l'esprit critique : Ne pas se limiter à la gestion de la "vie physique" mais interroger activement les normes incorporées par les élèves.

L'enjeu final pour l'Éducation Physique et Sportive (EPS) est de s'assurer que les choix des élèves résultent d'une véritable expérience motrice et non d'un a priori social préexistant.

L'Intérêt en Situation des Élèves en Musculation : Analyse des Formats d'Autorégulation

Résumé Exécutif

Ce document synthétise les résultats d'une étude menée par Arthur Lefebvre dans le cadre du projet REFPS, portant sur l'intérêt en situation des élèves de lycée lors de séances de musculation.

L'objectif central était de déterminer s'il existe un format d'autorégulation de la charge idéal pour favoriser l'engagement des élèves selon leur niveau d'expertise (novice, intermédiaire, expert).

Les conclusions majeures indiquent que :

• L'hétérogénéité est mieux gérée par le format RPE 8 (Échelle de perception de l'effort), qui s'avère être le format le plus inclusif, ne créant quasiment aucune différence d'intérêt entre les niveaux.

• Les formats plus complexes (APRE 10, Temps 2, RIR 2) favorisent systématiquement les élèves experts, créant un écart significatif en termes de plaisir et d'intention d'exploration par rapport aux novices.

• Le défi perçu est plus élevé chez les novices, ce qui peut nuire à leur plaisir si la tâche est perçue comme trop complexe.

• Une progression pédagogique est préconisée, débutant par le format RPE pour engager les novices, avant d'introduire des formats plus exigeants comme l'APRE pour développer l'attention et la précision du rapport à la charge.

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1. Cadre Théorique et Objectifs de l'Étude

L'étude s'inscrit dans la continuité des travaux sur l'intérêt en situation, défini par Chen (2006) comme l'effet attractif des caractéristiques d'une tâche sur un individu.

Contrairement aux études précédentes focalisées sur le badminton (activité d'opposition et compétitive), cette recherche explore la musculation, une activité autoréférencée et non compétitive.

Les Dimensions de l'Intérêt en Situation

L'analyse s'appuie sur quatre des cinq dimensions du modèle de Tienen (2014) :

1. Le plaisir instantané : La satisfaction immédiate liée à la pratique.

2. Le défi : La complexité perçue de la tâche.

3. La demande d'attention : La concentration requise par l'activité.

4. L'intention d'exploration : La volonté de découvrir et d'apprendre de nouveaux éléments.

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2. Méthodologie de la Recherche

L'étude a suivi un protocole rigoureux sur une séquence complète de 9 leçons :

• Participants : 164 élèves (moyenne d'âge 17 ans) répartis en 5 classes de lycée et des étudiants de STAPS.

• Classification par expertise : 47 novices, 68 intermédiaires, 49 experts (déterminés par un questionnaire d'intérêt individuel).

• Formats testés : Quatre formats basés sur l'autorégulation de la charge :

◦ APRE 10 : Régulation progressive basée sur la performance.  

◦ Temps 2 : Format basé sur le temps de travail.    ◦ RIR 2 (Repetitions in Reserve) : Évaluation subjective des répétitions restantes possibles.  

◦ RPE 8 (Rate of Perceived Exertion) : Évaluation de l'effort perçu sur une échelle de 1 à 10.

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3. Analyse Comparative des Formats de Pratique

L'analyse des résultats montre que l'intérêt des élèves varie considérablement selon le format utilisé et leur niveau initial.

| Format | Impact sur les Experts | Impact sur les Novices | Conclusion Pédagogique | | --- | --- | --- | --- | | APRE 10 | Très favorable (Plaisir, Attention, Exploration élevés). | Moins favorable ; écart significatif avec les experts. | Convient aux élèves expérimentés. | | Temps 2 | Intérêt soutenu. | Différences significatives en faveur des experts. | Format exigeant pour les novices. | | RIR 2 | Plaisir et exploration élevés. | Écart marqué avec les experts. | Favorise l'expertise. | | RPE 8 | Intérêt élevé et constant. | Intérêt quasi identique à celui des experts. | Format idéal pour l'hétérogénéité. |

Le cas spécifique du format RPE 8

Le format RPE 8 se distingue comme le "format qui épouse le mieux l'hétérogénéité". Il ne présente qu'une seule différence significative entre novices et experts sur les quatre dimensions étudiées. C'est le format qui "parle le plus à tout le monde", indépendamment du niveau.

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4. Analyse par Dimensions de l'Intérêt

Le Défi et le Plaisir

Il existe une corrélation entre le plaisir et le défi. L'étude révèle que la dimension "défi" est significativement plus élevée chez les novices (2,91 contre 2,54 pour les experts).

Si le défi est trop grand, la tâche n'est plus optimale et le plaisir diminue.

L'Intention d'Exploration

Le format RPE est identifié comme un excellent point d'entrée pour les novices dans l'exploration de l'activité.

Cependant, il semble insuffisant à lui seul pour maintenir cette dynamique de progression sur le long terme, nécessitant le passage vers d'autres formats plus complexes au fur et à mesure que l'expertise augmente.

La Demande d'Attention

Le format APRE 10 est celui qui génère la plus grande différence d'attention entre experts et novices.

Les résultats suggèrent que pour développer l'attention, il est nécessaire de travailler spécifiquement sur le rapport à la charge et le rapport à l'échec.

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5. Perspectives et Innovations Pédagogiques

Proposition d'une Innovation : Le "Format au Tonnage"

Pour pallier l'absence d'un format unique idéal, Arthur Lefebvre propose une hybridation entre l'APRE et le RPE :

• Principe : 5 séries de 10 répétitions avec le tonnage le plus élevé possible.

• Contrainte : Si l'élève ne parvient pas à réaliser les 10 répétitions (échec ou arrêt prématuré), le score de la série est de 0 kg.

• Objectif : Allier le ressenti sensoriel (RPE) et la rigueur cognitive de la charge (APRE).

Recommandations pour la Séquence d'Enseignement

1. Début de séquence : Privilégier le format RPE 8 pour garantir l'engagement de tous les élèves, particulièrement des novices.

2. Milieu de séquence : Introduire progressivement des formats plus subjectifs ou objectifs (RIR, Temps).

3. Fin de séquence : Utiliser des formats type APRE ou des formats hybrides pour affiner l'expertise et la concentration sur la performance.

Limites de l'Étude

L'auteur souligne que l'échantillon (164 participants) et l'absence de mesure de la temporalité (l'évolution de l'intérêt sur le long terme selon le modèle de Hidi et Renninger) constituent des limites à prendre en compte pour les recherches futures.

why

Synthèse de Pratiques Pédagogiques : Créer du Lien et de la Bienveillance en Maternelle

Résumé Exécutif

Ce document synthétise les réflexions et les pratiques de Mélanie, enseignante en maternelle à Strasbourg forte de 12 ans d'expérience. Le point central de son approche est la création d'un lien affectif profond avec ses élèves, un élément qu'elle considère comme le socle indispensable à tout apprentissage. En rompant délibérément avec la distance professionnelle traditionnelle, elle préconise une « communication bienveillante » et une « fermeté bienveillante » pour instaurer un climat de confiance.

Le document détaille comment cette posture se traduit concrètement par l'abandon des systèmes de notation du comportement, l'adoption de classes à niveaux multiples pour favoriser la douceur sociale, et une organisation spatiale et pédagogique axée sur l'autonomie. L'objectif ultime est de transformer la classe en un « cocon » serein où l'enfant, apaisé et respecté dans son rythme, développe un rapport positif durable avec l'institution scolaire.

1. La Centralité du Lien Affectif

Pour Mélanie, l'attachement entre l'enseignant et l'élève n'est pas un obstacle, mais un moteur pédagogique. Cette vision a évolué au cours de sa carrière, passant d'une réserve initiale à une affirmation assumée de l'affection envers ses élèves.

Déconstruction des barrières professionnelles

• Contestation du dogme de la distance : L'enseignante s'oppose à l'idée, souvent transmise lors de la formation initiale ou par certains tuteurs, qu'il faut maintenir une distance stricte. Elle cite l'ouvrage Chagrin d'école de Daniel Pennac pour illustrer cette interdiction tacite d'aimer ses élèves.

• Expression de l'affection : Elle assume l'utilisation de surnoms et n'hésite pas à dire « je t'aime » à ses élèves. Selon elle, cette proximité ne nuit pas au respect des règles ; au contraire, la connexion établie renforce l'autorité naturelle et le respect mutuel.

• L'importance de la stabilité : Le lien est plus difficile à tisser pour les enseignants remplaçants ou à temps partiel (comme les stagiaires en quart de décharge). Avoir sa propre classe à temps plein est présenté comme un facteur déterminant pour l'épanouissement professionnel et relationnel.

L'impact sur le climat scolaire

Le but est de créer un « cocon » où la sérénité est palpable. En début d'année, l'enseignante privilégie délibérément la relation, le cadre de travail et l'autonomie au détriment immédiat des apprentissages purement académiques, afin de garantir une fluidité pour le reste de l'année.

2. Une Gestion de Classe Basée sur la Communication

La communication dans la classe de Mélanie repose sur une compréhension profonde de l'enfant et un rejet des méthodes de coercition classiques.

Rejet des systèmes de comportement

L'enseignante a abandonné les outils traditionnels de gestion du comportement (lions de couleur, systèmes de points, etc.) car elle a constaté qu'ils aggravaient souvent les difficultés des élèves les plus fragiles. Elle privilégie désormais :

• La discussion systématique : Même si cela peut sembler répétitif ou aboutir parfois à des impasses, le dialogue reste l'outil principal.

• La compréhension des besoins : L'effort est mis sur l'analyse de la cause du comportement plutôt que sur la sanction immédiate.

• La posture physique : Elle souligne l'importance de parler à « hauteur d'enfant », une pratique inspirée des modèles scandinaves.

La « Fermeté Bienveillante »

Cette approche ne signifie pas l'absence de règles. Elle a été qualifiée par un inspecteur de « bienveillante fermeté ».

• Exemple de gestion de conflit : Face à une bévue (ex: écrire sur une table par inadvertance), l'enseignante dédramatise (« tu n'as pas besoin d'avoir l'air triste ») tout en responsabilisant l'enfant (nettoyer avec un papier et de l'eau).

• Limites de la patience : L'agacement survient principalement lors de comportements nuisant aux relations sociales (moqueries, phrases méchantes répétées), plutôt que lors d'accidents matériels.

3. Organisation Pédagogique et Structure de Classe

La forme scolaire elle-même est pensée pour soutenir cette bienveillance et s'adapter au rythme biologique et psychologique des enfants.

Les bénéfices des niveaux multiples

Mélanie préconise la mixité des âges (Petits, Moyens, Grands) pour plusieurs raisons :

• Atténuation des effets de groupe : Le mélange casse les dynamiques de groupes trop soudés et potentiellement conflictuels qui se suivent depuis la crèche.

• Instauration d'une douceur naturelle : La présence de « petits » incite les plus grands à la protection et au calme, créant une ambiance de type « familial ».

• Bénéfice social : Placer un enfant difficile avec des plus petits peut s'avérer bénéfique pour son propre apaisement.

Autonomie et différenciation

Le fonctionnement en autonomie permet d'éviter la standardisation des tâches :

• Les enfants ne sont pas obligés d'exécuter tous le même travail en même temps.

• Cela réduit le stress lié à des tâches inadaptées (trop complexes ou trop simples).

• L'apaisement qui en découle rend les élèves plus disponibles pour les apprentissages.

Aménagement de l'espace de travail

L'espace physique est segmenté en zones spécifiques pour favoriser différents types d'activités :

| Espace | Fonction / Caractéristiques | | --- | --- | | L'Ellipse | Un tracé au sol au milieu de la classe pour les regroupements (préféré aux tables). | | Espaces à scénario | Zones dédiées à des jeux de rôle ou situations thématiques (ex: yoga, pressing). | | Ateliers autonomes | Meubles de rangement organisés par domaines (phonologie, motricité fine, etc.). | | Sous le bureau | Utilisation de l'espace sous le bureau de l'enseignante pour créer un « coin écoute » avec des boîtes à histoires. | | Tables spécifiques | Table en U pour les ateliers dirigés, petite table pour la peinture. |

4. Posture et Défis de l'Enseignant

L'enseignement en maternelle exige un investissement personnel et une vigilance constante sur sa propre santé.

• Évolution de la sérénité : La confiance en soi s'acquiert avec les années et la stabilité du poste, permettant d'investir davantage dans la dimension relationnelle.

• Santé vocale : Mélanie souligne la pénibilité du métier pour la voix et les oreilles (bruit de la cour, appels). Elle s'efforce de ne pas crier pour préserver ses cordes vocales et maintenir le calme ambiant.

• Compétences annexes : L'usage d'instruments, comme la guitare (apprise de manière autodidacte), est utilisé comme un outil de lien supplémentaire, très apprécié par les élèves malgré un niveau technique qu'elle juge modeste.

Conclusion

L'approche décrite dans ce document montre que la réussite scolaire en maternelle repose sur un équilibre entre une structure pédagogique flexible (autonomie, multi-niveaux) et une relation humaine forte. En cassant la barrière de la distance traditionnelle, l'enseignante crée un environnement sécurisant qui favorise l'appétence des enfants pour l'école dès leurs premières années.

Trajectoires des Jeunes Protégés et Facteurs de Résilience : Note de Synthèse

Résumé Exécutif

Ce document synthétise les interventions de Laëtitia Sauvage, chercheuse en anthropologie de l'éducation et membre du Conseil national de la protection de l'enfance, concernant les parcours de résilience des jeunes issus de la protection de l'enfance.

La thèse centrale établit que la résilience n'est pas une compétence individuelle intrinsèque, mais un processus complexe, dynamique et systémique qui se construit dans l'interaction entre l'individu et son environnement.

L'institution scolaire est identifiée comme un « tuteur de résilience » potentiel, à condition qu'elle dépasse le cadre strictement disciplinaire pour investir la dimension psychosociale.

Le rapport au savoir agit comme un levier de mentalisation essentiel, permettant au jeune de se projeter au-delà de ses traumatismes.

La réussite de ce processus repose sur une approche pluridisciplinaire coordonnée (école, famille, travailleurs sociaux) et sur la capacité des professionnels à décoder les comportements de « résistance » (agressivité, provocation) comme des appels au lien éducatif plutôt que comme de simples manquements disciplinaires.

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1. Redéfinition Théorique de la Résilience

La résilience doit être comprise non pas comme une capacité fixe, mais comme un phénomène psychosociologique en constante redéfinition.

Un processus dynamique : la métaphore du « flipper »

L'individu est comparé à une bille de flipper, ballotée par les traumatismes. Son parcours de résilience se divise en étapes clés :

• Résistance : Réaction immédiate pour éviter l'effondrement ou la désorganisation mentale.

• Reconstruction : Mécanismes de réparation à moyen terme.

• Remaniement psychique (Néo-développement) : Transformation durable et continue tout au long de la vie.

Distinction entre les mécanismes de réaction

Il est crucial de ne pas confondre la résilience avec d'autres modalités de réaction aux traumatismes :

• Résistance : Confrontation nécessaire à l'autorité, souvent perçue à tort comme de l'agressivité gratuite.

• Désilience : Incapacité totale à se mobiliser, pouvant mener à des addictions ou au retrait social.

• Désistance : Abandon d'une sphère spécifique (ex: décrochage scolaire) tout en maintenant un investissement dans d'autres domaines (social, associatif).

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2. Analyse Systémique et Environnementale

Le développement de l'enfant s'inscrit dans le modèle écologique de Bronfenbrenner, complété par la notion d'ontosystème.

| Système | Définition | Rôle dans la Résilience | | --- | --- | --- | | Ontosystème | Monde sensible, psyché et valeurs intimes de l'enfant. | Siège de la sensibilité et des affects traumatiques. | | Microsystème | Sphère immédiate (famille, substituts parentaux). | Souvent le lieu des « fracas » initiaux en protection de l'enfance. | | Mésosystème | Interactions entre les milieux (école, sport, associations). | L'école y joue un rôle pivot de décloisonnement. | | Macrosystème | Normes institutionnelles et politiques nationales. | Évolue vers une meilleure prise en compte de la vulnérabilité. |

Citation clé : « La résilience est un tricot qui noue une laine développementale avec une laine affective et sociale. Ce n'est pas une substance, c'est un maillage. »

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3. Le Rôle de l'Institution Scolaire

L'école peut agir comme un tuteur de résilience en offrant un cadre sécurisant et des opportunités de mentalisation.

Le rapport au savoir comme levier

Le rapport au savoir ne se limite pas à l'acquisition de connaissances ; il soutient les capacités de projection de soi.

Pour les jeunes protégés, l'institution du savoir peut être le seul espace de « sécurité pleine et totale ».

L'importance de l'« autrui significatif »

Des gestes simples et humanisants, comme le sourire d'une gardienne ou l'accueil d'un chauffeur de bus, constituent des ancrages fondamentaux.

Ces interactions valident l'existence de l'enfant et soutiennent son sentiment d'appartenance.

Défis et statistiques alarmantes

Le système actuel présente des failles majeures dans l'accompagnement des jeunes confiés :

• Accès aux études supérieures : Seulement 8 % des jeunes issus de la protection de l'enfance (contre 52 % en population générale).

• Retard scolaire : 40 % des enfants de 11 ans accueillis sont encore en primaire (contre 10 % en population générale).

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4. Facteurs de Risque et de Protection

L'analyse doit porter sur l'équilibre entre les vulnérabilités et les ressources disponibles.

Facteurs de risque (Freins)

• Manque de coordination entre enseignants, familles et travailleurs sociaux.

• Orientations scolaires contraintes par des impératifs d'autonomie financière rapide.

• Instabilité géographique (déplacements fréquents de lieux d'accueil).

• Réunions institutionnelles organisées durant le temps scolaire, entravant la scolarité.

Facteurs de protection (Leviers)

• Relations stables : Présence d'adultes référents non-jugeants.

• Espaces sécures : Accès aux bibliothèques, foyers ou salles de repos.

• Renforcement positif : Valorisation systématique des forces de caractère et des efforts de l'élève.

• Compétences psychosociales : Développement de l'estime de soi et de la capacité d'agir.

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5. Stratégies et Outils Opérationnels

Pour transformer un établissement en environnement porteur de résilience, trois étapes de professionnalisation sont proposées :

1. Identifier et dissocier : Apprendre à distinguer les mécanismes de défense (souvent inconscients, comme la sur-intellectualisation) des stratégies d'adaptation (recherche active d'informations).

2. Décoder la résistance : Comprendre que l'agressivité d'un jeune peut être une marque de confiance, une « porte ouverte à la relation éducative » dans un lieu où il s'autorise enfin à exprimer son traumatisme.

3. Valoriser les ressources psychologiques : S'appuyer sur des modèles comme les 24 forces de caractère de Seligman ou les ressources de Pourtois (affectives, sociales, cognitives, conatives).

Programmes de « résilience assistée » mentionnés :

• Spark : Utilisation de supports ludiques pour la mentalisation.

• Care Commites (Pays-Bas) : Approche communautaire intégrée.

• Mentorat (Espagne) : Accompagnement par les pairs ou des tuteurs externes.

• Projets personnels d'accompagnement : Création d'une alliance éducative entre le jeune, un enseignant de son choix et son éducateur.

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Conclusion

La promotion de la résilience en milieu scolaire exige un changement de paradigme : il ne s'agit plus de se focaliser uniquement sur le traumatisme ou les lacunes disciplinaires, mais d'adopter une approche inclusive et systémique.

En identifiant les forces intrinsèques des jeunes et en sécurisant leur rapport au savoir, l'école devient le terreau d'un nouveau développement, permettant à l'élève de transformer son « fracas » initial en un épanouissement original et durable.

Graag de (vanuit PZH aangeleverde) definities inclusief bronvermelding in een kolom meenemen bij de tabellen. In deze versie is de bronvermelding weggelaten.

Zo krijgt de lezer een eerste indruk waar en hoe het model zich verhoudt tot standaarden @Elisabeth.de.Vries

Question mal formulée ? si la reponse est "?" alors la question aurait du etre "un ou plusieurs parametres" si non la logique veut qu"on réponde "&"

Ne le font-ils pas fondamentalement ? Je trouve que poser cela comme hypothèse n'est pas correct.

"Qui" sont-ils ? S'il existe des "non lieux", c'est qu'ils sont localisés, localisables, vécus donc il est possible de les nommer et de les lister - sans faire toutefois un inventaire ... à côté de "hauts-lieux" (terme peut-être à reprendre) comme le Père Lachaise, etc.

La ville, "notre" ville est donc à "notre mesure" ? Cette proposition de "'mesure" aurait un écho avec le sentiment d'être "petit" ailleurs.

Est-ce la seule et unique chose que permette cette "intertextualité " urbaine ?

L'écho ne renvoie-t-il qu'à la comparaison ?

Les "conséquences" sur cette idée de protection seraient interessantes à développer. Je pense notamment à la figure urbaine du flâneur (Benjamin et Baudelaire) qui est "protégé" car noyé dans le flot des individus et maitrisant quelques codes de la ville, peut se soustraire au regard des autres et être, en quelque sorte, protégé.

Ce propos fait écho à un élément que vous laissiez plutôt en suspend dans les premières lignes du chapitre 5.

N'est-ce pas plutôt la même histoire où la "face cachée" de ce rapport ? Une ville est habitée autant qu'elle nous habite, non ?

Rapport de Synthèse : Conclusion du Grand Témoin – Julien Gagnebien

Résumé Exécutif

Ce document synthétise l'intervention de Julien Gagnebien, Inspecteur Général, lors d'un séminaire à l'INSPÉ Lille.

L'analyse souligne un changement de paradigme nécessaire dans l'enseignement de l'Éducation Physique et Sportive (EPS). Les points clés incluent :

• L'Éthique au cœur du métier : L'enseignement doit équilibrer l'éthique relationnelle et l'éthique conceptuelle pour répondre aux besoins fondamentaux des élèves.

• La mutation du Champ d'Apprentissage 5 (CA5) : Bien que populaire, le CA5 (musculation, step, etc.) doit se réinventer pour aider les élèves à développer un regard critique face à l'influence croissante des réseaux sociaux et des influenceurs fitness.

• De l'exécution à la conception : Les futurs enseignants sont encouragés à privilégier le « quoi » et le « pourquoi » pédagogique avant le « comment », en s'éloignant des formats d'enseignement exclusifs ou obsolètes.

• La posture du « Chercheur de solutions » : L'institution ne demande pas des enseignants conformistes, mais des praticiens capables de douter, d'expérimenter et de collaborer pour favoriser la réussite de tous les élèves.

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1. Posture Professionnelle et Analyse de la Pratique

L'intervention met en avant l'importance de l'observation et de la collaboration entre la recherche et le terrain pour l'évolution des pratiques en EPS.

L'importance de l'observation in situ

L'observation n'est pas une perte de temps, mais un levier de transformation majeure. Julien Gagnebien souligne que :

• L'observation outillée permet de réinterroger les méthodes de l'enseignant.

• Elle aide à mesurer le lien de cause à effet entre le contexte créé par l'enseignant et l'engagement réel des élèves.

• Pour les candidats aux concours (CAPEPS), cette phase nourrit directement les propositions pour les épreuves orales.

La relation Recherche-Praticiens

L'Inspection Générale accorde une valeur significative aux enseignants-chercheurs. Leur travail est perçu comme un service essentiel pour faire évoluer les praticiens, malgré les contraintes financières des laboratoires. Cette synergie permet de nourrir l'institution et d'impulser de nouvelles dynamiques pédagogiques.

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2. Éthique et Engagement : La « Fleur des Besoins »

Le métier d'enseignant repose sur une double responsabilité : marquer positivement la vie des élèves et adopter une posture juste.

L'équilibre des éthiques

Les enseignants d'excellence se situent à l'équilibre entre deux piliers :

1. L'éthique relationnelle : La qualité du lien avec les élèves (point fort actuel des enseignants d'EPS).

2. L'éthique conceptuelle : La capacité à concevoir des contextes d'apprentissage pertinents.

La satisfaction des besoins fondamentaux

S'appuyant sur les travaux d'André Canvel et Damien Tessier (théories de l'autorégulation), l'intervention présente la « fleur des besoins ». L'engagement de l'élève dépend de la capacité de l'enseignant à nourrir ces bulles :

| Catégorie de besoins | Éléments clés | | --- | --- | | Sécurité et Confiance | Création d'un climat de classe serein. | | Justice et Respect | Évaluations transparentes et équitables. | | Autonomie et Choix | Possibilité pour l'élève de s'exprimer et de décider. | | Appartenance et Estime | Sentiment de faire partie du groupe et valorisation de soi. |

Constat : Dans une leçon, le désengagement survient souvent après le premier quart d'heure, lorsque l'élève perçoit que le « menu » proposé ne répond pas à ces besoins.

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3. Analyse Critique des Formats de Pratique

L'enseignant a le devoir de questionner les formats sportifs traditionnels qui peuvent devenir des vecteurs d'exclusion.

• Le paradoxe du cross scolaire : Le format classique (course de distance par catégorie d'âge) devient souvent insignifiant dès la classe de 5ème pour les élèves connaissant déjà leur classement. Ce format exclut les trois quarts des élèves alors même que l'EPS prône l'inclusion.

• La nécessité de réinvention : Il est impératif de concevoir des formats qui conservent l'enjeu de performance (Champ d'Apprentissage 1) tout en garantissant l'accessibilité et l'inclusion scolaire.

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4. Le Champ d'Apprentissage 5 (CA5) : Enjeux et Paradoxes

Le CA5 (activités de développement des ressources personnelles) occupe une place prépondérante mais fait face à des défis inédits.

Un succès institutionnel et matériel

• Les activités comme la musculation sont parmi les plus choisies par les élèves (voie professionnelle et GT).

• Les collectivités territoriales ont investi massivement dans des salles dédiées.

• Ces activités favorisent l'autonomie et le réinvestissement à long terme dans la vie adulte.

Le défi de la légitimité face aux influenceurs

L'enseignant de CA5 est désormais en concurrence avec les influenceurs YouTube.

• Le conflit de crédibilité : Un élève peut contester l'enseignement d'un professeur en s'appuyant sur le discours d'un influenceur dont le morphotype lui semble plus légitime.

• L'enjeu du regard critique : Le véritable défi de 2025 est de former des élèves capables d'analyser de manière critique les programmes d'entraînement extérieurs plutôt que de subir l'influence de modèles esthétiques ou commerciaux.

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5. Directives pour les Futurs Enseignants (Concours et Carrière)

Julien Gagnebien livre des conseils stratégiques pour les candidats aux concours (notamment l'Oral 3) et pour la pratique professionnelle.

Priorités de conception

Les jurys attendent une hiérarchisation claire des intentions pédagogiques :

1. Le Quoi et le Pourquoi : Définir avec précision ce que l'élève doit construire et les raisons du scénario pédagogique. C'est le « ticket d'entrée » dans la profession.

2. Le Comment : Les modalités pratiques (exercices, situations) viennent en second lieu. Une plus grande tolérance est accordée aux erreurs sur le « comment » car il relève de l'expérience en construction.

Sortir de l'éparpillement

Il est crucial de renoncer à vouloir « tout faire ». Une séquence (en musculation ou badminton) doit cibler des apprentissages fondamentaux spécifiques à chaque niveau (6ème vs Terminale) pour éviter le syndrome de « l'éternel débutant ».

Travailler par « Dilemmes »

Une piste innovante consiste à entrer dans les champs d'apprentissage par les dilemmes (ex: s'engager vs se préserver). Amener l'élève à traiter ces compromis en classe le prépare à faire des choix éclairés en autonomie hors de l'école.

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Conclusion : L'Injonction à la Liberté

Le document conclut sur une déconstruction du mythe de « l'injonction institutionnelle ».

En dehors de la sécurité, de l'évaluation et de l'équité, les programmes offrent une grande liberté.

« On n'a pas besoin d'enseignants qui se conforment, on a besoin d'enseignants qui cherchent des solutions et qui en trouvent. »

La mission ultime de l'enseignant est de devenir un « chercheur de solutions », capable de douter et d'expérimenter pour répondre à la complexité des contextes scolaires et assurer la réussite de tous les élèves.

De l'Éducation des Parents au Soutien à la Parentalité: Tensions et Controverses

I. Introduction: Interactions Enfant-Parent-École et la Question Parentale

Interactions Enfant-Parent-École : Un Système d'Attentes Réciproques (5:00):

La relation entre parents et école est marquée par des attentes mutuelles, notamment en ce qui concerne la réussite scolaire.

Cette interaction est fortement influencée par des sujets partagés comme la réussite scolaire, le comportement et le bien-être des enfants.

L'Emprise Scolaire et la Transformation des Parents en Coachs (6:15):

La massification de l'accès à l'école a engendré une "emprise scolaire", où la question de l'école domine les interactions parents-enfants.

Les parents se transforment en "coachs scolaires", centrés sur la performance de leurs enfants, ce qui peut avoir un impact négatif sur la relation parent-enfant.

Symptômes Émergents et Mal-être des Enfants (10:00):

L'augmentation des troubles psychiques chez les enfants et adolescents, manifestée par des symptômes comme le retrait scolaire (hikikomori) et la surconsommation de psychotropes, met en lumière les difficultés croissantes rencontrées par les jeunes et interroge le rôle des parents et des institutions dans leur bien-être.

II. Histoire de la Relation entre Pouvoirs Publics et Parents

L'Émergence de l'Éducation des Parents (15:00):

Dès le 19ème siècle, l'idée d'éduquer les parents à leur rôle, notamment en matière de maternage, prend forme pour lutter contre la mortalité infantile et garantir le bien-être des enfants.

Cette préoccupation s'intensifie au 20ème siècle, avec la création d'institutions dédiées à l'éducation des parents.

L'École des Parents et la Défense du Rôle Parental (19:00):

Créée dans un contexte de crise idéologique dans les années 30, l'École des Parents vise à soutenir les parents face à l'intrusion perçue de l'État dans l'éducation des enfants.

Elle est initialement portée par une élite catholique et conservatrice, défendant une vision traditionnelle de la famille.

L'Après-Guerre et le Marché du Conseil aux Parents (24:00):

Après la Seconde Guerre mondiale, un véritable marché du conseil aux parents se développe, avec des figures comme Benjamin Spock, Françoise Dolto et Laurence Pernoud, qui publient des ouvrages et donnent des conseils aux parents.

L'accent est mis sur la valorisation des connaissances des mères et l'importance de l'écoute et de la compréhension de l'enfant.

III. Le Tournant de la Parentalité et l'Émergence d'une Politique Publique

L'Apparition du Concept de "Parentalité" (27:50):

Dans les années 90, le concept de "parentalité" émerge, influencé par la Convention Internationale des Droits de l'Enfant et le rôle croissant des institutions internationales dans la promotion du bien-être des enfants.

Le Soutien à la Parentalité : Définition et Objectifs (31:20):

Le soutien à la parentalité est défini comme un ensemble de mesures visant à informer, soutenir, conseiller et former les parents dans leur rôle.

Il se distingue des politiques de l'enfance en ciblant les parents plutôt que les enfants.

Diversité des Mesures et Tensions Idéologiques (34:00):

Le soutien à la parentalité se traduit par une variété de mesures, allant de l'information générale au conseil individuel en passant par des programmes de formation.

Cependant, des tensions idéologiques émergent entre des approches universalistes et des initiatives ciblant les parents en difficulté.

IV. Controverses et Débats Autour de la Parentalité

Le Déterminisme Parental et la Responsabilisation des Parents (46:00):

Une vision déterministe de la parentalité tend à attribuer la responsabilité des problèmes rencontrés par les enfants aux déficits parentaux.

Cette approche risque d'individualiser et de psychologiser les difficultés sociales, en négligeant les contextes socio-économiques dans lesquels les familles évoluent.

Débats Autour des Neurosciences et de la Psychologie Positive (48:00):

L'influence croissante des neurosciences et de la psychologie positive dans le domaine de la parentalité suscite des débats.

La focalisation sur les trois premières années de l'enfant et l'insistance sur l'importance des interactions précoces peuvent occulter les influences sociales et culturelles qui façonnent la parentalité.

Parentalité Positive vs Autorité Parentale (50:00):

La promotion de la parentalité positive, prônant la bienveillance et l'écoute, est parfois confrontée à des discours valorisant l'autorité et la discipline.

La question de la limite et de la punition dans l'éducation des enfants divise les experts et les parents.

V. Conclusion : Penser les Cultures de la Parentalité et les Inégalités

Le Double Bind de la Parentalité (58:00):

Les parents sont confrontés à un "double bind" : ils sont encouragés à s'investir intensément dans l'éducation de leurs enfants, mais risquent d'être critiqués s'ils en font "trop" ou "pas assez".

Il est essentiel de reconnaître la diversité des cultures de la parentalité et de ne pas imposer un modèle unique.

L'Importance des Contextes Socio-économiques (59:00):

Les conditions de vie des familles, leurs ressources économiques, leurs conditions de travail et de logement, influencent profondément la manière dont les parents exercent leur rôle.

Il est crucial de tenir compte de ces inégalités et de ne pas responsabiliser les parents sans prendre en considération les contextes dans lesquels ils évoluent.

Réceptivité des Formats de Pratique en Musculation et Développement de l’Intérêt

Résumé Exécutif

Ce document synthétise les recherches de Mehdi Belhouchat concernant l'engagement psychologique des élèves en musculation scolaire.

L'étude s'appuie sur le modèle de développement de l'intérêt en quatre phases pour évaluer comment différents formats de pratique influencent la motivation des élèves. Les conclusions majeures révèlent une corrélation directe entre le niveau d'intérêt initial d'un élève et sa réceptivité à un format spécifique.

Alors que les élèves experts autogénèrent leur intérêt quelle que soit la tâche, les élèves novices (phases 1 et 2) sont extrêmement dépendants du design pédagogique.

Les formats favorisant un guidage externe (APRE) ou interne (RPE) s'avèrent les plus efficaces pour déclencher l'engagement chez les débutants, tandis que le format "au temps" doit être utilisé de manière stratégique et ponctuelle pour favoriser des sauts qualitatifs de progression.

Cadre Théorique et Problématique

La recherche s'inscrit dans le cadre de la théorie de l'intérêt, notamment développée par Cédric Roure en contexte francophone, et le design de tâches d'apprentissage (Olivier Dieu).

Le Constat de Départ

• Expansion de la musculation : Une activité en forte croissance depuis 20 ans en milieu scolaire et sociétal.

• Hétérogénéité des profils : Les classes se composent d'élèves aux profils variés, allant de l'expert inscrit en salle de sport (intérêt individuel développé) au décrocheur sédentaire (intérêt faible ou nul).

• Décalage des formats : Il existe une rupture entre les formats scolaires traditionnels (souvent basés sur le ressenti subjectif/RPE) et les pratiques sociales plus objectives, guidantes et intenses.

Le Modèle de l'Intérêt en Quatre Phases

Le développement de l'intérêt est analysé comme un passage d'un état psychologique éphémère à un trait de personnalité intégré :

1. Phase 1 : Intérêt individuel très faible.

2. Phase 2 : Intérêt individuel faible.

3. Phase 3 : Intérêt individuel émergent.

4. Phase 4 : Intérêt individuel bien développé.

L'intérêt en situation est mesuré par trois facteurs : le déclenchement, le maintien au ressenti (valence affective) et le maintien aux valeurs (ancrage profond).

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Analyse des Formats de Pratique

L'étude identifie trois types de guidage dans l'autorégulation de la charge de travail :

| Format | Nature du Guidage | Caractéristiques | | --- | --- | --- | | APRE (Autoregulation Progressive Resistance Exercise) | Externe | Protocole normatif strict (tableaux). L'élève a peu de choix ; l'environnement dicte l'action. | | Au Temps | Mixte | Équilibre entre l'individu et l'environnement. Repère temporel imposé, mais décision de charge laissée à l'élève. | | RPE (Rating of Perceived Exertion) | Interne | Poids de l'environnement très faible. L'élève est au cœur des décisions de régulation selon son ressenti. |

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Résultats Clés de la Recherche

L'étude, menée auprès de 319 participants (10 classes de lycée et étudiants), met en évidence plusieurs phénomènes critiques :

1. L'Indépendance des Experts

Les élèves situés en phases 3 et 4 (intérêt émergent ou développé) ne montrent aucune réceptivité spécifique aux formats.

Ils projettent leur propre intérêt dans n'importe quelle situation et sont capables de redéfinir le but de la tâche pour s'impliquer. Ils sont psychologiquement indépendants du design pédagogique.

2. La Sensibilité des Novices

Pour les élèves en phases 1 et 2, le format est déterminant :

• Le format APRE (guidage externe) est dominant pour les novices les plus éloignés de la pratique (Phase 1). Il agit comme un environnement "puissant" qui stimule l'affect et l'intensité physique.

• Le format RPE (guidage interne) est également efficace en Phase 2, car il permet à l'élève de connecter ses propres expériences aux connaissances à acquérir.

• Le format "Au Temps" est le moins efficace pour déclencher l'intérêt chez les novices.

3. Dynamique de Développement de l'Intérêt

• Linéarité du RPE : Ce format favorise un développement constant de l'intérêt à travers toutes les phases. Il est idéal pour gérer l'hétérogénéité d'une classe.

• Non-linéarité du format "Au Temps" : Ce format ne produit des effets que lors d'une transition spécifique entre l'intérêt faible et l'intérêt émergent. Il provoque un "saut" qualitatif.

• Instabilité de l'APRE : Son impact est décrit comme "désordonné et fluctuant", suggérant qu'il doit être utilisé de façon percutante mais espacée.

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Préconisations Pédagogiques pour l'Enseignant

L'objectif pour l'enseignant est de devenir un "designer pédagogique" capable d'agencer les formats pour maximiser l'engagement, particulièrement chez les élèves les moins motivés.

Séquence Type Recommandée

Plutôt que d'utiliser un format unique, l'étude suggère un agencement stratégique durant le cycle de musculation :

1. Début de cycle (Novices) : Prioriser des formats hybrides ou le RPE. Cela permet d'épouser l'hétérogénéité de la classe et d'enclencher le processus de développement de l'intérêt.

2. Milieu de cycle : Introduire ponctuellement le format APRE pour injecter de l'intensité et stimuler les facteurs externes de l'intérêt.

3. Fin de cycle : Utiliser le format Au Temps. Ce format, moins adapté aux débutants complets, devient pertinent plus tard pour valider un saut qualitatif dans l'intérêt émergent.

4. Évaluation : Utiliser des formats hybrides pour stabiliser les acquis.

"Une bonne séquence pédagogique en musculation, c'est la capacité à agencer convenablement des formats, notamment pour les novices." — Mehdi Belhouchat

Good. This one sounds scary but the idea is actually simple.

Let’s go slow.

The sentence:

“If localities are insufficiently homogeneous…”

Translation:

If local areas are not all made up of similar people.

So inside one town or region, there might be:

• Different ethnic groups
• Different religions
• Different economic classes
• Different political views

In other words: each locality is internally divided.

“…decentralisation may paradoxically diminish accountability…”

Paradoxically = surprisingly.

Diminish accountability = make government less responsive to some people.

So it’s saying:

Giving power to local governments can sometimes make government LESS responsive.

That sounds backwards, right? We usually think local = more responsive.

“…to interests that are marginalised in localities…”

Marginalised = small minority, ignored group.

So imagine:

In each town, Group X is only 10% of the population.

They are too small locally to influence elections.

So local politicians can ignore them.

“…but enjoy some degree of cohesiveness at central level.”

Here’s the key.

Even though Group X is small in each town…

Across the whole country, they might be:

• 20% or 25% of the total population
• United
• Organized
• Able to influence national elections

So nationally, they matter.

Locally, they don’t.

What’s the big idea?

If you decentralise power:

• Decisions move to the local level.
• In each locality, the minority group is too small to matter.
• They get ignored everywhere.

But if power is centralised:

• That same minority group can band together nationally.
• Their total size becomes politically important.
• National politicians have to pay attention to them.

So decentralisation can actually make minorities worse off.

Super simple example

Imagine a country with:

• 10 regions
• In each region, Group A = 90%, Group B = 10%

If power is local:

• Group B loses everywhere.
• They are too small in every region.

If power is central:

• Group B is now 10% × 10 regions = 10% of the whole country.
• That might be enough to swing national elections.
• National politicians might listen to them.

One-sentence idiot version:

If a group is small in every town but bigger when added up nationally, giving power to towns can make them weaker — not stronger.

That’s the “paradox.”

poor want high taxes rich want low taxes so govt make low taxes

politicians are self interested. they want centralization if it reduces accountability. they don't want centralization if it

Yes. Good. Let’s go painfully slow and do it sentence by sentence, very plainly.

Here’s the paragraph again in pieces.

Sentence 1:

“The interest of citizens in one or other form of government is not necessarily shared by their political representatives.”

Translation:

What regular people want is not always what politicians want.

Citizens care about good policy. Politicians care about their careers.

Sentence 2:

“Local politicians will lose, and central politicians gain, from centralisation, whatever the benefits to their citizens.”

Translation:

If power moves to the center:

• Local politicians lose power.
• National politicians gain power.

And this is true even if centralisation is good for citizens.

So politicians judge it by who gets power, not just by whether it helps people.

Sentence 3:

“Furthermore, if some politicians are potentially mobile, in the sense that they have a significant probability of forming the government under either central or local arrangements, their incentives are perverse.”

Break this down.

“Potentially mobile” = Some politicians could end up in power under either system (local or central).

So they don’t care which level they rule at — they just want to rule somewhere.

“Incentives are perverse” = Their motivations don’t line up nicely with what’s good for society.

Why? We’re about to see.

Sentence 4 (the big one):

“They will have an interest in arguing for centralisation only when its impact in reducing accountability outweighs its beneficial impact on internalising spillovers…”

Slow down.

Centralisation does two things:

1. Reduces accountability → Harder to remove politicians → Good for politicians

2. Fixes spillovers → Makes policy more efficient → Good for society

The sentence says:

These mobile politicians support centralisation only if the reduction in accountability is more important (to them) than the spillover benefit.

In other words:

They support centralisation when it helps them personally more than it improves policy.

Final part:

“…since under these circumstances the level of effort they have to undertake will be reduced.”

This is the key.

Politicians have to put in effort to:

• Make good policies
• Satisfy voters
• Win elections

If accountability is high → they must work hard.

If accountability drops → they can slack more.

So they support centralisation when it reduces how hard they have to work.

The whole paragraph in idiot language:

Citizens might want the system that produces better policy.

Politicians want the system that:

• Gives them more power
• Makes it harder to fire them
• Lets them work less

So some politicians will only support centralisation if it mainly makes their jobs safer and easier — not just because it improves coordination.

When decisions require many places to agree, extreme situations (very secure or very insecure governments) get exaggerated under centralisation — but moderate situations don’t.

normative govt?

OMG ITS THE ELECTORAL COLLAGE!

"what weakens accountability of centralized governnment to the localities is the risk that localities will be differentially satisfied with whatever policies they have"

spillover = should centralize

Second best theory “holds that where it is not possible to satisfy all the conditions necessary for [a] . . . system to reach an overall optimum, it is not generally desirable to satisfy as many of those conditions as possible.” Adrian Vermeule, Foreword: System Effects and the Constitution, 123 Harv. L. Rev. 4, 17 (2009). In other words, if you are not moving all the way to the ideal state of affairs, it is unclear whether partial moves that seem to go in the direction of the ideal make the world “better” or “worse”—with “better” or “worse” defined by the same criteria (whatever they may be) that defined your ideal

centralized govt can differentiate policy

why this matters

accountability for accountability's sake

non verifiable welfare = cannot reward or punish govt performance

acountability definition

centralization = more coordination less accountability

decentralization = matter of which groups of electors decide re-election. accountability

political application

contracts incomplete = self interested actors

the reason why politicians r inefficient is because contracts r incomplete

L’Utilisation du Gym Aware Flex pour la Mesure de l’Engagement Physique en Musculation

Résumé Exécutif

Ce document présente une analyse du dispositif Gym Aware Flex, un encodeur linéaire utilisé pour quantifier l'activité et l'engagement physique des élèves en musculation.

Validé scientifiquement, cet outil permet de passer d'une évaluation subjective de l'effort à une mesure objective basée sur la vitesse propulsive et la trajectoire de la barre.

Les principaux enseignements montrent que le dispositif permet non seulement de distinguer précisément les niveaux d'expertise (novice vs expert) par l'analyse de la variabilité motrice, mais aussi d'optimiser l'entraînement en corrélant la vitesse aux zones de force spécifiques. Au-delà de la recherche, son intégration en Éducation Physique et Sportive (EPS) favorise une interaction pédagogique riche en confrontant le ressenti de l'élève aux données réelles, tout en sécurisant la pratique par l'estimation du maximum théorique (1RM) sans passage à l'échec.

Présentation du Dispositif et Fonctionnement Technique

Le Gym Aware Flex est défini comme un outil de mesure robuste et fiable, destiné à quantifier l'engagement physique en musculation. Son fonctionnement repose sur une technologie de précision :

• Composants du système :

◦ Un encodeur linéaire fixé sur le manchon au bout d'une barre de musculation.    ◦ Un tapis réflecteur qui capte le signal et suit les oscillations de la barre.    ◦ Une application mobile dédiée (Flex Stronger ou Gymware) pour le stockage et la réception des données.

• Données collectées en temps réel :

◦ Trajectoire de la barre et vitesse propulsive.    ◦ Vitesse moyenne (mètres par seconde) affichée à chaque répétition.    ◦ Nombre de répétitions.    ◦ Indicateurs de puissance et de dépenses énergétiques (kilojoules) calculés par algorithmes.    ◦ Distinction entre les phases concentriques et excentriques.

Identification de l'Expertise par la Variabilité

L'un des apports majeurs du dispositif est l'identification du niveau réel d'expertise des pratiquants, basée sur le principe de variabilité des trajectoires et des vitesses.

| Profil | Caractéristiques de la trajectoire | Contrôle de la vitesse | Phase excentrique | | --- | --- | --- | --- | | Novice | Trajectoire désordonnée et variable. | Vitesses propulsives irrégulières. | Incapacité ou faible capacité à contrôler la charge lors de la descente. | | Expert | Trajectoire linéaire et rectiligne. | Vitesse constante ou légère déclinaison régulière. | Maîtrise et contrôle précis du mouvement de retenue. |

L'application permet de visualiser ces différences via des codes couleurs illustrant l'accélération et la décélération, facilitant ainsi la classification des élèves.

Mesure de l'Engagement et Registres de Force

L'indicateur central retenu pour mesurer l'intensité de l'engagement physique est la moyenne de vélocité dans la phase propulsive. Cette donnée est corrélée à des objectifs de développement spécifiques :

• Zones de vitesse et projets d'entraînement :

◦ Vitesse-Force : Travail entre 1,0 et 1,3 m/s.    ◦ Force d'accélération (Hypertrophie / Résistance) : Travail entre 0,5 et 0,75 m/s.

• Corrélation avec la charge : La vitesse moyenne propulsive permet d'estimer le pourcentage de la charge maximale engagée. Par exemple, une vitesse de 0,72 m/s correspond approximativement à 60 % du maximum (1RM) de l'individu.

Applications Pédagogiques en Milieu Scolaire

L'intégration de cet outil en EPS transforme la dynamique de la leçon en favorisant une approche métacognitive :

• Confrontation Perçu/Réel : L'élève effectue sa série sans voir la tablette, puis compare son ressenti (ex: "Ai-je ralenti sur les deux dernières ?") avec les données objectives de l'histogramme de vitesse (forme d'escalier descendant).

• Interaction Sociale : Le dispositif encourage la discussion entre le pratiquant, l'observateur (pareur) et les données numériques, créant des interactions pédagogiques ayant du sens.

• Éducation Numérique : L'usage de l'outil s'inscrit dans les objectifs actuels de l'école concernant la littératie numérique.

Avantages Sécuritaires et Économiques

Le document souligne l'efficacité du Gym Aware Flex par rapport aux méthodes traditionnelles :

• Fiabilité du Maximum Théorique : Contrairement à la méthode indirecte de Brisky, souvent peu fiable, le dispositif utilise un algorithme basé sur des charges moyennes et les vitesses propulsives associées pour calculer le 1RM théorique. Cela évite aux élèves d'aller jusqu'à l'échec physique, sécurisant ainsi la pratique.

• Accessibilité Financière : Bien que représentant un investissement, le coût du dispositif a fortement baissé, passant de 2 300 € à environ 650 € l'unité.

• Stratégie d'Équipement : Il n'est pas nécessaire d'équiper l'ensemble d'une salle ; l'achat de deux ou trois unités pour des ateliers spécifiques est présenté comme une option viable pour un budget d'équipe EPS standard.

Guide de Référence Solidatech : Solutions Numériques pour les Associations

Synthèse Opérationnelle

Solidatech est un programme de solidarité numérique créé en 2008, porté par les Ateliers du Bocage, une coopérative d'utilité sociale membre d'Emmaüs. Sa mission principale est de renforcer l'impact des associations, fondations et fonds de dotation par le biais du numérique.

Le programme repose sur deux piliers stratégiques : permettre aux structures de réaliser des économies significatives sur leurs équipements (logiciels et matériel) et les accompagner dans leur montée en compétences.

Avec plus de 45 000 structures accompagnées, Solidatech s'impose comme un intermédiaire clé entre le secteur technologique et le monde associatif.

Le programme traverse actuellement une phase de transition importante suite à la fin de son partenariat historique avec le réseau international TechSoup, entraînant une restructuration interne et une autonomisation de son catalogue de solutions.

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1. Identité et Gouvernance de Solidatech

L'organisation se distingue par son ancrage dans l'économie sociale et solidaire (ESS).

• Structure porteuse : Les Ateliers du Bocage, une entreprise d'insertion et entreprise adaptée située dans les Deux-Sèvres (79).

• Affiliation : Membre du mouvement Emmaüs.

• Écosystème : Accompagne environ 45 000 associations, fonds de dotation et fondations reconnues d'utilité publique.

• Accessibilité : L'inscription au programme est entièrement gratuite pour les structures éligibles.

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2. Le Pilier Économique : Équipements et Logiciels

Solidatech facilite l'accès à des ressources technologiques à tarifs préférentiels via une boutique en ligne dédiée.

Solutions Logicielles

Le catalogue est en cours de reconstruction pour privilégier des solutions françaises, sécurisées et, de plus en plus, issues du logiciel libre.

• Domaines couverts : Travail collaboratif, communication, sécurité informatique, comptabilité et gestion.

• Modèle tarifaire : Les associations s'acquittent d'un coupon (frais de gestion) auprès de Solidatech pour obtenir des remises importantes (souvent 30 % à 50 %) sur les abonnements annuels ou mensuels des partenaires.

• Exemples d'offres : AssoConnect (gestion associative), Kaspersky (sécurité).

Matériel Informatique

Le matériel est majoritairement reconditionné en France, au sein des Ateliers du Bocage.

• Gamme "Les Cabossés" : Une offre spécifique de matériel présentant des défauts esthétiques mineurs (rayures) mais parfaitement fonctionnel, proposée à des tarifs encore plus réduits.

• Diversité des équipements : Ordinateurs portables, unités centrales, écrans, tablettes, smartphones et accessoires.

• Garantie : Tout le matériel est garanti 1 an, avec une option d'extension d'un an supplémentaire.

• Systèmes d'exploitation : Possibilité d'équiper les machines avec Windows, Linux (dont PrimTux pour les enfants) ou ChromeOS Flex.

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3. Le Pilier Compétences : Formation et Accompagnement

Au-delà de l'équipement, Solidatech propose un écosystème de services pour professionnaliser les usages numériques.

Formation Professionnelle

• Certification : Organisme certifié Qualiopi, permettant le financement des formations via les crédits OPCO (équivalent du CPF pour les structures employeuses).

• Thématiques : Intelligence Artificielle (IA), Canva, Microsoft 365, RGPD, communication digitale et outils de travail collaboratif.

Accompagnement et Diagnostic

• Diagnostic Numérique : Un outil gratuit d'auto-évaluation basé sur sept piliers de maturité numérique pour identifier les priorités d'action.

• Services de Migration : Aide au passage vers des environnements Cloud (Microsoft 365, Google Workspace) pour sécuriser les données et favoriser la collaboration.

• Prestatech : Une plateforme répertoriant des prestataires de confiance sélectionnés par Solidatech, pratiquant souvent des tarifs solidaires pour les associations.

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4. Évolutions Stratégiques et Changements Structurels

Le paysage opérationnel de Solidatech a été modifié de manière significative à la fin de l'année 2023.

| Aspect | Ancienne Situation | Situation Actuelle (Post-31/12/2023) | | --- | --- | --- | | Partenariat majeur | TechSoup Global (depuis 2008) | Fin du partenariat (décision de TechSoup) | | Support utilisateur | Équipe support interne dédiée | Suppression de l'équipe support (6 départs) | | Gestion des licences | Centralisée via TechSoup | Directe via les partenaires ou le nouveau catalogue Solidatech | | Catalogue | Partagé internationalement | Catalogue autonome en cours de repeuplement |

Conséquence pour les utilisateurs : Pour les licences historiques acquises via TechSoup (ex: anciennes licences Microsoft ou Adobe), les associations doivent désormais s'adresser directement à TechSoup Europe (basé en Pologne) ou aux éditeurs concernés, Solidatech n'ayant plus accès aux données de ces anciens comptes.

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5. Ressources et Pilotage de la Maturité Numérique

Solidatech produit et diffuse des connaissances pour éclairer le secteur associatif.

• Étude Nationale : Publication triennale de l'enquête "La place du numérique dans le projet associatif" (5ème édition disponible), coproduite avec Recherches & Solidarités.

• Centre de Ressources : Articles conseils, replays de webinaires et guides pratiques (ex: alternatives libres à la suite Adobe).

• Veille et Information : Une newsletter mensuelle et des webinaires réguliers (format court d'une heure) sur des enjeux d'actualité comme LinkedIn ou l'IA.

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6. Modalités Pratiques d'Inscription

Pour bénéficier des services, une structure doit suivre un processus simple :

1. Inscription sur solidatech.fr : Nécessite le téléchargement des documents officiels de l'association.

2. Création de compte boutique : Une étape unique pour accéder au catalogue matériel et logiciel.

3. Mise à jour des contacts : Il est recommandé de renseigner plusieurs contacts pour assurer la continuité des échanges malgré le turn-over associatif.

Solidatech encourage activement les associations à faire remonter leurs besoins spécifiques via des questionnaires pour orienter les futurs partenariats du catalogue en reconstruction.

Should be a curly bracket

I think this is key. Even without a productivist mindset, some games can get frustratingly slow. Too challenging, in a way. Too painful perhaps. Too minimal to encourage reflection. Is a game like Mini Metro a reflective one? I don't think much. But then again, is a game like Magnet Block? Not either, probably.

I feel the focus on walking simulators is nice as it gives many examples of what could be, but it misses genres like puzzles, or visual novels, that may sometimes be blurry! Coffee Talk or The Red Strings may be somewhat reflective, but is Doki Doki Literature Club or Gods Will be Watching? The story... the story and the intensity therein, its agressiveness, its fear, violence, etc. I'd argue contribute to the perception as well.

Te has dado cuenta que esa persona que trata su propio cumpleaños como un día cualquiera no lo hace por desinterés? Lo hace porque aprendió a no depender de fechas para sentirse importante.

Es alguien que ya pasó por suficientes silencios, responsabilidades y golpes como para saber que el tiempo no se celebra, se aprovecha. Cumplir años para él no es sumar velas, es hacer cuentas: qué logró, qué perdió, qué sigue debiendo.

No espera mensajes, ni fiestas, ni atención. Está acostumbrado a caminar solo, a cargar lo suyo y a seguir adelante aunque nadie esté mirando. Por eso su cumpleaños no es un evento… es una pausa breve antes de volver al camino.

Esa persona suele ser la misma que no presume lo que aguanta, que no pide reconocimiento por lo que sostiene, y que entiende que el verdadero respeto no llega con felicitaciones, sino con coherencia.

Tratar el cumpleaños como un día cualquiera no es frialdad. Es madurez. Es haber entendido que el valor no lo da una fecha, sino lo que haces con tu tiempo. Ver menos

If someone is not religious, it does not mean that they are immediately a bad person

The Idea of sperate parties based on views is something inevitable, and has been shown in different governments in different ways.

eLife assessment

The manuscript presents important findings with theoretical or practical implications beyond a single subfield. The work is overall solid, and the methods, data, and analyses broadly support the claims. Although the novelty of this study and the work put into it are appreciated, there are also clearly some weaknesses that should be addressed.

Reviewer #1 (Public review):

Summary:

This manuscript by Lin et al. presents a timely, technically strong study that builds patient-specific midbrain-like organoids (MLOs) from hiPSCs carrying clinically relevant GBA1 mutations (L444P/P415R and L444P/RecNcil). The authors comprehensively characterize nGD phenotypes (GCase deficiency, GluCer/GluSph accumulation, altered transcriptome, impaired dopaminergic differentiation), perform CRISPR correction to produce an isogenic line, and test three therapeutic modalities (SapC-DOPS-fGCase nanoparticles, AAV9-GBA1, and SRT with GZ452). The model and multi-arm therapeutic evaluation are important advances with clear translational value.

My overall recommendation is that the work undergo a major revision to address the experimental and interpretive gaps listed below.

Strengths:

(1) Human, patient-specific midbrain model: Use of clinically relevant compound heterozygous GBA1 alleles (L444P/P415R and L444P/RecNcil) makes the model highly relevant to human nGD and captures patient genetic context that mouse models often miss.

(2) Robust multi-level phenotyping: Biochemical (GCase activity), lipidomic (GluCer/GluSph by UHPLC-MS/MS), molecular (bulk RNA-seq), and histological (TH/FOXA2, LAMP1, LC3) characterization are thorough and complementary.

(3) Use of isogenic CRISPR correction: Generating an isogenic line (WT/P415R) and demonstrating partial rescue strengthens causal inference that the GBA1 mutation drives many observed phenotypes.

(4) Parallel therapeutic testing in the same human platform: Comparing enzyme delivery (SapC-DOPS-fGCase), gene therapy (AAV9-GBA1), and substrate reduction (GZ452) within the same MLO system is an elegant demonstration of the platform's utility for preclinical evaluation.

(5) Good methodological transparency: Detailed protocols for MLO generation, editing, lipidomics, and assays allow reproducibility

Weaknesses:

(1) Limited genetic and biological replication

(a) Single primary disease line for core mechanistic claims. Most mechanistic data derive from GD2-1260 (L444P/P415R); GD2-10-257 (L444P/RecNcil) appears mainly in therapeutic experiments. Relying primarily on one patient line risks conflating patient-specific variation with general nGD mechanisms.

(b) Unclear biological replicate strategy. It is not always explicit how many independent differentiations and organoid batches were used (biological replicates vs. technical fields of view).

(c) A significant disadvantage of employing brain organoids is the heterogeneity during induction and potential low reproducibility. In this study, it is unclear how many independent differentiation batches were evaluated and, for each test (for example, immunofluorescent stain and bulk RNA-seq), how many organoids from each group were used. Please add a statement accordingly and show replicates to verify consistency in the supplementary data.

(d) Isogenic correction is partial. The corrected line is WT/P415R (single-allele correction); residual P415R complicates the interpretation of "full" rescue and leaves open whether the remaining pathology is due to incomplete correction or clonal/epigenetic effects.

(e) The authors tested week 3, 4, 8, 15, and 28 old organoids in different settings. However, systematic markers of maturation should be analyzed, and different maturation stages should be compared, for example, comparing week 8 organoids to week 28 organoids, with immunofluorescent marker staining and bulk RNAseq.

(f) The manuscript frequently refers to Wnt signaling dysregulation as a major finding. However, experimental validation is limited to transcriptomic data. Functional tests, such as the use of Wnt agonist/inhibitor, are needed to support this claim (see below).

(g) Suggested fixes/experiments

Add at least one more independent disease hiPSC line (or show expanded analysis from GD2-10-257) for key mechanistic endpoints (lipid accumulation, transcriptomics, DA markers)

Generate and analyze a fully corrected isogenic WT/WT clone (or a P415R-only line) if feasible; at minimum, acknowledge this limitation more explicitly and soften claims.

Report and increase independent differentiations (N = biological replicates) and present per-differentiation summary statistics.

(2) Mechanistic validation is insufficient

(a) RNA-seq pathways (Wnt, mTOR, lysosome) are not functionally probed. The manuscript shows pathway enrichment and some protein markers (p-4E-BP1) but lacks perturbation/rescue experiments to link these pathways causally to the DA phenotype.

(b) Autophagy analysis lacks flux assays. LC3-II and LAMP1 are informative, but without flux assays (e.g., bafilomycin A1 or chloroquine), one cannot distinguish increased autophagosome formation from decreased clearance.

(c) Dopaminergic dysfunction is superficially assessed. Dopamine in the medium and TH protein are shown, but no neuronal electrophysiology, synaptic marker co-localization, or viability measures are provided to demonstrate functional recovery after therapy.

(d) Suggested fixes/experiments

Perform targeted functional assays:

(i) Wnt reporter assays (TOP/FOP flash) and/or treat organoids with Wnt agonists/antagonists to test whether Wnt modulation rescues DA differentiation.

(ii)Test mTOR pathway causality using mTOR inhibitors (e.g., rapamycin) or 4E-BP1 perturbation and assay effects on DA markers and autophagy.

Include autophagy flux assessment (LC3 turnover with bafilomycin), and measure cathepsin activity where relevant.

Add at least one functional neuronal readout: calcium imaging, MEA recordings, or synaptic marker quantification (e.g., SYN1, PSD95) together with TH colocalization.

(3) Therapeutic evaluation needs greater depth and standardization

(a) Short windows and limited durability data. SapC-DOPS and AAV9 experiments range from 48 hours to 3 weeks; longer follow-up is needed to assess durability and whether biochemical rescue translates into restored neuronal function.

(b) Dose-response and biodistribution are under-characterized. AAV injection sites/volumes are described, but transduction efficiency, vg copies per organoid, cell-type tropism quantification, and SapC-DOPS penetration/distribution are not rigorously quantified.

(c) Specificity controls are missing. For SapC-DOPS, inclusion of a non-functional enzyme control (or heat-inactivated fGCase) would rule out non-specific nanoparticle effects. For AAV, assessment of off-target expression and potential cytotoxicity is needed.

(d) Comparative efficacy lacking. It remains unclear which modality is most effective in the long term and in which cellular compartments.

(e) Suggested fixes/experiments

Extend follow-up (e.g., 6+ weeks) after AAV/SapC dosing and evaluate DA markers, electrophysiology, and lipid levels over time.

Quantify AAV transduction by qPCR for vector genomes and by cell-type quantification of GFP+ cells (neurons vs astrocytes vs progenitors).

Include SapC-DOPS control nanoparticles loaded with an inert protein and/or fluorescent cargo quantitation to show distribution and uptake kinetics.

Provide head-to-head comparative graphs (activity, lipid clearance, DA restoration, and durability) with statistical tests.

(4) Model limitations not fully accounted for in interpretation

(a) Absence of microglia and vasculature limits recapitulation of neuroinflammatory responses and drug penetration, both of which are important in nGD. These absences could explain incomplete phenotypic rescues and must be emphasized when drawing conclusions about therapeutic translation.

(b) Developmental vs degenerative phenotype conflation. Many phenotypes appear during differentiation (patterning defects). The manuscript sometimes interprets these as degenerative mechanisms; the distinction must be clarified.

(c) Suggested fixes

Tone down the language throughout (Abstract/Results/Discussion) to avoid overstatement that MLOs fully recapitulate nGD neuropathology.

Add plans or pilot data (if available) for microglia incorporation or vascularization to indicate how future work will address these gaps.

(5) Statistical and presentation issues

(a) Missing or unclear sample sizes (n). For organoid-level assays, report the number of organoids and the number of independent differentiations.

(b) Statistical assumptions not justified. Tests assume normality; where sample sizes are small, consider non-parametric tests and report exact p-values.

(c) Quantification scope. Many image quantifications appear to be from selected fields of view, which are then averaged across organoids and differentiations.

(d) RNA-seq QC and deposition. Provide mapping rates, batch correction details, and ensure the GEO accession is active. Include these in Methods/Supplement.

(e) Suggested fixes

Add a table summarizing biological replicates, technical replicates, and statistical tests used for each figure panel.

Recompute statistics where appropriate (non-parametric if N is small) and report effect sizes and confidence intervals.

(6) Minor comments and clarifications

(a) The authors should validate midbrain identity further with additional regional markers (EN1, OTX2) and show absence/low expression of forebrain markers (FOXG1) across replicates.

(b) Extracellular dopamine ELISA should be complemented with intracellular dopamine or TH+ neuron counts normalized per organoid or per total neurons.

(c) For CRISPR editing: the authors should report off-target analysis (GUIDE-seq or targeted sequencing of predicted off-targets) or at least in-silico off-target score and sequencing coverage of the edited locus.

(d) It should be clarified as to whether lipidomics normalization is to total protein per organoid or per cell, and include representative LC-MS chromatograms or method QC.

(e) Figure legends should be improved in order to state the number of organoids, the number of differentiations, and the exact statistical tests used (including multiple-comparison corrections).

(f) In the title, the authors state "reveal disease mechanisms", but the studies mainly exhibit functional changes. They should consider toning down the statement.

(7) Recommendations

This reviewer recommends a major revision. The manuscript presents substantial novelty and strong potential impact but requires additional experimental validation and clearer, more conservative interpretation. Key items to address are:

(a) Strengthening genetic and biological replication (additional lines or replicate differentiations).

(b) Adding functional mechanistic validation for major pathways (Wnt/mTOR/autophagy) and providing autophagy flux data.

(c) Including at least one neuronal functional readout (calcium imaging/MEA/patch) to demonstrate functional rescue.

(d) Deepening therapeutic characterization (dose, biodistribution, durability) and including specificity controls.

(e) Improving statistical reporting and explicitly stating biological replicate structure.

Reviewer #2 (Public review):

Sun et al. have developed a midbrain-like organoid (MLO) model for neuronopathic Gaucher disease (nGD). The MLOs recapitulate several features of nGD molecular pathology, including reduced GCase activity, sphingolipid accumulation, and impaired dopaminergic neuron development. They also characterize the transcriptome in the MLO nGD model. CRISPR correction of one of the GBA1 mutant alleles rescues most of the nGD molecular phenotypes. The MLO model was further deployed in proof-of-principle studies of investigational nGD therapies, including SapC-DOPS nanovesicles, AAV9-mediated GBA1 gene delivery, and substrate-reduction therapy (GZ452). This patient-specific 3D model provides a new platform for studying nGD mechanisms and accelerating therapy development. Overall, only modest weaknesses are noted.

Reviewer #3 (Public review):

Summary:

In this study, the authors describe modeling of neuronopathic Gaucher disease (nGD) using midbrain-like organoids (MLOs) derived from hiPSCs carrying GBA1 L444P/P415R or L444P/RecNciI variants. These MLOs recapitulate several disease features, including GCase deficiency, reduced enzymatic activity, lipid substrate accumulation, and impaired dopaminergic neuron differentiation. Correction of the GBA1 L444P variant restored GCase activity, normalized lipid metabolism, and rescued dopaminergic neuronal defects, confirming its pathogenic role in the MLO model. The authors further leveraged this system to evaluate therapeutic strategies, including: (i) SapC-DOPS nanovesicles for GCase delivery, (ii) AAV9-mediated GBA1 gene therapy, and (iii) GZ452, a glucosylceramide synthase inhibitor. These treatments reduced lipid accumulation and ameliorated autophagic, lysosomal, and neurodevelopmental abnormalities.

Strengths:

This manuscript demonstrates that nGD patient-derived MLOs can serve as an additional platform for investigating nGD mechanisms and advancing therapeutic development.

Comments:

(1) It is interesting that GBA1 L444P/P415R MLOs show defects in midbrain patterning and dopaminergic neuron differentiation (Figure 3). One might wonder whether these abnormalities are specific to the combination of L444P and P415R variants or represent a general consequence of GBA1 loss. Do GBA1 L444P/RecNciI (GD2-10-257) MLOs also exhibit similar defects?

(2) In Supplementary Figure 3, the authors examined GCase localization in SapC-DOPS-fGCase-treated nGD MLOs. These data indicate that GCase is delivered to TH⁺ neurons, GFAP⁺ glia, and various other unidentified cell types. In fruit flies, the GBA1 ortholog, Gba1b, is only expressed in glia (PMID: 35857503; 35961319). Neuronally produced GluCer is transferred to glia for GBA1-mediated degradation. These findings raise an important question: in wild-type MLOs, which cell type(s) normally express GBA1? Are they dopaminergic neurons, astrocytes, or other cell types?

(3) The authors may consider switching Figures 2 and 3 so that the differentiation defects observed in nGD MLOs (Figure 3) are presented before the analysis of other phenotypic abnormalities, including the various transcriptional changes (Figure 2).

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

This manuscript by Lin et al. presents a timely, technically strong study that builds patientspecific midbrain-like organoids (MLOs) from hiPSCs carrying clinically relevant GBA1 mutations (L444P/P415R and L444P/RecNcil). The authors comprehensively characterize nGD phenotypes (GCase deficiency, GluCer/GluSph accumulation, altered transcriptome, impaired dopaminergic differentiation), perform CRISPR correction to produce an isogenic line, and test three therapeutic modalities (SapC-DOPS-fGCase nanoparticles, AAV9GBA1, and SRT with GZ452). The model and multi-arm therapeutic evaluation are important advances with clear translational value.

My overall recommendation is that the work undergo a major revision to address the experimental and interpretive gaps listed below.

Strengths:

(1) Human, patient-specific midbrain model: Use of clinically relevant compound heterozygous GBA1 alleles (L444P/P415R and L444P/RecNcil) makes the model highly relevant to human nGD and captures patient genetic context that mouse models often miss.

(2) Robust multi-level phenotyping: Biochemical (GCase activity), lipidomic (GluCer/GluSph by UHPLC-MS/MS), molecular (bulk RNA-seq), and histological (TH/FOXA2, LAMP1, LC3) characterization are thorough and complementary.

(3) Use of isogenic CRISPR correction: Generating an isogenic line (WT/P415R) and demonstrating partial rescue strengthens causal inference that the GBA1 mutation drives many observed phenotypes.

(4) Parallel therapeutic testing in the same human platform: Comparing enzyme delivery (SapC-DOPS-fGCase), gene therapy (AAV9-GBA1), and substrate reduction (GZ452) within the same MLO system is an elegant demonstration of the platform's utility for preclinical evaluation.

(5) Good methodological transparency: Detailed protocols for MLO generation, editing, lipidomics, and assays allow reproducibility

Weaknesses:

(1) Limited genetic and biological replication

(a) Single primary disease line for core mechanistic claims. Most mechanistic data derive from GD2-1260 (L444P/P415R); GD2-10-257 (L444P/RecNcil) appears mainly in therapeutic experiments. Relying primarily on one patient line risks conflating patient-specific variation with general nGD mechanisms.

We thank the reviewer for highlighting the importance of genetic and biological replication. An additional patient-derived iPSC line was included in the manuscript, therefore, our study includes two independent nGD patient-derived iPSC lines, GD2-1260 (GBA1^L444P/P415R) and GD2-10-257 (GBA1^{L444P/RecNcil}), both of which carry the severe mutations associated with nGD. These two lines represent distinct genetic backgrounds and were used to demonstrate the consistency of key disease phenotypes (reduced GCase activity, elevated substrate, impaired dopaminergic neuron differentiation, etc.) across different patient’s MLOs. Major experiments (e.g., GCase activity assays, substrate, immunoblotting for DA marker TH, and therapeutic testing with SapC-DOPS-fGCase, AAV9-GBA1) were performed using both patient lines, with results showing consistent phenotypes and therapeutic responses (see Figs. 2-6, and Supplementary Figs. 4-5). To ensure clarity and transparency, a new Supplementary Table 2 summarizes the characterization of both the GD2-1260 and GD2-10-257 lines.

(b) Unclear biological replicate strategy. It is not always explicit how many independent differentiations and organoid batches were used (biological replicates vs. technical fields of view).

Biological replication was ensured in our study by conducting experiments in at least 3 independent differentiations per line, and technical replicates (multiple organoids/fields per batch) were averaged accordingly. We have clarified biological replicates and differentiation in the figure legends. 

(c) A significant disadvantage of employing brain organoids is the heterogeneity during induction and potential low reproducibility. In this study, it is unclear how many independent differentiation batches were evaluated and, for each test (for example, immunofluorescent stain and bulk RNA-seq), how many organoids from each group were used. Please add a statement accordingly and show replicates to verify consistency in the supplementary data.

In the revision, we have clarified biological replicates and differentiation in the figure legend in Fig.1E; Fig.2B,2G; Fig.3F, 3G; Fig.4B-C,E,H-J, M-N; Fig.6D; and Fig.7A-C, I.

(d) Isogenic correction is partial. The corrected line is WT/P415R (single-allele correction); residual P415R complicates the interpretation of "full" rescue and leaves open whether the remaining pathology is due to incomplete correction or clonal/epigenetic effects.

We attempted to generate an isogenic iPSC line by correcting both GBA1 mutations (L444P and P415R). However, this was not feasible because GBA1 overlaps with a highly homologous pseudogene (PGBA), which makes precise editing technically challenging. Consequently, only the L444P mutation was successfully corrected, and the resulting isogenic line retains the P415R mutation in a heterozygous state. Because Gaucher disease is an autosomal recessive disorder, individuals carrying a single GBA1 mutation (heterozygous carriers) do not develop clinical symptoms. Therefore, the partially corrected isogenic line, which retains only the P415R allele, represents a clinically relevant carrier model. Consistent with this, our results show that GCase activity was restored to approximately 50% of wild-type levels (Fig.4B-C), supporting the expected heterozygous state. These findings also make it unlikely that the remaining differences observed are due to clonal variation or epigenetic effects.

(e) The authors tested week 3, 4, 8, 15, and 28 old organoids in different settings. However, systematic markers of maturation should be analyzed, and different maturation stages should be compared, for example, comparing week 8 organoids to week 28 organoids, with immunofluorescent marker staining and bulk RNAseq.

We agree that a systematic analysis of maturation stages is essential for validating the MLO model. Our data integrated a longitudinal comparison across multiple developmental windows (Weeks 3 to 28) to characterize the transition from progenitors to mature/functional states for nGD phenotyping and evaluation of therapeutic modalities: 1) DA differentiation (Wks 3 and 8 in Fig. 3): qPCR analysis demonstrated the progression of DA-specific programs. We observed a steady increase in the mature DA neuron marker TH and ASCL1. This was accompanied by a gradual decrease in early floor plate/progenitor markers FOXA2 and PLZF, indicating a successful differentiation path from progenitors to differentiated/mature DA neurons. 2) Glycosphingolipid substrates accumulation (Wks 15 and 28 in Fig 2): To assess late-stage nGD phenotyping, we compared GluCer and GluSph at Week 15 and Week 28. This comparison highlights the progressive accumulation of substrates in nGD MLOs, reflecting the metabolic consequences of the disease at different mature stage. 3) Organoid growth dynamics (Wks 4, 8, and 15 in new Fig. 4): The new Fig. 4 tracks physical maturation through organoid size and growth rates across three key time points, providing a macro-scale verification of consistent development between WT and nGD groups. By comparing these early (Wk 3-8) and late (Wk 15-28) stages, we confirmed that our MLOs transition from a proliferative state to a post-mitotic, specialized neuronal state, satisfied the requirement for comparing distinct maturation stages.

(f) The manuscript frequently refers to Wnt signaling dysregulation as a major finding. However, experimental validation is limited to transcriptomic data. Functional tests, such as the use of Wnt agonist/inhibitor, are needed to support this claim (see below).

We agree that the suggested experiments could provide additional mechanistic insights into this study and will consider them in future work.

(g) Suggested fixes / experiments

Add at least one more independent disease hiPSC line (or show expanded analysis from GD2-10-257) for key mechanistic endpoints (lipid accumulation, transcriptomics, DA markers).

Additional line iPSC GD2-10-257 derived MLO was included in the manuscript. This was addressed above [see response to Weaknesses (1)-a]. 

Generate and analyze a fully corrected isogenic WT/WT clone (or a P415R-only line) if feasible; at minimum, acknowledge this limitation more explicitly and soften claims.

We attempted to generate an isogenic iPSC line by correcting both GBA1 mutations (L444P and P415R). However, this was unsuccessful because the GBA1 gene overlaps with a pseudogene (PGBA) located 16 kb downstream of GBA1, which shares 96-98% sequence similarity with GBA1 (Ref#1, #2), which complicates precise editing. GBA1 is shorter (~5.7 kb) than PGBA (~7.6 kb). The primary exonic difference between GBA1 and PGBA is a 55-bp deletion in exon 9 of the pseudogene. As a result, the isogenic line we obtained carries only the P415R mutation, and L444P was corrected to the normal sequence. We have included this limitation in the Methods as “This gene editing strategy is expected to also target the GBA1 pseudogene due to the identical target sequence, which limits the gene correction on certain mutations (e.g., P415R)”. 

References:

(1) Horowitz M., Wilder S., Horowitz Z., Reiner O., Gelbart T., Beutler E. The human glucocerebrosidase gene and pseudogene: structure and evolution. Genomics (1989). 4, 87–96. doi:10.1016/0888-7543(89)90319-4

(2) Woo EG, Tayebi N, Sidransky E. Next-Generation Sequencing Analysis of GBA1: The Challenge of Detecting Complex Recombinant Alleles. Front Genet. (2021). 12:684067. doi:10.3389/fgene.2021.684067. PMCID: PMC8255797.

Report and increase independent differentiations (N = biological replicates) and present per-differentiation summary statistics.

This was addressed above [see response to Weaknesses (1)-b, (1)-c]. 

(2) Mechanistic validation is insufficient

(a) RNA-seq pathways (Wnt, mTOR, lysosome) are not functionally probed. The manuscript shows pathway enrichment and some protein markers (p-4E-BP1) but lacks perturbation/rescue experiments to link these pathways causally to the DA phenotype.

(b) Autophagy analysis lacks flux assays. LC3-II and LAMP1 are informative, but without flux assays (e.g., bafilomycin A1 or chloroquine), one cannot distinguish increased autophagosome formation from decreased clearance.

(c) Dopaminergic dysfunction is superficially assessed. Dopamine in the medium and TH protein are shown, but no neuronal electrophysiology, synaptic marker co-localization, or viability measures are provided to demonstrate functional recovery after therapy.

(d) Suggested fixes/experiments

Perform targeted functional assays:

(i) Wnt reporter assays (TOP/FOP flash) and/or treat organoids with Wnt agonists/antagonists to test whether Wnt modulation rescues DA differentiation.

(ii) Test mTOR pathway causality using mTOR inhibitors (e.g., rapamycin) or 4E-BP1 perturbation and assay effects on DA markers and autophagy.

Include autophagy flux assessment (LC3 turnover with bafilomycin), and measure cathepsin activity where relevant.

Add at least one functional neuronal readout: calcium imaging, MEA recordings, or synaptic marker quantification (e.g., SYN1, PSD95) together with TH colocalization.

We thank the reviewer for these valuable suggestions. We agree that the suggested experiments could provide additional mechanistic insights into this study and will consider them in future work. Importantly, the primary conclusions of our manuscript, that GBA1 mutations in nGD MLOs resulted in nGD pathologies such as diminished enzymatic function, accumulation of lipid substrates, widespread transcriptomic changes, and impaired dopaminergic neuron differentiation, which can be corrected by several therapeutic strategies in this study, are supported by the evidence presented. The suggested experiments represent an important direction for future research using brain organoids.

(3) Therapeutic evaluation needs greater depth and standardization

(a) Short windows and limited durability data. SapC-DOPS and AAV9 experiments range from 48 hours to 3 weeks; longer follow-up is needed to assess durability and whether biochemical rescue translates into restored neuronal function.

We agree with the reviewer. Because this is a proof-of-principle study, the treatment was designed within a short time window. Long-term studies with more comprehensive outcome assessments will be conducted in future work.

(b) Dose-response and biodistribution are under-characterized. AAV injection sites/volumes are described, but transduction efficiency, vg copies per organoid, cell-type tropism quantification, and SapC-DOPS penetration/distribution are not rigorously quantified.

We appreciate the reviewer’s concerns. This study was intended to demonstrate the feasibility and initial response of MLOs to AAV therapy. A comprehensive evaluation of AAV biodistribution will be considered in future studies.

The penetration and distribution of SapC-DOPS have been extensively characterized in prior studies. In vivo biodistribution of SapC–DOPS coupled CellVue Maroon, a fluorescent cargo, was examined in mice bearing human tumor xenografts using real-time fluorescence imaging, where CellVue Maroon fluorescence in tumor remained for 48 hours (Ref. #3: Fig. 4B, mouse 1), 100 hours (Ref. #4: Fig. 5), up to 216 hours (Ref. #5: Fig. 3). Uptake kinetics were also demonstrated in cells, with flow cytometry quantification showing that fluorescent cargo coupled SapC-DOPS nanovesicles, were incorporated into human brain tumor cell membranes within minutes and remained stably incorporated into the cells for up to one hour (Ref. # 6: Fig. 1a and Fig. 1b). Building on these findings, the present study focuses on evaluating the restoration of GCase function rather than reexamining biodistribution and uptake kinetics.

References:

(3) X. Qi, Z. Chu, Y.Y. Mahller, K.F. Stringer, D.P. Witte, T.P. Cripe. Cancer-selective targeting and cytotoxicity by liposomal-coupled lysosomal saposin C protein. Clin. Cancer Res. (2009) 15, 5840-5851. PMID: 19737950.

(4) Z. Chu, S. Abu-Baker, M.B. Palascak, S.A. Ahmad, R.S. Franco, and X. Qi. Targeting and cytotoxicity of SapC-DOPS nanovesicles in pancreatic cancer. PLOS ONE (2013) 8, e75507. PMID: 24124494.

(5) Z. Chu, K. LaSance, V.M. Blanco, C.-H. Kwon, B., Kaur, M., Frederick, S., Thornton, L., Lemen, and X. Qi. Multi-angle rotational optical imaging of brain tumors and arthritis using fluorescent SapC-DOPS nanovesicles. J. Vis. Exp. (2014) 87, e51187, 17. PMID: 24837630.

(6) J. Wojton, Z. Chu, C-H. Kwon, L.M.L. Chow, M. Palascak, R. Franco, T. Bourdeau, S. Thornton, B. Kaur, and X. Qi. Systemic delivery of SapC-DOPS has antiangiogenic and antitumor effects against glioblastoma. Mol. Ther. (2013) 21, 1517-1525. PMID: 23732993.

(c) Specificity controls are missing. For SapC-DOPS, inclusion of a non-functional enzyme control (or heat-inactivated fGCase) would rule out non-specific nanoparticle effects. For AAV, assessment of off-target expression and potential cytotoxicity is needed.

Including inactive fGCase would confound the assessment of fGCase in MLOs by immunoblot and immunofluorescence; therefore, saposin C–DOPS was used as the control instead. 

We agree that assessment of Off-target expression and potential cytotoxicity for AAV is important; this will be included in future studies.

(d) Comparative efficacy lacking. It remains unclear which modality is most effective in the long term and in which cellular compartments.

To address this comment, we have added a new table (Supplementary Table 2) comparing the four therapeutic modalities and summarizing their respective outcomes. While this study focused on short-term responses as a proof-of-principle, future work will explore long-term therapeutic effects. 

(e) Suggested fixes/experiments

Extend follow-up (e.g., 6+ weeks) after AAV/SapC dosing and evaluate DA markers, electrophysiology, and lipid levels over time.

We appreciate the reviewer’s suggestions. The therapeutic testing in patient-derived MLOs was designed as a proof-of-principle study to demonstrate feasibility and the primary response (rescue of GCase function) to the treatment. A comprehensive, long-term therapeutic evaluation of AAV and SapC-DOPS-fGCase is indeed important for a complete assessment; however, this represents a separate therapeutic study and is beyond the scope of the current work.

Quantify AAV transduction by qPCR for vector genomes and by cell-type quantification of GFP+ cells (neurons vs astrocytes vs progenitors).

For the AAV-treated experiments, we agree that measuring AAV copy number and GFP expression would provide additional information. However, the primary goal of this study was to demonstrate the key therapeutic outcome, rescue of GCase function by AAV-delivered normal GCase, which is directly relevant to the treatment objective.

Include SapC-DOPS control nanoparticles loaded with an inert protein and/or fluorescent cargo quantitation to show distribution and uptake kinetics.

As noted above [see response to Weakness (3)-c], using inert GCase would confound the assessment of fGCase uptake in MLOs; therefore, it was not suitable for this study. See response above for the distribution and uptake kinetics of SapC-DOPS [see response to Weaknesses (3)-b].

Provide head-to-head comparative graphs (activity, lipid clearance, DA restoration, and durability) with statistical tests.

We have added a new table (Supplementary Table 2) providing a head-to-head comparison of the treatment effects. 

(4) Model limitations not fully accounted for in interpretation

(a) Absence of microglia and vasculature limits recapitulation of neuroinflammatory responses and drug penetration, both of which are important in nGD. These absences could explain incomplete phenotypic rescues and must be emphasized when drawing conclusions about therapeutic translation.

We agree that the absence of microglia and vasculature in midbrain-like organoids represents a limitation, as we have discussed in the manuscript. In this revision, we highlighted this limitation in the Discussion section and clarified that it may contribute to incomplete phenotyping and phenotypic rescue observed in our therapeutic experiments. Additionally, we have outlined future directions to incorporate microglia and vascularization into the organoid system to better recapitulate the in vivo environment and improve translational relevance (see 7th paragraph in the Discussion).

(b) Developmental vs degenerative phenotype conflation. Many phenotypes appear during differentiation (patterning defects). The manuscript sometimes interprets these as degenerative mechanisms; the distinction must be clarified.

We appreciate the reviewer’s comments. In the revised manuscript, we have clarified that certain abnormalities, such as patterning defects observed during early differentiation, likely reflect developmental consequences of GBA1 mutations rather than degenerative processes. Conversely, phenotypes such as substrate accumulation, lysosomal dysfunction, and impaired dopaminergic maturation at later stages are interpreted as degenerative features. We have updated the Results and Discussion sections to avoid conflating developmental defects with neurodegenerative mechanisms.

(c) Suggested fixes

Tone down the language throughout (Abstract/Results/Discussion) to avoid overstatement that MLOs fully recapitulate nGD neuropathology.

The manuscript has been revised to avoid overstatements.

Add plans or pilot data (if available) for microglia incorporation or vascularization to indicate how future work will address these gaps.

The manuscript now includes further plans to address the incorporation of microglia and vascularization, described in the last two paragraphs in the Discussion. Pilot study of microglia incorporation will be reported when it is completed.

(5) Statistical and presentation issues

(a) Missing or unclear sample sizes (n). For organoid-level assays, report the number of organoids and the number of independent differentiations.

We have clarified biological replicates and differentiation in the figure legend [see response to Weaknesses (1)-b, (1)-c]. 

(b) Statistical assumptions not justified. Tests assume normality; where sample sizes are small, consider non-parametric tests and report exact p-values.

We have updated Statistical analysis in the methods as described below:

“For comparisons between two groups, data were analyzed using unpaired two-tailed Student’s t-tests when the sample size was ≥6 per group and normality was confirmed by the Shapiro-Wilk test. When the normality assumption was not met or when sample sizes were small (n < 6), the non-parametric Mann-Whitney U test was used instead. For comparisons involving three or more groups, one-way ANOVA followed by Tukey’s multiple comparison test was applied when data were normally distributed; otherwise, the nonparametric Dunn’s multiple comparison test was used. Exclusion of outliers was made based on cut-offs of the mean ±2 standard deviations. All statistical analyses were performed using GraphPad Prism 10 software. Exact p-values are reported throughout the manuscript and figures where feasible. A p-value < 0.05 was considered statistically significant.”

(c) Quantification scope. Many image quantifications appear to be from selected fields of view, which are then averaged across organoids and differentiations.

In this work, quantitative immunofluorescence analyses (e.g., cell counts for FOXP1+, FOXG1+, SOX2+ and Ki67+ cells, as well as marker colocalization) were performed on at least 3–5 randomly selected non-overlapping fields of view (FOVs) per organoid section, with a minimum of 3 organoids per differentiation batch. Each FOV was imaged at consistent magnification (60x) and z-stack depth to ensure comparable sampling across conditions. Data from individual FOVs were first averaged within each organoid to obtain an organoid-level mean, and then biological replicates (independent differentiations, n ≥ 3) were averaged to generate the final group mean ± SEM. This multilevel averaging approach minimizes bias from regional heterogeneity within organoids and accounts for variability across differentiations. Representative confocal images shown in the figures were selected to accurately reflect the quantified data. We believe this standardized quantification strategy ensures robust and reproducible results while appropriately representing the 3D architecture of the organoids.

In the revision, we have clarified the method used for image analysis of sectioned MLOs as below:

“Quantitative immunofluorescence analyses (e.g., cell counts for FOXP1+, FOXG1+, SOX2+ and Ki67+ cells, as well as marker colocalization) were performed using ImageJ (NIH) on at least 3–5 randomly selected non-overlapping fields of view (FOVs) per organoid section, with a minimum of 3 organoids per differentiation batch. Each FOV was imaged at consistent magnification (60x) and z-stack depth to ensure comparable sampling across conditions. Data from individual FOVs were first averaged within each organoid to obtain an organoid-level mean, and then biological replicates (independent differentiations, n ≥ 3) were averaged to generate the final group mean ± SEM.”

(d) RNA-seq QC and deposition. Provide mapping rates, batch correction details, and ensure the GEO accession is active. Include these in Methods/Supplement.

RNA-seq data are from the same batch. The mapping rate is >90%. GEO accession will be active upon publication. These were included in the Methods.

(e) Suggested fixes

Add a table summarizing biological replicates, technical replicates, and statistical tests used for each figure panel.

We have revised the figure legends to include replicates for each figure and statistical tests [see response in weaknesses (1)-b, (1)-c].

Recompute statistics where appropriate (non-parametric if N is small) and report effect sizes and confidence intervals.

Statistical analysis method is provided in the revision [see response in Weaknesses (5)-b].

(6) Minor comments and clarifications

(a) The authors should validate midbrain identity further with additional regional markers (EN1, OTX2) and show absence/low expression of forebrain markers (FOXG1) across replicates.

We validated the MLO identity by 1) FOXG1 and 2) EN1. FOXG1 was barely detectable in Wk8 75.1_MLO but highly present in ‘age-matched’ cerebral organoid (CO), suggesting our culturing method is midbrain region-oriented. In nGD MLO, FOXG1 expression is significantly higher than 75.1_MLO, indicating that there was aberrant anterior-posterior brain specification, consistent with the transcriptomic dysregulation observed in our RNA-seq data.

To further confirm midbrain identity, we examined the expression of EN1, an established midbrain-specific marker. Quantitative RT-PCR analysis demonstrated that EN1 expression increased progressively during differentiation in both WT-75.1 and nGD2-1260 MLOs at weeks 3 and 8 (Author response image 1). EN1 reached 34-fold and 373-fold higher levels than in WT-75.1 iPSCs at weeks 3 and 8, respectively, in WT-75.1 MLOs. In nGD MLOs, although EN1 expression showed a modest reduction at week 8, the levels were not significantly different from those observed in age-matched WT-75.1 MLOs (p > 0.05, ns).

Author response image 1.

qRT-PCR quantification of midbrain progenitor marker EN1 expression in WT-75.1 and GD2-1260 MLOs at Wk3 and Wk8. Data was normalized to WT-75.1 hiPSC cells and presented as mean ± SEM (n = 3-4 MLOs per group).ns, not significant.<br />

(b) Extracellular dopamine ELISA should be complemented with intracellular dopamine or TH+ neuron counts normalized per organoid or per total neurons.

We quantified TH expression at both the mRNA level (Fig. 3F) and the protein level (Fig. 3G/H) from whole-organoid lysates, which provides a more consistent and integrative measure across samples. These TH expression levels correlated well with the corresponding extracellular (medium) dopamine concentrations for each genotype. In contrast, TH⁺ neuron counts may not reliably reflect total cellular dopamine levels because the number of cells captured on each organoid section varies substantially, making normalization difficult. Measuring intracellular dopamine is an alternative approach that will be considered in future studies.

(c) For CRISPR editing: the authors should report off-target analysis (GUIDE-seq or targeted sequencing of predicted off-targets) or at least in-silico off-target score and sequencing coverage of the edited locus. (off-target analysis (GUIDE-seq or targeted sequencing of predicted off-targets) or at least in-silico off-target score and sequencing coverage of the edited locus). 

The off-target effect was analyzed during gene editing and the chance to target other off-targets is low due to low off-target scores ranked based on the MIT Specificity Score analysis. The related method was also updated as stated below:

“The chance to target other Off-targets is low due to low Off-target scores ranked based on the MIT Specificity Score analysis (Hsu, P., Scott, D., Weinstein, J. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31, 827–832 (2013).https://doi.org/10.1038/nbt.2647).”

(d) It should be clarified as to whether lipidomics normalization is to total protein per organoid or per cell, and include representative LC-MS chromatograms or method QC.

The normalization was to the protein of the organoid lysate. This was clarified in the Methods section in the revision as stated below:

“The GluCer and GluSph levels in MLO were normalized to total MLO protein (mg) that were used for glycosphingolipid analyses. Protein mass was determined by BCA assay and glycosphingolipid was expressed as pmol/mg protein. Additionally, GluSph levels in the culture medium were quantified and normalized to the medium volume (pmol/mL).”

Representative LC-MS chromatograms for both normal and GD MLOs have been included in a new figure, Supplementary Figure 2.

(e) Figure legends should be improved in order to state the number of organoids, the number of differentiations, and the exact statistical tests used (including multiplecomparison corrections).

This was addressed above [see response to Weaknesses (1)-b and (5)-b].

(f) In the title, the authors state "reveal disease mechanisms", but the studies mainly exhibit functional changes. They should consider toning down the statement.

The title was revised to: Patient-Specific Midbrain Organoids with CRISPR Correction Recapitulate Neuronopathic Gaucher Disease Phenotypes and Enable Evaluation of Novel Therapies

(7) Recommendations

This reviewer recommends a major revision. The manuscript presents substantial novelty and strong potential impact but requires additional experimental validation and clearer, more conservative interpretation. Key items to address are:

(a) Strengthening genetic and biological replication (additional lines or replicate differentiations).

This was addressed above [see response to Weaknesses (1)-a, (1)-b, (1)-c].

(b) Adding functional mechanistic validation for major pathways (Wnt/mTOR/autophagy) and providing autophagy flux data.

(c) Including at least one neuronal functional readout (calcium imaging/MEA/patch) to demonstrate functional rescue.

As addressed above [see response to Weaknesses (2)], the suggested experiments in b) and c) would provide additional insights into this study and we will consider them in future work. 

(d) Deepening therapeutic characterization (dose, biodistribution, durability) and including specificity controls.

This was addressed above [see response to Weaknesses (3)-a to e].

(e) Improving statistical reporting and explicitly stating biological replicate structure.

This was addressed above [see response to Weaknesses (1)-b, (5)-b].

Reviewer #2 (Public review):

Sun et al. have developed a midbrain-like organoid (MLO) model for neuronopathic Gaucher disease (nGD). The MLOs recapitulate several features of nGD molecular pathology, including reduced GCase activity, sphingolipid accumulation, and impaired dopaminergic neuron development. They also characterize the transcriptome in the MLO nGD model. CRISPR correction of one of the GBA1 mutant alleles rescues most of the nGD molecular phenotypes. The MLO model was further deployed in proof-of-principle studies of investigational nGD therapies, including SapC-DOPS nanovesicles, AAV9-mediated GBA1 gene delivery, and substrate-reduction therapy (GZ452). This patient-specific 3D model provides a new platform for studying nGD mechanisms and accelerating therapy development. Overall, only modest weaknesses are noted.

We thank the reviewer for the supportive remarks.

Reviewer #3 (Public review):

Summary:

In this study, the authors describe modeling of neuronopathic Gaucher disease (nGD) using midbrain-like organoids (MLOs) derived from hiPSCs carrying GBA1 L444P/P415R or L444P/RecNciI variants. These MLOs recapitulate several disease features, including GCase deficiency, reduced enzymatic activity, lipid substrate accumulation, and impaired dopaminergic neuron differentiation. Correction of the GBA1 L444P variant restored GCase activity, normalized lipid metabolism, and rescued dopaminergic neuronal defects, confirming its pathogenic role in the MLO model. The authors further leveraged this system to evaluate therapeutic strategies, including: (i) SapC-DOPS nanovesicles for GCase delivery, (ii) AAV9-mediated GBA1 gene therapy, and (iii) GZ452, a glucosylceramide synthase inhibitor. These treatments reduced lipid accumulation and ameliorated autophagic, lysosomal, and neurodevelopmental abnormalities.

Strengths:

This manuscript demonstrates that nGD patient-derived MLOs can serve as an additional platform for investigating nGD mechanisms and advancing therapeutic development.

Comments:

(1) It is interesting that GBA1 L444P/P415R MLOs show defects in midbrain patterning and dopaminergic neuron differentiation (Figure 3). One might wonder whether these abnormalities are specific to the combination of L444P and P415R variants or represent a 

general consequence of GBA1 loss. Do GBA1 L444P/RecNciI (GD2-10-257) MLOs also exhibit similar defects?

We observed reduced dopaminergic neuron marker TH expression in GBA1 L444P/RecNciI (GD2-10-257) MLOs, suggesting that this line also exhibits defects in dopaminergic neuron differentiation. These data are provided in a new Supplementary Fig. 4E, and are summarized in new Supplementary Table 2 in the revision.

(2) In Supplementary Figure 3, the authors examined GCase localization in SapC-DOPSfGCase-treated nGD MLOs. These data indicate that GCase is delivered to TH⁺ neurons, GFAP⁺ glia, and various other unidentified cell types. In fruit flies, the GBA1 ortholog, Gba1b, is only expressed in glia (PMID: 35857503; 35961319). Neuronally produced GluCer is transferred to glia for GBA1-mediated degradation. These findings raise an important question: in wild-type MLOs, which cell type(s) normally express GBA1? Are they dopaminergic neurons, astrocytes, or other cell types?

All cell types in wild-type MLOs are expected to express GBA1, as it is a housekeeping gene broadly expressed across neurons, astrocytes, and other brain cell types. Its lysosomal function is essential for cellular homeostasis and is therefore not restricted to any specific lineage. (https://www.proteinatlas.org/ENSG00000177628GBA1/brain/midbrain). 

(3) The authors may consider switching Figures 2 and 3 so that the differentiation defects observed in nGD MLOs (Figure 3) are presented before the analysis of other phenotypic abnormalities, including the various transcriptional changes (Figure 2).

We appreciate the reviewer’s suggestion; however, we respectfully prefer to retain the current order of Figures 2 and 3, as we believe this structure provides the clearest narrative flow. Figure 2 establishes the core biochemical hallmarks: reduced GCase activity, substrate accumulation, and global transcriptomic dysregulation (1,429 DEGs enriched in neural development, WNT signaling, and lysosomal pathways), which together provide essential molecular context for studying the specific cellular differentiation defects presented in Figure 3. Presenting the broader disease landscape first creates a coherent mechanistic link to the subsequent analyses of midbrain patterning and dopaminergic neuron impairment.

To enhance readability, we have added a brief transitional sentence at the start of the Figure 3 paragraph: “Building on the molecular and transcriptomic hallmarks of GCase deficiency observed in nGD MLOs (Figure 2), we next investigated the impact on midbrain patterning and dopaminergic neuron differentiation (Figure 3).”

why should always the machine gets the best ideas applied

independent units called actors

but thee actors are actual human beings

So we get the Human Actor Model

for interpersonal computing on the web

:do.search.google?actor+model

dweb

eLife Assessment

This modelling study tests several hypotheses describing how seasonality and migration drive the epidemiology of Rift Valley Fever Virus among transhumant cattle in The Gambia. The work is methodologically solid, and the findings offer valuable insights into how the movement of cattle in and out of the Gambia River and Sahel ecoregions could lead to source-sink transmission dynamics among cattle subpopulations, sustaining endemic transmission.

Reviewer #1 (Public review):

Summary:

This study uses data from a recent RVFV serosurvey among transhumant cattle in The Gambia to inform the development of an RVFV transmission model. The model incorporates several hypotheses that capture the seasonal nature of both vector-borne RVFV transmission and cattle migration. These natural phenomena are driven by contrasting wet and dry seasons in The Gambia's two main ecoregions and are purported to drive cyclical source-sink transmission dynamics. Although the Sahel is hypothesized to be unsuitable for year-long RVFV transmission, findings suggest that cattle returning from the Gambia River to the Sahel at the beginning of the wet season could drive repeated RVFV introductions and ensuing seasonal outbreaks. Upon review, the authors have removed an additional analysis evaluating the potential impacts of cattle movement bans on transmission dynamics, which was poorly supported by the methodological approach.

Strengths:

Like most infectious diseases in animal systems in low- and middle-income countries, the transmission dynamics of RVFV in cattle in The Gambia are poorly understood. This study harnesses important data on RVFV seroepidemiology to develop and parameterize a novel transmission model, providing plausible estimates of several epidemiological parameters and transmission dynamic patterns.

This study is well written and easy to follow.

The authors consider both deterministic and stochastic formulations of their model, demonstrating potential impacts of random events (e.g. extinctions) and providing confidence regarding model robustness.

The authors use well-established Bayesian estimation techniques for model fitting and confront their transmission model with a seroepidemiological model to assess model fit.

Elasticity analyses help to understand the relative importance of competing demographic and epidemiological drivers of transmission in this system.

Weaknesses:

The model does not include an impact of infection on cattle birth rates, but the authors justify that this parameter should have limited impact on dynamics given predicted low-level circulation patterns, as opposed to explosive outbreaks, in this region.

The importance of the LVFV positivity decay rate is highlighted but loss of immunity is not considered in the SIR model. The authors do discuss uncertainty regarding model structure and a need for future data collection to begin to answer this question.

The model's structure, including homogenous mixing within each ecoregion and step-change seasonality, allows for estimation of generalized transmission rates at a macro scale. However, it greatly simplifies the movement process itself and assumes that transhumant cattle movement is the only mechanism for RVF reintroduction into the Sahel region. The authors discuss that integration of more finely-scaled movement and contact data may help to address this limitation in future work.

This model seems well-suited to be exploited in future work to explore for e.g. impacts of cattle vaccination, and potential differential efficiency when targeting T herds relative to M or L.

Comments on revisions:

I thank the authors for thoughtfully and thoroughly addressing my concerns. I have no further comments.

Author response:

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

Joint Public reviews:

(1) Stable annual dynamics vs. episodic outbreaks

We agree that RVF is classically described as producing periodic epidemics interspersed with long inter-epidemic periods, often linked to extreme rainfall events. Our model predicts more regular seasonal dynamics, which reflects the endemic transmission patterns we have observed in The Gambia through serological surveys. In this revision, we have:

- clarified that while epidemics occur in other parts of sub-Saharan Africa, our results are consistent with the epidemiological narrative of RVF in The Gambia, characterised by sustained, moderate transmission without resulting in substantial outbreaks (hyperendemicity).

- discussed how model assumptions (e.g. seasonality, homogenous mixing) may bias our results toward an endemic quasi-equilibrium dynamic.

- highlighted the implications of this for interpretation and for public health decision-making.

(2) Use of network analysis

We acknowledge the reviewer’s concern. The network analysis was conducted descriptively to characterize cattle movement patterns and the structure of herd connections, but it was not formally incorporated into the model. In this revision we have:

- clarified this distinction in the manuscript to avoid overinterpretation.

- emphasized the need for future modelling work using finer-scale movement data, which could support more realistic herd metapopulation dynamics and better capture heterogeneity in transmission.

(3) RVFV reproductive impacts

While RVF outbreaks are known to cause substantial abortions and neonatal deaths, these events occur during sporadic epidemics. In the Gambian context, where we’re not observing large outbreaks but rather low-level circulation, the annual impact of RVF infection on births is likely modest compared to baseline herd turnover. Moreover, cattle demography is partly managed, with replacement and movement buffering birth rates against short-term losses.

Our model includes birth as a constant demographic process, it’s reasonable to assume stable population since we are not explicitly modelling outbreak-scale reproductive losses. This approach is consistent with other RVF transmission models that adopt a similar simplifying assumption. However, we have acknowledged this simplification as a limitation in the revised manuscript.

(4) Missing ODEs for M herds in the dry season

We thank the reviewer for identifying this omission. The ODEs for the M subpopulation in the dry season were not included in the appendix due to an oversight, though demographic turnover was implemented in the model code. We have now added the missing equations to the appendix.

(5) Role of immunity loss and model structure (SIR vs. SIRS)

We acknowledge that the decline of detectable antibodies over time (seropositivity decay) is an important consideration in RVFV serology; however, whether this decline reflects a true loss of protective immunity following natural infection remains unknown. Available evidence suggests that infected cattle likely develop long-lasting immunity, and findings in humans further support this assumption, although longitudinal field data regarding RVFV-specific antibody durability in animals are not available to the best of our knowledge. From a modelling perspective, our objective was to estimate FOI and use it to predict an age-seroprevalence curve consistent with the observed cross-sectional age-seroprevalence patterns. We therefore adopted a parsimonious SIR framework, interpreting loss of seropositivity as a potential explanation for discrepancies between observed and predicted age-seroprevalence rather than explicitly modelling waning immunity. We have now:

- clarified this rationale, emphasising that there is no direct evidence for waning immunity following natural RVFV infection in cattle, although evidence of seropositivity decay has been suggested in human.

- highlighted that while an SEIS/SIRS framework could theoretically generate different long-term dynamics, evaluating this approach requires stronger evidence for true immunity loss.

(6) RVFV induced mortality in serocatalytic model

We thank the reviewer for this comment and for raising an important conceptual point. However, the force of infection in our study is not estimated using a serocatalytic framework. Instead, FOI is estimated mechanistically within the transmission model as a function of the number of infectious cattle, rather than from age-stratified seroprevalence data.

RVF-induced mortality is accounted for through its effect on the infectious compartment, where increased mortality reduces the number and duration of infectious cattle and therefore indirectly reduces FOI. Consequently, RVF-related cattle death does not need to be explicitly incorporated into the FOI expression itself. Seroreversion similarly does not influence FOI estimation under this modelling framework. We have clarified this distinction in the Methods section to avoid confusion between mechanistic transmission models and serocatalytic approaches.

(7) Clarifying previous vs. current study components

We have revised the Methods and Appendix to make clearer distinctions between our previous work (e.g. household survey data collection, seroprevalence estimates) and the analyses undertaken for this manuscript (e.g. model development and fitting).

(8) Limitations paragraph

We have expanded the limitations section to identify the sparse household movement data as contributing most to uncertainty. We have outlined how these limitations may have implications for our conclusions, and may lead to under- or over-estimation of periods of heightened transmission risk.

(9) Movement ban simulations & suitability of model for vaccination interventions

We appreciate the reviewer’s concerns regarding the movement ban simulation. On reassessment, we agree that our model structure might not ideally be suited to exploring a movement ban. In this revised manuscript, we have removed this analysis. We are currently developing separate work focused on RVF vaccination strategies in cattle, where this model structure might be more directly applicable, and will reserve a deeper investigation of vaccination interventions for that forthcoming publication.

Reviewer #1 (Recommendations for the authors):

We thank the reviewer for the recommendations regarding the Introduction, Methods, Results, and Supplementary Figures. We have addressed these points below and revised the manuscript accordingly.

(1) Introduction: Should avoid describing as "inaccessible" the regions that are inhabited by nomadic and transhumant pastoralists.

We have revised the wording to “hard-to-reach” regions.

(2) Methods: Can the authors state what share of the animals included in the household survey data were cattle as opposed to other small ruminants? It would be helpful to understand what share of the data is "excluded"

We have now included the total number of cattle sampled, providing clarity on the proportion of data used in the analyses.

(3) Methods: When introducing the deterministic model, it seems unnecessary to mention the initialization conditions (i.e., introduction of a single infected individual at time 0) when this is later repeated in the Estimation of model parameters section, where it seems simulations were first conducted.

We have removed the redundant description.

(4) Results: Could the negative correlation between geographic distance of connected herds and mean seroprevalence simply indicate proximal exposure rather than common risk factors?

We acknowledge that both mechanisms are plausible. RVFV transmission is strongly influenced by share environmental factors that shape mosquito dynamics; however, direct transmission between proximal cattle herds may also occur through close contact with infectious tissues, bodily fluids, or contaminated materials. We have clarified this interpretation in the Results section.

(5) Figure S5: inconsistent notation for the scaling factor parameter (tau), which is expressed in equations and tables as psi.

We thank the reviewer for identifying this issue and have corrected all instances to ensure consistent use of tau throughout the manuscript.

(6) Figure S6: Why a density plot, isn't the number of temporary extinctions (x-axis) discrete?

We have replaced the density plot with a bar plot in Figure S6.

eLife Assessment

This useful study examines whether the sugar trehalose, coordinates energy supply with the gene programs that build muscle in the cotton bollworm (Helicoverpa armigera). The evidence for this currently is incomplete. The central claim - that trehalose specifically regulates an E2F/Dp-driven myogenic program - is not supported by the specificity of the data: perturbations and sequencing are systemic, alternative explanations such as general energy or amino-acid scarcity remain plausible, and mechanistic anchors are also limited. The work will interest researchers in insect metabolism and development; focused, tissue-resolved measurements together with stronger mechanistic controls would substantially strengthen the conclusions.

Reviewer #1 (Public review):

Summary:

In this work by Mohite et al., they have used transcriptomic and metabolic profiling of H. armigera, muscle development, and S. frugiperda to link energy trehalose metabolism and muscle development. They further used several different bioinformatics tools for network analysis to converge upon transcriptional control as a potential mechanism of metabolite-regulated transcriptional programming for muscle development. The authors have also done rescue experiments where trehalose was provided externally by feeding, which rescues the phenotype. Though the study is exciting, there are several concerns and gaps that lead to the current results as purely speculative. It is difficult to perform any genetic experiments in non-model insects; the authors seem to suggest a similar mechanism could also be applicable in systems like Drosophila; it might be possible to perform experiments to fill some missing mechanistic details.

A few specific comments below:

The authors used N-(phenylthio) phthalimide (NPP), a trehalose-6-phosphate phosphatase (TPP) inhibitor. They also find several genes, including enzymes of trehalose metabolism, that change. Further, several myogenic genes are downregulated in bulk RNA sequencing. The major caveat of this experiment is that the NPP treatment leads to reduced muscle development, and so the proportion of the samples from the muscles in bulk RNA sequencing will be relatively lower, which might have led to the results. So, a confirmatory experiment has to be performed where the muscle tissues are dissected and sequenced, or some of the interesting targets could be validated by qRT-PCR. Further to overcome the off-target effects of NPP, trehalose rescue experiments could be useful.

Even the reduction in the levels of ADP, NAD, NADH, and NMN, all of which are essential for efficient energy production and utilization, could be due to the loss of muscles, which perform predominantly metabolic functions due to their mitochondria-rich environment. So it becomes difficult to judge if the levels of these energy molecules' reduction are due to a cause or effect.

The authors have used this transcriptomic data for pathway enrichment analysis, which led to the E2F family of transcription factors and a reduction in the level of when trehalose metabolism is perturbed. EMSA experiments, though, confirm a possibility of the E2F interaction with the HaTPS/TPP promoter, but it lacks proper controls and competition to test the actual specificity of this interaction. Several transcription factors have DNA-binding domains and could bind any given DNA weakly, and the specificity is ideally known only from competitive and non-competitive inhibition studies.

The work seems to have connected the trehalose metabolism with gene expression changes, though this is an interesting idea, there are no experiments that are conclusive in the current version of the manuscript. If the authors can search for domains in the E2F family of transcription factors that can bind to the metabolite, then, if not, a chip-seq is essential to conclusively suggest the role of E2F in regulating gene expression tuned by the metabolites.

Some of the above concerns are partially addressed in experiments where silencing of E2F/Dp shows similar phenotypes as with NPP and dsRNA. It is also notable that silencing any key transcription factor can have several indirect effects, and delayed pupation and lethality could not be definitely linked to trehalose-dependent regulation.

Trehalose rescue experiments that rescue phenotype and gene expression are interesting. But is it possible that the fed trehalose is metabolized in the gut and might not reach the target tissue? In which case, the role of trehalose in directly regulating transcription factors becomes questionable. So, a confirmatory experiment is needed to demonstrate that the fed trehalose reaches the target tissues. This could possibly be done by measuring the trehalose levels in muscles post-rescue feeding. Also, rescue experiments need to be done with appropriate control sugars.

No experiments are performed with non-target control dsRNA. All the experiments are done with an empty vector. But an appropriate control should be a non-target control.

Reviewer #2 (Public review):

Summary:

This study shows that the knockdown of the effects of TPS/TPP in Helicoverpa armigera and Spodoptera frugiperda can be rescued by trehalose treatment. This suggests that trehalose metabolism is necessary for development in the tissues that NPP and dsRNA can reach.

Strengths:

This study examines an important metabolic process beyond model organisms, providing a new perspective on our understanding of species-specific metabolism equilibria, whether conserved or divergent.

Weaknesses:

While the effects observed may be truly conserved across Lepidopterans and may be muscle-specific, the study largely relies on one species and perturbation methods that are not muscle-specific. The technical limitations arising from investigations outside model systems, where solid methods are available, limit the specificity of inferences that may be drawn from the data.

Reviewer #3 (Public review):

The hypothesis is that Trehalose metabolism regulates transcriptional control of muscle development in lepidopteran insects.

The manuscript investigates the role of Trehalose metabolism in muscle development. Through sequencing and subsequent bioinformatics analysis of insects with perturbed trehalose metabolism (knockdown of TPS/TPP), the authors have identified transcription factor E2F, which was validated through RT-PCR. Their hypothesis is that trehalose metabolism regulates E2F, which then controls the myogenic genes. Counterintuitive to this hypothesis, the investigators perform EMSAs with the E2F protein and promoter of the TPP gene and show binding. Their knockdown experiments with Dp, the binding partner of E2F, show direct effect on several trehalose metabolism genes. Similar results are demonstrated in the trehalose feeding experiment, where feeding trehalose leads to partial rescue of the phenotype observed as a result of Dp knockdown. This seems contradictory to their hypothesis. Even more intriguing is a similar observation between paramyosin, a structural muscle protein, and E2F/Dp - they show that paramyosin regulates E2F/Dp and E2F/Dp regulated paramyosin. The only plausible way to explain the results is the existence of a feed-forward loop between TPP-E2F/Dp and paramyosin-E2F/Dp. But the authors have mentioned nothing in this line. Additionally, I think trehalose metabolism impacts amino acid content in insects, and that will have a direct bearing on muscle development. The sequencing analysis and follow-up GSEA studies have demonstrated enrichment of several amino acid biosynthetic genes. Yet authors make no efforts to measure amino acid levels or correlate them with muscle development. Any study aiming to link trehalose metabolism and muscle development and not considering the above points will be incomplete.

The result section of the manuscript is quite concise, to my understanding (especially the initial few sections), which misses out on mentioning details that would help readers understand the paper better. While technical details of the methods should be in the Materials and Methods section, the overall experimental strategy for the experiments performed should be explained in adequate detail in the results section itself or in figure legends. I would request authors to include more details in the results section. As an extension of the comment above, many times, abbreviations have been used without introducing them. A thorough check of the manuscript is required regarding this.

The Spodoptera experiments appear ad hoc and are insufficient to support conservation beyond Helicoverpa. To substantiate this claim, please add a coherent, minimal set of Spodoptera experiments and present them in a dedicated subsection. Alternatively, consider removing these data and limiting the conclusions (and title) to H. armigera.

In order to check the effects of E2F/Dp, a dsRNA-mediated knockdown of Dp was performed. Why was the E2F protein, a primary target of the study, not chosen as a candidate? The authors should either provide justification for this or perform the suggested experiments to come to a conclusion. I would like to point out that such experiments were performed in Drosophila.

Silencing of HaDp resulted in a significant decrease in HaE2F expression. I find this observation intriguing. DP is the cofactor of E2F, and they both heterodimerise and sit on the promoter of target genes to regulate them. I would request authors to revisit this result, as it contradicts the general understanding of how E2F/Dp functions in other organisms. If Dp indeed controls E2F expression, then further experiments should be conducted to come to a conclusion convincingly. Additionally, these results would need thorough discussion with citations of similar results observed for other transcription factor-cofactor complexes.

I consider the overall bioinformatics analysis to remain very poorly described. What is specifically lacking is clear statements about why a particular dry lab experiments were conducted.

In my judgement, the EMSA analysis presented is technically poor in quality. It lacks positive and negative controls, does not show mutation analysis or super shifts. Also, it lacks any competition assays that are important to prove the binding beyond doubt. I am not sure why protein is not detected at all in lower concentrations. Overall, the EMSA assays need to be redone; I find the current results to be unacceptable.

GSEA studies clearly indicate enrichment of the amino acid synthesis gene in TPP knockdown samples. This supports the plausible theory that a lack of Trehalose means a lack of enough nutrients, therefore less of that is converted to amino acids, and therefore muscle development is compromised. Yet the authors make no effort to measure amino acid levels. While nutrients can be sensed through signalling pathways leading to shut shutdown of myogenic genes, a simple and direct correlation between less raw material and deformed muscle might also be possible.

The authors are encouraged to stick to one color palette while demonstrating sequencing results. Choosing a different color palette for representing results from the same sequencing analysis confuses readers.

Expression of genes, as understood from sequencing analysis in Figure 1D, Figure 2F, and Figure 3D, appears to be binary in nature. This result is extremely surprising given that the qRT-PCR of these genes have revealed a checker and graded expression.

In several graphs, non-significant results have been interpreted as significant in the results section. In a few other cases, the reported changes are minimal, and the statistical support is unclear; please recheck the analyses and include exact statistics. In the results section, fold changes observed should be discussed, as well as the statistical significance of the observed change.

Finally, I would add that trehaolse metabolism regulates cell cycle genes, and muscle development genes establish correlation and causation. The authors should ensure that any comments they make are backed by evidence.

Author response:

eLife Assessment

This useful study examines whether the sugar trehalose, coordinates energy supply with the gene programs that build muscle in the cotton bollworm (Helicoverpa armigera). The evidence for this currently is incomplete. The central claim - that trehalose specifically regulates an E2F/Dp-driven myogenic program - is not supported by the specificity of the data: perturbations and sequencing are systemic, alternative explanations such as general energy or amino-acid scarcity remain plausible, and mechanistic anchors are also limited. The work will interest researchers in insect metabolism and development; focused, tissue-resolved measurements together with stronger mechanistic controls would substantially strengthen the conclusions.

We thank the reviewer for the thoughtful and constructive evaluation of our work and for recognizing its potential relevance to researchers working on insect metabolism and development. We fully agree that our current evidence is preliminary and that the mechanistic link between trehalose and the E2F/Dp‑driven myogenic program needs to be strengthened.

Our intention was to present trehalose-E2F/Dp coupling as a working model emerging from our data, rather than as a fully established pathway. We agree that systemic manipulations of trehalose and whole‑larval RNA‑seq cannot fully differentiate global metabolic stress from specific effects on myogenic programs. In the revision, we plan to include additional metabolic readouts (e.g., ATP/AMP ratio, key amino acids where available) to better discuss the overall energetic and nutritional state. We will reanalyze our RNA‑seq data to more clearly distinguish broad stress/metabolic signatures from cell‑cycle/myogenic signatures. Furthermore, we will reframe our discussion to explicitly state that we cannot completely rule out a contribution of general energy or amino‑acid scarcity at this stage.

We acknowledge that, with our current experiments, the specificity for an E2F/Dp‑driven program is inferred mainly from enrichment of E2F targets among differentially expressed genes, and expression changes in canonical E2F partners and downstream cell‑cycle/myogenic regulators. To address this more rigorously, we are performing targeted qRT-PCR for a panel of well‑characterized E2F/Dp target genes and myogenic markers in larval muscle versus non‑muscle tissues, following trehalose perturbation. Where technically feasible, testing whether partial knockdown of HaE2F or HaDp modifies the effect of trehalose manipulation on selected myogenic markers. These data, even if limited, will help to provide a more direct functional link, and we will include them in the manuscript if completed in time. In parallel, we will soften statements that imply a fully established, trehalose‑specific regulation of E2F/Dp and instead present this as a strong candidate pathway suggested by the current data.

We fully agree that tissue‑resolved analyses are essential to move from systemic correlations to causality in muscle. We are in the process of standardizing larval muscle dissections and isolating thoracic/abdominal body wall muscle for trehalose, glycogen, and expression assays. Comparing expression of key metabolic and myogenic genes in muscle versus fat body and midgut, under trehalose manipulation. These tissue‑resolved data will directly address whether the transcriptional changes we report are preferentially localized to muscle.

We are grateful for the reviewer’s critical but encouraging comments. We will moderate our central claims, also explicitly consider and discuss alternative explanations. Further, we will add tissue‑resolved and more focused mechanistic data as far as possible within the current revision. We believe these changes will substantially strengthen the manuscript and better align our conclusions with the evidence we presently have.

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this work by Mohite et al., they have used transcriptomic and metabolic profiling of H. armigera, muscle development, and S. frugiperda to link energy trehalose metabolism and muscle development. They further used several different bioinformatics tools for network analysis to converge upon transcriptional control as a potential mechanism of metabolite-regulated transcriptional programming for muscle development. The authors have also done rescue experiments where trehalose was provided externally by feeding, which rescues the phenotype. Though the study is exciting, there are several concerns and gaps that lead to the current results as purely speculative. It is difficult to perform any genetic experiments in non-model insects; the authors seem to suggest a similar mechanism could also be applicable in systems like Drosophila; it might be possible to perform experiments to fill some missing mechanistic details.

A few specific comments below:

The authors used N-(phenylthio) phthalimide (NPP), a trehalose-6-phosphate phosphatase (TPP) inhibitor. They also find several genes, including enzymes of trehalose metabolism, that change. Further, several myogenic genes are downregulated in bulk RNA sequencing. The major caveat of this experiment is that the NPP treatment leads to reduced muscle development, and so the proportion of the samples from the muscles in bulk RNA sequencing will be relatively lower, which might have led to the results. So, a confirmatory experiment has to be performed where the muscle tissues are dissected and sequenced, or some of the interesting targets could be validated by qRT-PCR. Further to overcome the off-target effects of NPP, trehalose rescue experiments could be useful.

Thank you for this valuable comment. We will validate the gene expression data using qRT-PCR on muscle tissue samples from both treated and control groups. This will help determine whether the gene expression patterns observed in the RNA-seq data are muscle-specific or systemic.

Even the reduction in the levels of ADP, NAD, NADH, and NMN, all of which are essential for efficient energy production and utilization, could be due to the loss of muscles, which perform predominantly metabolic functions due to their mitochondria-rich environment. So it becomes difficult to judge if the levels of these energy molecules' reduction are due to a cause or effect.

We thank the reviewer for this thoughtful comment and agree that reduced levels of ADP, NAD, NADH, and NMN could arise either from a disturbance of energy metabolism or from loss of mitochondria‑rich muscles. Our current data cannot fully separate these two possibilities. Still, several studies support the interpretation that perturbing trehalose metabolism causes a primary systemic energy deficit that is coupled to mitochondrial function, not merely a passive consequence of tissue loss.

For example:

(1) Our previous study in H. armigera showed that chemical inhibition of trehalose synthesis results in depletion of trehalose, glucose, glucose‑6‑phosphate, and suppression of the TCA cycle, indicating reduced energy levels and dysregulated fatty‑acid oxidation (Tellis et al., 2023).

(2) Chang et al. (2022) showed that trehalose catabolism and mitochondrial ATP production are mechanistically linked. HaTreh1 localizes to mitochondria and physically interacts with ATP synthase subunit α. 20‑hydroxyecdysone increases HaTreh1 expression, enhances its binding to ATP synthase, and elevates ATP content, while knockdown of HaTreh1 or HaATPs‑α reduces ATP levels.

(3) Similarly, our previous study inhibition of Treh activity in H. armigera generates an “energy‑deficient condition” characterized by deregulation of carbohydrate, protein, fatty‑acid, and mitochondria‑related pathways, and a concomitant reduction in key energy metabolites (Tellis et al., 2024).

(4) The starvation study in H. armigera has shown that reduced hemolymph trehalose is associated with respiratory depression and large‑scale reprogramming of glycolysis and fatty‑acid metabolism (Jiang et al., 2019).

These findings support a direct coupling between trehalose availability and systemic energy/redox state. Therefore, the coordinated decrease in ADP, NAD, NADH, and NMN following TPS/TPP silencing is consistent with a primary disturbance of systemic energy and mitochondrial metabolism rather than exclusively a secondary consequence of muscle loss. We agree, however, that the present whole‑larva metabolite measurements do not allow a quantitative partitioning between changes due to altered muscle mass and those due to intrinsic metabolic impairment at the cellular level. Thus, tissue-specific quantification of these metabolites would allow us to directly test whether altered energy metabolites are a cause or consequence of muscle loss.

References:

(1) Tellis, M. B., Mohite, S. D., Nair, V. S., Chaudhari, B. Y., Ahmed, S., Kotkar, H. M., & Joshi, R. S. (2024). Inhibition of Trehalose Synthesis in Lepidoptera Reduces Larval Fitness. Advanced Biology, 8(2), 2300404.

(2) Chang, Y., Zhang, B., Du, M., Geng, Z., Wei, J., Guan, R., An, S. and Zhao, W., 2022. The vital hormone 20-hydroxyecdysone controls ATP production by upregulating the binding of trehalase 1 with ATP synthase subunit α in Helicoverpa armigera. Journal of Biological Chemistry, 298(2).

(3) Tellis, M., Mohite, S. and Joshi, R., 2024. Trehalase inhibition in Helicoverpa armigera activates machinery for alternate energy acquisition. Journal of Biosciences, 49(3), p.74.

(4) Jiang, T., Ma, L., Liu, X.Y., Xiao, H.J. and Zhang, W.N., 2019. Effects of starvation on respiratory metabolism and energy metabolism in the cotton bollworm Helicoverpa armigera (Hübner)(Lepidoptera: Noctuidae). Journal of Insect Physiology, 119, p.103951.

The authors have used this transcriptomic data for pathway enrichment analysis, which led to the E2F family of transcription factors and a reduction in the level of when trehalose metabolism is perturbed. EMSA experiments, though, confirm a possibility of the E2F interaction with the HaTPS/TPP promoter, but it lacks proper controls and competition to test the actual specificity of this interaction. Several transcription factors have DNA-binding domains and could bind any given DNA weakly, and the specificity is ideally known only from competitive and non-competitive inhibition studies.

We thank the reviewer for this important comment and fully agree that EMSA alone, without appropriate competition and control reactions, cannot establish the specificity or functional relevance of a transcription factor-DNA interaction. In our study, we found the E2F family from GRN analysis of the RNA seq data obtained upon HaTPS/TPP silencing, suggesting a potential regulatory connection. After that, we predicted E2F binding sites on the promoter of HaTPS/TPP. The EMSA experiments were intended as preliminary evidence that E2F can associate with the HaTPS/TPP promoter in vitro. We will clarify this in the manuscript by softening our conclusion to indicate that our data support a “possible E2F-HaTPS/TPP interaction”. We also perform EMSA with specific and non‑specific competitors to confirm the E2F binding to the HaTPS/TPP promoter.

The work seems to have connected the trehalose metabolism with gene expression changes, though this is an interesting idea, there are no experiments that are conclusive in the current version of the manuscript. If the authors can search for domains in the E2F family of transcription factors that can bind to the metabolite, then, if not, a chip-seq is essential to conclusively suggest the role of E2F in regulating gene expression tuned by the metabolites.

A previous study in D. melanogaster, Zappia et al., (2016) showed vital role of E2F in skeletal muscle required for animal viability. They have shown that Dp knockdown resulted in reduced expression of genes encoding structural and contractile proteins, such as Myosin heavy chain (Mhc), fln, Tropomyosin 1 (Tm1), Tropomyosin 2 (Tm2), Myosin light chain 2 (Mlc2), sarcomere length short (sals) and Act88F, and myogenic regulators, such as held out wings (how), Limpet (Lmpt), Myocyte enhancer factor 2 (Mef2) and spalt major (salm). Also, ChiP-qRT-PCR showed upstream regions of myogenic genes, such as how, fln, Lmpt, sals, Tm1 and Mef2, were specifically enriched with E2f1, E2f2, and Dp antibodies in comparison with a nonspecific antibody. Further, Zappia et al. (2019) reported a chip-seq dataset that suggests that E2F/Dp directly activates the expression of glycolytic and mitochondrial genes during muscle development. Zappia et al., (2023) showed the regulation of one of the glycolytic genes, Phosphoglycerate kinase (Pgk) by E2F during Drosophila development.

However, the regulation of trehalose metabolic genes by E2F/Dp and vice versa was not studied previously. So here in our study, we tried to understand the correlation of trehalose metabolism and E2F/Dp in the muscle development of H. armigera.

References:

(1) Zappia, M.P. and Frolov, M.V., 2016. E2F function in muscle growth is necessary and sufficient for viability in Drosophila. Nature Communications, 7(1), p.10509.

(2) Zappia, M.P., Rogers, A., Islam, A.B. and Frolov, M.V., 2019. Rbf activates the myogenic transcriptional program to promote skeletal muscle differentiation. Cell reports, 26(3), pp.702-719.

(3) Zappia, M. P., Kwon, Y.-J., Westacott, A., Liseth, I., Lee, H. M., Islam, A. B., Kim, J., & Frolov, M. V. (2023a). E2F regulation of the Phosphoglycerate kinase gene is functionally important in Drosophila development. Proceedings of the National Academy of Sciences, 120(15), e2220770120.

Some of the above concerns are partially addressed in experiments where silencing of E2F/Dp shows similar phenotypes as with NPP and dsRNA. It is also notable that silencing any key transcription factor can have several indirect effects, and delayed pupation and lethality could not be definitely linked to trehalose-dependent regulation.

Yes. It’s true that silencing of any key transcription factor can have several indirect effects. Our intention was not to argue that delayed pupation and lethality are exclusively due to trehalose-dependent regulation, but that E2F/Dp and HaTPS/TPP silencing showed a consistent set of phenotypes and molecular changes, such as (i) transcriptomic enrichment of E2F targets upon trehalose perturbation, (ii) reduced HaTPS/TPP expression following E2F/Dp silencing, (iii) reduced myogenic gene expression that parallels the phenotypes observed with HaTPS/TPP silencing and (iv) restoration of E2F and Dp expression in E2F/Dp‑silenced insects upon trehalose feeding in the rescue assay. Together, these findings support a functional association between E2F/Dp and trehalose homeostasis. At the same time, we fully acknowledge that these results do not exclude additional, trehalose‑independent roles of E2F/Dp in development.

Trehalose rescue experiments that rescue phenotype and gene expression are interesting. But is it possible that the fed trehalose is metabolized in the gut and might not reach the target tissue? In which case, the role of trehalose in directly regulating transcription factors becomes questionable. So, a confirmatory experiment is needed to demonstrate that the fed trehalose reaches the target tissues. This could possibly be done by measuring the trehalose levels in muscles post-rescue feeding. Also, rescue experiments need to be done with appropriate control sugars.

Yes, it’s possible that, to some extent, trehalose is metabolized in the gut. Even though trehalase is present in the insect gut, some of the trehalose will be absorbed via trehalose transporters on the gut lining. Trehalose feeding was not rescued in insects fed with the control diet (empty vector and dsHaTPP), which contains chickpea powder, which is composed of an ample amount of amino acids and carbohydrates. Insects fed exclusively on a trehalose-containing diet are rescued, but not on a control diet that contains other carbohydrates. We agree that direct measurement of trehalose in target tissues will provide important confirmation. In the manuscript, we will measure trehalose levels in muscle, gut, and haemolymph after trehalose feeding.

No experiments are performed with non-target control dsRNA. All the experiments are done with an empty vector. But an appropriate control should be a non-target control.

Yes, there was no experiment with non-target dsRNA. Earlier, we have optimized a protocol for dsRNA delivery and its effectiveness in target knockdown (concentration, time) experiment, and published several research articles using a similar protocol:

(1) Chaudhari, B.Y., Nichit, V.J., Barvkar, V.T. and Joshi, R.S., 2025. Mechanistic insights in the role of trehalose transporter in metabolic homeostasis in response to dietary trehalose. G3: Genes, Genomes, Genetics, p. jkaf303.

(2) Barbole, R.S., Sharma, S., Patil, Y., Giri, A.P. and Joshi, R.S., 2024. Chitinase inhibition induces transcriptional dysregulation altering ecdysteroid-mediated control of Spodoptera frugiperda development. Iscience, 27(3).

(3) Patil, Y.P., Wagh, D.S., Barvkar, V.T., Gawari, S.K., Pisalwar, P.D., Ahmed, S. and Joshi, R.S., 2025. Altered Octopamine synthesis impairs tyrosine metabolism affecting Helicoverpa armigera vitality. Pesticide Biochemistry and Physiology, 208, p.106323.

(4) Tellis, M.B., Chaudhari, B.Y., Deshpande, S.V., Nikam, S.V., Barvkar, V.T., Kotkar, H.M. and Joshi, R.S., 2023. Trehalose transporter-like gene diversity and dynamics enhances stress response and recovery in Helicoverpa armigera. Gene, 862, p.147259.

(5) Joshi, K.S., Barvkar, V.T., Hadapad, A.B., Hire, R.S. and Joshi, R.S., 2025. LDH-dsRNA nanocarrier-mediated spray-induced silencing of juvenile hormone degradation pathway genes for targeted control of Helicoverpa armigera. International Journal of Biological Macromolecules, p.148673.

The same vector backbone and preparation procedures were used for both control and experimental constructs, allowing us to specifically compare the effects of the target dsRNA. The phenotypes and gene expression changes we observed were specific to the target genes and were not seen in the empty vector controls, suggesting that the effects are not due to nonspecific responses of dsRNA delivery or vector components.<br /> We acknowledge your suggestions, and in future studies, we will keep non-target dsRNA as a control in silencing assays.

Reviewer #2 (Public review):

Summary:

This study shows that the knockdown of the effects of TPS/TPP in Helicoverpa armigera and Spodoptera frugiperda can be rescued by trehalose treatment. This suggests that trehalose metabolism is necessary for development in the tissues that NPP and dsRNA can reach.

Strengths:

This study examines an important metabolic process beyond model organisms, providing a new perspective on our understanding of species-specific metabolism equilibria, whether conserved or divergent.

Weaknesses:

While the effects observed may be truly conserved across Lepidopterans and may be muscle-specific, the study largely relies on one species and perturbation methods that are not muscle-specific. The technical limitations arising from investigations outside model systems, where solid methods are available, limit the specificity of inferences that may be drawn from the data.

Thank you for this potting out this experimental weakness. We will validate the gene expression data using qRT-PCR on muscle tissue samples from both treated and control groups. We will also perform metabolite analysis with muscle samples. This will help to determine whether the observed gene expression patterns and metabolite changes are muscle-specific or systemic.

Reviewer #3 (Public review):

The hypothesis is that Trehalose metabolism regulates transcriptional control of muscle development in lepidopteran insects.

The manuscript investigates the role of Trehalose metabolism in muscle development. Through sequencing and subsequent bioinformatics analysis of insects with perturbed trehalose metabolism (knockdown of TPS/TPP), the authors have identified transcription factor E2F, which was validated through RT-PCR. Their hypothesis is that trehalose metabolism regulates E2F, which then controls the myogenic genes. Counterintuitive to this hypothesis, the investigators perform EMSAs with the E2F protein and promoter of the TPP gene and show binding. Their knockdown experiments with Dp, the binding partner of E2F, show direct effect on several trehalose metabolism genes. Similar results are demonstrated in the trehalose feeding experiment, where feeding trehalose leads to partial rescue of the phenotype observed as a result of Dp knockdown. This seems contradictory to their hypothesis. Even more intriguing is a similar observation between paramyosin, a structural muscle protein, and E2F/Dp - they show that paramyosin regulates E2F/Dp and E2F/Dp regulated paramyosin. The only plausible way to explain the results is the existence of a feed-forward loop between TPP-E2F/Dp and paramyosin-E2F/Dp. But the authors have mentioned nothing in this line. Additionally, I think trehalose metabolism impacts amino acid content in insects, and that will have a direct bearing on muscle development. The sequencing analysis and follow-up GSEA studies have demonstrated enrichment of several amino acid biosynthetic genes. Yet authors make no efforts to measure amino acid levels or correlate them with muscle development. Any study aiming to link trehalose metabolism and muscle development and not considering the above points will be incomplete.

We appreciate the reviewer’s efforts in the careful evaluation of this manuscript and constructive comments. From our and earlier data we found it was difficult to consider linear pathway “trehalose → E2F → muscle,” but rather a regulatory module in which trehalose metabolism and E2F/Dp form an interdependent circuit controlling myogenic genes. E2F/Dp binds and activates trehalose metabolism genes (TPS/TPP, Treh1) and myogenic structural genes, consistent with EMSA (TPS/TPP-E2F) and predicted binding sites of E2F on metabolic genes, Treh1, Pgk, and myogenic genes such as Act88F, Prm, Tm1, Fln, etc. At the same time, perturbing trehalose synthesis reduces E2F/Dp expression and myogenic gene expression, and trehalose feeding partially restores all three. This bidirectional influence is similar to E2F‑dependent control of carbohydrate metabolism and systemic sugar homeostasis described in D. melanogaster, where E2F/Dp both regulates metabolic genes and is itself constrained by metabolic state (Zappia et al., 2023a; Zappia et al., 2021).

The reciprocal regulation between Prm and E2F/Dp is indeed intriguing. Rather than a paradox, we interpret this as evidence that E2F/Dp couples metabolic genes and structural muscle genes within a shared module, and that key sarcomeric components (such as paramyosin) feed back on this transcriptional program. Similar cross‑talk between E2F‑controlled metabolic programs and tissue function has been documented in D. melanogaster muscle and fat body, where E2F loss in one tissue elicits systemic changes in the other (Zappia et al., 2021). For further confirmation of E2F-regulated Prm, we will perform EMSA on the Prm promoter with appropriate controls.

We fully agree that amino‑acid metabolism is a critical missing piece. In the manuscript, we will quantify the amino acid levels and include the results: “Amino acids display differential levels showing cysteine, leucine, histidine, valine, and proline showed significant reductions, while isoleucine and lysine showed non-significant reductions upon trehalose metabolism perturbation. These results are consistent with previous reports published by Tellis et al. (2024) and Shi et al. (2016)”. We will reframe our conclusions more cautiously as establishing a trehalose-E2F/Dp-muscle development, while stating that “definitive causal links via amino‑acid metabolism remain to be demonstrated”.

Reference:

(1) Zappia, M. P., Kwon, Y.-J., Westacott, A., Liseth, I., Lee, H. M., Islam, A. B., Kim, J., & Frolov, M. V. (2023a). E2F regulation of the Phosphoglycerate kinase gene is functionally important in Drosophila development. Proceedings of the National Academy of Sciences, 120(15), e2220770120.

(2) Zappia, M.P., Guarner, A., Kellie-Smith, N., Rogers, A., Morris, R., Nicolay, B., Boukhali, M., Haas, W., Dyson, N.J. and Frolov, M.V., 2021. E2F/Dp inactivation in fat body cells triggers systemic metabolic changes. elife, 10, p.e67753.

(3)Tellis, M., Mohite, S. and Joshi, R., 2024. Trehalase inhibition in Helicoverpa armigera activates machinery for alternate energy acquisition. Journal of Biosciences, 49(3), p.74.

(4) Shi, J.F., Xu, Q.Y., Sun, Q.K., Meng, Q.W., Mu, L.L., Guo, W.C. and Li, G.Q., 2016. Physiological roles of trehalose in Leptinotarsa larvae revealed by RNA interference of trehalose-6-phosphate synthase and trehalase genes. Insect Biochemistry and Molecular Biology, 77, pp.52-68.

Author response image 1.

The result section of the manuscript is quite concise, to my understanding (especially the initial few sections), which misses out on mentioning details that would help readers understand the paper better. While technical details of the methods should be in the Materials and Methods section, the overall experimental strategy for the experiments performed should be explained in adequate detail in the results section itself or in figure legends. I would request authors to include more details in the results section. As an extension of the comment above, many times, abbreviations have been used without introducing them. A thorough check of the manuscript is required regarding this.

Thank you very much for pointing out this issue. We will revise the manuscript content according to these suggestions.

The Spodoptera experiments appear ad hoc and are insufficient to support conservation beyond Helicoverpa. To substantiate this claim, please add a coherent, minimal set of Spodoptera experiments and present them in a dedicated subsection. Alternatively, consider removing these data and limiting the conclusions (and title) to H. armigera.

We thank the reviewer for this helpful comment. We agree that, in this current version of the manuscript, the S. frugiperda experiments are not sufficiently systematic to support strong claims about conservation beyond H. armigera. Our primary focus in this study is indeed on H. armigera, and the addition of the S. frugiperda data was intended only as preliminary, supportive evidence rather than a central component of our conclusions. To avoid over‑interpretation and to keep the manuscript focused and coherent, we will remove all S. frugiperda data from the revised version, including the corresponding text and figures. We will also adjust the title, abstract, and conclusion to clearly state that our findings are limited to H. armigera.

In order to check the effects of E2F/Dp, a dsRNA-mediated knockdown of Dp was performed. Why was the E2F protein, a primary target of the study, not chosen as a candidate? The authors should either provide justification for this or perform the suggested experiments to come to a conclusion. I would like to point out that such experiments were performed in Drosophila.

Thank you for this thoughtful comment and the specific suggestion. We agree that directly targeting E2F would, in principle, be an informative complementary approach. In our study, however, we prioritized Dp knockdown for two main reasons. First, E2F is a large family, and E2F-Dp functions as an obligate heterodimer. Previous work in D. melanogaster has shown that depletion of Dp is sufficient to disrupt E2F-dependent transcription broadly, often with more efficient loss of complex activity than targeting individual E2F isoforms (Zappia et al., 2021; Zappia et al., 2016). Second, in our preliminary trials, we performed a dsRNA feeding assay with dsHaE2F, dsHaDp, and combined dsHaE2F plus dsHaDp. In that assay, we did not achieve silencing of E2F in dsRNA targeting HaE2F (dsHaE2F). So here, as E2F is a large family, other E2F isoforms may be compensating for the silencing effect of targeted HaE2F. However, HaE2F showed significantly reduced expression upon dsHaDp and combined dsHaE2F plus dsHaDp feeding (Figure A), whereas HaDp showed a significant reduction in its expression in all three conditions (Figure B).  As we observed reduced expression of both HaE2F and HaDp upon combined feeding of dsHaE2F and dsHaDp, we further performed a rescue assay by exogenous feeding of trehalose. We observed the significant upregulation of HaE2F, HaDp, trehalose metabolic genes (HaTPS/TPP and HaTreh1), and myogenic genes (HaPrm and HaTm2) (Figure C). For these reasons, we focused on Dp silencing as a more reliable way to impair E2F/Dp complex function in H. armigera.

Author response image 2.

References:

(1) Zappia, M.P. and Frolov, M.V., 2016. E2F function in muscle growth is necessary and sufficient for viability in Drosophila. Nature Communications, 7(1), p.10509.

(2) Zappia, M.P., Guarner, A., Kellie-Smith, N., Rogers, A., Morris, R., Nicolay, B., Boukhali, M., Haas, W., Dyson, N.J. and Frolov, M.V., 2021. E2F/Dp inactivation in fat body cells triggers systemic metabolic changes. elife, 10, p.e67753.

Silencing of HaDp resulted in a significant decrease in HaE2F expression. I find this observation intriguing. DP is the cofactor of E2F, and they both heterodimerise and sit on the promoter of target genes to regulate them. I would request authors to revisit this result, as it contradicts the general understanding of how E2F/Dp functions in other organisms. If Dp indeed controls E2F expression, then further experiments should be conducted to come to a conclusion convincingly. Additionally, these results would need thorough discussion with citations of similar results observed for other transcription factor-cofactor complexes.

Thank you for highlighting this point and for prompting us to examine these data more carefully. Silencing HaDp leading to reduced HaE2F mRNA is indeed unexpected if one only considers the canonical view of E2F/Dp as a heterodimer that co-occupies target promoters without strongly regulating each other’s expression. However, several lines of work suggest that transcription factor-cofactor networks frequently include feedback loops in which cofactors influence the expression of their partner TFs. First, in multiple systems, transcription factors and their cofactors are known to regulate each other’s transcription, forming positive or negative feedback loops. For example, in hematopoietic cells, the transcription factor Foxp3 controls the expression of many of its own cofactors, and some of these cofactors in turn facilitate or stabilize Foxp3 expression, forming an interconnected regulatory network rather than a simple one‑way interaction (Rudra et al., 2012). Second, E2F/Dp complexes exhibit non‑canonical regulatory mechanisms and can regulate broad sets of targets, including other transcriptional regulators. Several studies show that E2F/Dp proteins not only control classical cell‑cycle genes but also participate in diverse processes such as DNA damage signaling, mitochondrial function, and differentiation (Guarner et al., 2017; Ambrus et al., 2013; Sánchez-Camargo et al., 2021). In D. melanogaster, complete loss of dDP alters the expression of direct targets E2F/DP, including dATM (Guarner et al., 2017).

All these reports indicate that the E2F-Dp complex sits at the top of multi‑layer regulatory hierarchies. Such architectures make it plausible that Dp silencing in H. armigera could modulate HaE2F expression in a non-canonical way.

References:

(1) Rudra, D., DeRoos, P., Chaudhry, A., Niec, R.E., Arvey, A., Samstein, R.M., Leslie, C., Shaffer, S.A., Goodlett, D.R. and Rudensky, A.Y., 2012. Transcription factor Foxp3 and its protein partners form a complex regulatory network. Nature immunology, 13(10), pp.1010-1019.

(2) Guarner, A., Morris, R., Korenjak, M., Boukhali, M., Zappia, M.P., Van Rechem, C., Whetstine, J.R., Ramaswamy, S., Zou, L., Frolov, M.V. and Haas, W., 2017. E2F/DP prevents cell-cycle progression in endocycling fat body cells by suppressing dATM expression. Developmental cell, 43(6), pp.689-703.

(3) Ambrus, A.M., Islam, A.B., Holmes, K.B., Moon, N.S., Lopez-Bigas, N., Benevolenskaya, E.V. and Frolov, M.V., 2013. Loss of dE2F compromises mitochondrial function. Developmental cell, 27(4), pp.438-451.

(4) Sánchez-Camargo, V.A., Romero-Rodríguez, S. and Vázquez-Ramos, J.M., 2021. Non-canonical functions of the E2F/DP pathway with emphasis in plants. Phyton, 90(2), p.307.

I consider the overall bioinformatics analysis to remain very poorly described. What is specifically lacking is clear statements about why a particular dry lab experiments were conducted.

We again thank the reviewer for advising us to give a biological context/motivation for every bioinformatics analysis performed. The bioinformatics analyses devised here, try to explain the systems-level perturbations of HaTPS/TPP silencing to explain the observed phenotype and to discover transcription factors potentially modulating the HaTPS/TPP induced gene regulatory changes.

(1) Gene set enrichment analyses:

Differential gene expression analyses of the bulk RNA sequencing data followed by qRT-PCR confirmed the transcriptional changes in myogenic genes and gene expression alterations in metabolic and cell cycle-related genes. These perturbations merely confirmed the effect induced by HaTPS/TPP silencing in obviously expected genes. We wanted to see whether using an “unbiased” system-level statistical analyses like gene set enrichment analyses (GSEA), can reveal both expected and novel biological processes that underlie HaTPS/TPP silencing. GSEA results revealed large-scale transcriptional changes in 11 enriched processes, including amino acid metabolism, energy metabolism, developmental regulatory processes, and motor protein activity. GSEA not only divulged overall transcriptionally enriched pathways but also identified the genes undergoing synchronized pathway-level transcriptional change upon HaTPS/TPP silencing.

(2) Gene regulatory network analysis:

Although GSEA uncovered potential pathway-level changes, we were also interested in identifying the gene regulatory network associated with such large-scale process-level transcriptional perturbations. Interestingly, the biological processes undergoing perturbations were also heterogeneous (e.g., motor protein activity, energy metabolism, amino acid metabolism, etc.). We hypothesized that the inference of a causal gene regulatory network associated with the genes associated with GSEA-enriched biological processes should predict core/master transcription factors that might synchronously regulate metabolic and non-metabolic processes related to HaTPS/TPP silencing, thereby providing a broad understanding of the perturbed phenotype. The gene regulatory network analysis statistically inferred an “active” gene regulatory network corresponding to the GSEA-enriched KEGG gene sets. Ranking the transcription factors (TFs) based on the number of outgoing connections (outdegree centrality) within the active gene regulatory network, E2F family TFs were identified to be top-ranking, highly connected transcription factors associated with the transcriptionally enriched processes. This suggests that E2F family TFs are central to controlling the flow of regulatory information within this network. Intriguingly, E2F has been previously implicated in muscle development in insects (Zappia et al., 2016). Further extracting the regulated targets of E2F family TFs within this network revealed the mechanistic connection with the 11 enriched processes. This GRN analysis was crucial in discovering and prioritizing E2F TFs as central transcription factors mediating HaTPS/TPP silencing effects, which was not apparent using trivial analyses like differential gene expression analysis.

As per the reviewer’s suggestions, we will add these outlined points in the text of the manuscript (Results section) to further give context and clarity to the bioinformatics analyses conducted in this study.

In my judgement, the EMSA analysis presented is technically poor in quality. It lacks positive and negative controls, does not show mutation analysis or super shifts. Also, it lacks any competition assays that are important to prove the binding beyond doubt. I am not sure why protein is not detected at all in lower concentrations. Overall, the EMSA assays need to be redone; I find the current results to be unacceptable.

Thank you for pointing out this issue. We will reperform the EMSA analysis with appropriate controls.  Although the gel image was not clear, there was a light band of protein (indicated by the white square) observed in well No. 8, where we used 8 μg of E2F protein and 75 ng of HaTPS/TPP promoter, upon gel stained with SYPRO Ruby protein stain, suggesting weak HaTPS/TPP-E2F complex formation.

GSEA studies clearly indicate enrichment of the amino acid synthesis gene in TPP knockdown samples. This supports the plausible theory that a lack of Trehalose means a lack of enough nutrients, therefore less of that is converted to amino acids, and therefore muscle development is compromised. Yet the authors make no effort to measure amino acid levels. While nutrients can be sensed through signalling pathways leading to shut shutdown of myogenic genes, a simple and direct correlation between less raw material and deformed muscle might also be possible.

We quantified amino acid levels as per the suggestion, and we observed differential levels of amino acids upon trehalose metabolism perturbation.

However, we observed that insect were failed to rescue when fed a control chickpea-based artificial diet that contained nutrients required for normal growth and development. Based on this observation, we conclude that trehalose deficiency is the only possible cause for the defect in muscle development.

The authors are encouraged to stick to one color palette while demonstrating sequencing results. Choosing a different color palette for representing results from the same sequencing analysis confuses readers.

Thank you for the comment. We will revise the color palette as per the suggestion.

Expression of genes, as understood from sequencing analysis in Figure 1D, Figure 2F, and Figure 3D, appears to be binary in nature. This result is extremely surprising given that the qRT-PCR of these genes have revealed a checker and graded expression.

Thank you for pointing out this issue. We will revise the scale range for these figures to get more insights about gene expression levels and include figures as per the suggestion.

In several graphs, non-significant results have been interpreted as significant in the results section. In a few other cases, the reported changes are minimal, and the statistical support is unclear; please recheck the analyses and include exact statistics. In the results section, fold changes observed should be discussed, as well as the statistical significance of the observed change.

We will revise the analyses and include exact statistics as per the suggestion.

Finally, I would add that trehalose metabolism regulates cell cycle genes, and muscle development genes establish correlation and causation. The authors should ensure that any comments they make are backed by evidence.

We thank the reviewer for this insightful comment.  Although direct evidence in insects is currently lacking, multiple independent studies in yeast, plants and mammalian systems support a regulatory link between trehalose metabolism and the cell cycle. In budding yeast Saccharomyces cerevisiae, neutral Treh (Nth1) is directly phosphorylated and activated by the major cyclin‑dependent kinase Cdk1 at G1/S, routing stored trehalose into glycolysis to fuel DNA replication and mitosis (Ewald et al., 2016). CDK‑dependent regulation of trehalase activity has also been reported in plants, where CDC28‑mediated phosphorylation channels glucose into biosynthetic pathways necessary for cell proliferation (Lara-núñez et al., 2025). Furthermore, budding yeast cells accumulate trehalose and glycogen upon entry into quiescence and subsequently mobilize these stores to generate a metabolic “finishing kick” that supports re‑entry into the cell cycle (Silljé et al., 1999; Shi et al., 2010). Exogenous trehalose that perturbs the trehalose cycle impairs glycolysis, reduces ATP, and delays cell cycle progression in S. cerevisiae, highlighting a dose‑ and context‑dependent control of growth versus arrest (Zhang, Zhang and Li, 2020). In mammalian systems, trehalose similarly modulates proliferation-differentiation decisions. In rat airway smooth muscle cells, low trehalose concentrations promote autophagy, whereas higher doses induce S/G2–M arrest, downregulate Cyclin A1/B1, and trigger apoptosis, indicating a shift from controlled growth to cell elimination at higher exposure (Xiao et al., 2021). In human iPSC‑derived neural stem/progenitor cells, low‑dose trehalose enhances neuronal differentiation and VEGF secretion, while higher doses are cytotoxic, again highlighting a tunable impact on cell‑fate outcomes (Roose et al., 2025). In wheat, exogenous trehalose under heat stress reduces growth, lowers auxin, gibberellin, abscisic acid and cytokinin levels, and represses CycD2 and CDC2 expression, suggesting that trehalose signalling integrates with hormone pathways and core cell‑cycle regulators to restrain proliferation during stress (Luo, Liu, and Li, 2021). Together, these studies showed the importance of trehalose metabolism in cell‑cycle regulation to decide whether cells and tissues proliferate, differentiate, or remain quiescent.

With respect to muscle development, previous work has implicated glycolytic metabolism in myogenesis and muscle growth. Tixier et al. (2013) showed that loss of key glycolytic genes results in abnormally thin muscles, while Bawa et al. (2020) demonstrated that loss of TRIM32 decreases glycolytic flux and reduces muscle tissue size. These findings indicate that carbohydrate and energy metabolism pathways are important determinants of muscle structure and growth. However, there are no previous studies about the role of trehalose metabolism in muscle development, other than as an energy source, so here we specifically set out to establish the involvement of trehalose metabolism in muscle development.

References:

(1) Ewald, J.C. et al. (2016) “The yeast cyclin-dependent kinase routes carbon fluxes to fuel cell cycle progression,” Molecular cell, 62(4), pp. 532–545.

(2) Lara-núñez, A. et al. (2025) “The Cyclin-Dependent Kinase activity modulates the central carbon metabolism in maize during germination,” (January), pp. 1–16.

(3) Silljé, H.H.W. et al. (1999) “Function of trehalose and glycogen in cell cycle progression and cell viability in Saccharomyces cerevisiae,” Journal of bacteriology, 181(2), pp. 396–400.

(4) Shi, L. et al. (2010) “Trehalose Is a Key Determinant of the Quiescent Metabolic State That Fuels Cell Cycle Progression upon Return to Growth,” 21, pp. 1982–1990.

(5) Zhang, X., Zhang, Y. and Li, H. (2020) “Regulation of trehalose, a typical stress protectant, on central metabolisms, cell growth and division of Saccharomyces cerevisiae CEN. PK113-7D,” Food Microbiology, 89, p. 103459.

(6) Xiao, B. et al. (2021) “Trehalose inhibits proliferation while activates apoptosis and autophagy in rat airway smooth muscle cells,” Acta Histochemica, 123(8), p. 151810.

(7) Roose, S.K. et al. (2025) “Trehalose enhances neuronal differentiation with VEGF secretion in human iPSC-derived neural stem / progenitor cells,” Regenerative Therapy, 30, pp. 268–277.

(8) Luo, Y., Liu, X. and Li, W. (2021) “Exogenously-supplied trehalose inhibits the growth of wheat seedlings under high temperature by affecting plant hormone levels and cell cycle processes,” Plant Signaling & Behavior, 16(6).

(9) Tixier, V., Bataillé, L., Etard, C., Jagla, T., Weger, M., DaPonte, J.P., Strähle, U., Dickmeis, T. and Jagla, K., 2013. Glycolysis supports embryonic muscle growth by promoting myoblast fusion. Proceedings of the National Academy of Sciences, 110(47), pp.18982-18987.

(10) Bawa, S., Brooks, D.S., Neville, K.E., Tipping, M., Sagar, M.A., Kollhoff, J.A., Chawla, G., Geisbrecht, B.V., Tennessen, J.M., Eliceiri, K.W. and Geisbrecht, E.R., 2020. Drosophila TRIM32 cooperates with glycolytic enzymes to promote cell growth. elife, 9, p.e52358.

Finally, we appreciate the meticulous review of this manuscript and constructive comments. We will perform the recommended experiments, data analysis, and revise the manuscript accordingly.

after his death he was lost, he did not go to heaven nor hell. Him being lost and trapped means he has not found peace = revenge being suggested.

immediately indicate that the character speaking on stage is clearly dead. So, the audience now knows that they have been introduced to a ghost.

character who has been serving others. expectation of either selflessness but "imprisoned" gives the idea it is unwelcome and forced upon him

eLife Assessment

The authors developed a fundamental computational method, which is intended to automatically process bioluminescence imaging-derived tumour images across anatomical regions and over time. This allows quantitative analysis of such data, and the authors applied it to describe the spatiotemporal distribution of tumour cells in response to CD19-targeted CAR-T cells that contained either CD28 or 4-1BB costimulatory domains. Some operational limitations were identified, which relate to the pipeline's reliance on predefined regions of interest instead of aligning signal sites with anatomical information, scaling, and limitations in taking animal pose into account. Overall, the authors provide compelling evidence for the functionality of their computational approach towards automated analysis of bioluminescence imaging data, while applying it to a current topic of wide interest in cell therapy research.

Reviewer #1 (Public review):

Summary:

This paper presents maRQup a Python pipeline for automating the quantitative analysis of preclinical cancer immunotherapy experiments using bioluminescent imaging in mice. maRQup processes images to quantify tumor burden over time and across anatomical regions, enabling large-scale analysis of over 1,000 mice. The study uses this tool to compare different CAR-T cell constructs and doses, identifying differences in initial tumor control and relapse rates, particularly noting that CD19.CD28 CAR-T cells show faster initial killing but higher relapse compared to CD19.4-1BB CAR-T cells. Furthermore, maRQup facilitates the spatiotemporal analysis of tumor dynamics, revealing differences in growth patterns based on anatomical location, such as the snout exhibiting more resistance to treatment than bone marrow.

Strengths:

(1) The maRQup pipeline enables the automatic processing of a large dataset of over 1,000 mice, providing investigators with a rapid and efficient method for analyzing extensive bioluminescent tumor image data.

(2) Through image processing steps like tail removal and vertical scaling, maRQup normalizes mouse dimensions to facilitate the alignment of anatomical regions across images. This process enables the reliable demarcation of nine distinct anatomical regions within each mouse image, serving as a basis for spatiotemporal analysis of tumor burden within these consistent regions by quantifying average radiance per pixel.

Weaknesses:

(1) While the pipeline aims to standardize images for regional assessment, the reliance on scaling primarily along the vertical axis after tail removal may introduce limitations to the quantitative robustness of the anatomically defined regions. This approach does not account for potential non-linear growth across dimensions in animals of different ages or sizes, which could result in relative stretching or shrinking of subjects compared to an average reference.

(2) Furthermore, despite excluding severely slanted images, the pipeline does not fully normalize for variations in animal pose during image acquisition (e.g., tucked body, leaning). This pose variability not only impacts the precise relative positioning of internal anatomical regions, potentially making their definition based on relative image coordinates more qualitative than truly quantitative for precise regional analysis, but it also means that the bioluminescent light signal from the tumor will not propagate equally to the camera as photons will travel differentially through the tissue. This differing light path through tissues due to variable positioning can introduce large variability in the measured radiance that was not accounted for in the analysis algorithm. Achieving more robust anatomical and quantitative normalization might require methods that control animal posture using a rigid structure during imaging.

Comments on revisions:

(1) Clarification of 2D Analysis. We strongly recommend that the authors explicitly define maRQup as a 2D spatiotemporal analysis technique. Since optical imaging quantification is inherently dependent on tissue type and signal depth, characterizing this as a 3D or volumetric method without tomographic correction is inaccurate. Please precede "spatiotemporal" with "2D" throughout the text to ensure precision regarding the method's capabilities.

(2) Data Validation and Scaling in Supplemental Figure g currently lacks the units necessary to support the assertion.

Non-Uniform Growth: The authors' method implies that mouse growth is linear and uniform in all directions (isotropic). However, murine growth is not akin to the inflation of a balloon; animals elongate and widen at different rates. The current scaling does not account for these physiological non-linearities.

Pose Variability: The scaling approach appears to neglect significant variability in animal positioning. Even under anesthesia, animal pose is rarely identical across subjects or time points.

Requirement for Evidence: Without quantitative data, there appears to be significant differences between the individual images and the merged image. If the authors assert that this is a "classical setting" where mouse positioning is 100% consistent and growth curves are identical in multiple dimensions, please provide specific references that validate these assumptions. Otherwise, the scaling must be corrected to account for anisotropic growth and pose differences or stated that scaling was only based on one dimension.

(3) Methodology of Spatial Regions The manuscript does not currently indicate how the nine distinct spatial regions were determined. Please expand the methods section to include the specific segmentation algorithms or anatomical criteria used to define these regions, as this is critical for reproducibility.

Reviewer #3 (Public review):

Summary:

The paper "The 1000+ mouse project: large-scale spatiotemporal parametrization and modeling of preclinical cancer immunotherapies" is focused on developing a novel methodology for automatic processing of bioluminescence imaging data. It provides quantitative and statistically robust insights on preclinical experiments that will contribute to optimizing cell-based therapies. There is an enormous demand for such methods and approaches that enable the spatiotemporal evaluation of cell monitoring in large cohorts of experimental animals.

Strengths:

The manuscript is generally well written, and the experiments are scientifically sound. The conclusions reflect the soundness of experimental data. This approach seems to be quite innovative and promising to improve the statistical accuracy of BLI data quantification.<br /> This methodology can be used as a universal quantification tool for BLI data for in vivo assessment of adoptively transferred cells due to the versatility of the technology.

Comments on revisions:

The critiques have been taken care of appropriately.

Aurhor response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

This paper presents maRQup, a Python pipeline for automating the quantitative analysis of preclinical cancer immunotherapy experiments using bioluminescent imaging in mice. maRQup processes images to quantify tumor burden over time and across anatomical regions, enabling large-scale analysis of over 1,000 mice. The study uses this tool to compare different CAR-T cell constructs and doses, identifying differences in initial tumor control and relapse rates, particularly noting that CD19.CD28 CAR-T cells show faster initial killing but higher relapse compared to CD19.4-1BB CAR-T cells. Furthermore, maRQup facilitates the spatiotemporal analysis of tumor dynamics, revealing differences in growth patterns based on anatomical location, such as the snout exhibiting more resistance to treatment than bone marrow.

Strengths:

(1) The maRQup pipeline enables the automatic processing of a large dataset of over 1,000 mice, providing investigators with a rapid and efficient method for analyzing extensive bioluminescent tumor image data.

(2) Through image processing steps like tail removal and vertical scaling, maRQup normalizes mouse dimensions to facilitate the alignment of anatomical regions across images. This process enables the reliable demarcation of nine distinct anatomical regions within each mouse image, serving as a basis for spatiotemporal analysis of tumor burden within these consistent regions by quantifying average radiance per pixel.

Weaknesses:

(1) While the pipeline aims to standardize images for regional assessment, the reliance on scaling primarily along the vertical axis after tail removal may introduce limitations to the quantitative robustness of the anatomically defined regions. This approach does not account for potential non-linear growth across dimensions in animals of different ages or sizes, which could result in relative stretching or shrinking of subjects compared to an average reference.

Our answer to this comment is included in the Supplemental Methods. The standard deviation of the mouse pixels was calculated to ensure that the image processing steps did not alter the shape or size of the mice. Such consistency is particularly striking because our dataset was accrued by nine lab members over the last five years, before we conceived and carried out our analysis (c.f., answer to point #2). In fact, it is the very consistency of this IVIS measurement that led us to conceive our pipeline. As seen from Supplemental Figure 4G, there is minimal difference in the shape or size of the mice across 7,534 images. A total of 99 images were removed either due to being too slanted (91/7663, 1.2%) or due to processing errors (8/7633, 0.1%). Also, the vertical scaling was conducted while keeping the aspect ratio unchanged to prevent any non-anatomical scaling. Hence, we did not record any nonlinear growth of the mice that would warrant more convoluted alignment and/or batch correction for our images.

(2) Furthermore, despite excluding severely slanted images, the pipeline does not fully normalize for variations in animal pose during image acquisition (e.g., tucked body, leaning). This pose variability not only impacts the precise relative positioning of internal anatomical regions, potentially making their definition based on relative image coordinates more qualitative than truly quantitative for precise regional analysis, but it also means that the bioluminescent light signal from the tumor will not propagate equally to the camera, as photons will travel differentially through the tissue. This differing light path through tissues due to variable positioning can introduce large variability in the measured radiance that was not accounted for in the analysis algorithm. Achieving more robust anatomical and quantitative normalization might require methods that control animal posture using a rigid structure during imaging.

Reviewer #1 is correct that different mouse postures would be an issue when aligning the images and normalizing for size. However, all experiments are conducted for luminescence measurements in the IVIS system (i.e., this requires anesthesia and long integration time for imaging). In our experience and in our 1000+ mouse dataset, we noticed that all experiments (n=37) did place the anesthetized mice in a stretched/elongated position. Of note, these experiments were conducted by nine different researchers who were not instructed on how to place the mice on the machine for ideal image processing, thus showing that the standard protocol of imaging mice on IVIS does not introduce large variations in animal pose during image acquisition. We think the issue raised by Reviewer #1 is moot in the context of classical settings for mouse luminescence imaging.

Reviewer #2 (Public review):

Summary:

The authors developed a method that automatically processes bioluminescent tumor images for quantitative analysis and used it to describe the spatiotemporal distribution of tumor cells in response to CD19-targeting CAR-T cells, comprising CD28 or 4-1BB costimulatory domains. The conclusion highlights the dependence of tumor decay and relapse on the number of injected cells, the type of cells, and the initial growth rate of tumors (where initial is intended from the first day of therapy). The authors also determined the spatiotemporal analysis of tumor response to CAR T therapy in different regions of the mouse body in a model of acute lymphoblastic leukemia (ALL).

Strengths:

The analysis is based on a large number of images and accounts for many variables. The results of the analysis largely support their claims that the kinetics of tumor decay and relapse are dependent on the CAR T co-stimulatory domain and number of cells injected and tumor growth rates. 

Weaknesses:

The study does not specify how a) differences in mouse positioning (and whether they excluded not-aligned mice) and b) tumor spread at the start of therapy influenced their data. The study does not take into account the potential heterogeneity of CAR T cells in terms of CAR T expression or T cell immunophenotype (differentiation, exhaustion, fitness...).

See answer #2 to Reviewer #1.

Author response image 1.

Author response image 1 shows the average tumor radiance on day zero (when CAR-T cell therapy was administered) for all mice. While there is some spread, most mice had tumor localized to the liver or bone marrow.

Reviewer #3 (Public review):

Summary:

The paper "The 1000+ mouse project: large-scale spatiotemporal parametrization and modeling of preclinical cancer immunotherapies" is focused on developing a novel methodology for automatic processing of bioluminescence imaging data. It provides quantitative and statistically robust insights into preclinical experiments that will contribute to optimizing cell-based therapies. There is an enormous demand for such methods and approaches that enable the spatiotemporal evaluation of cell monitoring in large cohorts of experimental animals.

Strengths:

The manuscript is generally well written, and the experiments are scientifically sound. The conclusions reflect the soundness of experimental data. This approach seems to be quite innovative and promising to improve the statistical accuracy of BLI data quantification. 

This methodology can be used as a universal quantification tool for BLI data for in vivo assessment of adoptively transferred cells due to the versatility of the technology.

Weaknesses: 

No weaknesses were identified by this Reviewer. 

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

In this paper, the authors propose a significant advancement in optical image data analysis by employing automation. They effectively demonstrate the valuable insights that can be gained from analyzing extensive datasets with a more unbiased methodology. At present, I do not have any specific suggestions for improvement.

However, it is important to note that this work is limited in its operational scope. Specifically, it relies on predefined ROIs rather than aligning the signal site with anatomical systems. The scaling model and image cropping are simplistic, animal pose is not taken into account, and the data output needs to be called semi-quantitative or qualitative, and would have been stronger utilizing an AI agent. Nevertheless, this work underscores the potential of automated systems in preclinical image analysis, which is a crucial step towards developing more sophisticated approaches to optical image data analysis.

While our analysis used predefined ROIs, the maRQup pipeline allows users to manually draw ROIs on the mouse image.

Reviewer #2 (Recommendations for the authors):

The writing and presentation of data are clear and accurate, but some additional information should be added regarding the imaging protocol used to acquire the original data. 

The authors mention fluorescence in Figure 1. I expected all the data to be generated from bioluminescent NALM-6 tumors, since bioluminescence is indeed measured in average radiance and can be per pixel (p/sec/cm2/sr/pixel). Fluorescence should be measured using radiance efficiency (p/sec/cm2/sr)/(µW/cm2), a unit that compensates for non-uniform excitation light pattern in the instrument. Would the author find different results if fluorescence data were analyzed separately?

Reviewer #2 is correct that the unit for fluorescence would be radiance efficiency. The word “fluorescent” was included in the label of Figure 1a  to highlight that our workflow could be applied to other types of light-generating methods (i.e., fluorescence vs. bioluminescence). However, in this study, measurements of bioluminescent tumors only were analyzed. If fluorescence measurements are to be analyzed, our methods of image acquisition and processing would be directly applicable.

Did the author ever check the signal of the snout in mice with no tumor?

In mice with no tumor, there is no detectable signal in the snout (or anywhere else, for that matter).

The urine of mice contains phosphor, and might give a background signal, especially if longer exposure is used at the end of the study.

For the mice with no tumor injection, the luminescence signal was below background (<10² p/sec/cm²/sr/pixel). In particular, we do not detect any signal in the bladder/urine. Additionally, as described in the Supplemental Methods and Figure 1b, only pixels that were on the mouse as determined from the brightfield image were used to calculate the tumor burden from the radiance of the luminescent image. This method ensures that any background signal (e.g., from phosphor in mouse urine) would be excluded in the radiance quantification and not bias the results.

Additionally, as described in the Methods, the exposure time was held constant at 30 seconds for each IVIS measurement across all 37 experiments.

The data using more than 2 million cells comes from only 10 mice, and maybe the biological relevance of this group is limited since it will not be achievable and translatable in humans (PMID: 33653113).

We appreciate Reviewer #2’s attention to this issue. The effect observed in our study is large enough to reach statistical significance despite the small number of mice. Note that the dosing regimen used was optimized for the murine NSG model and would require appropriate scaling before clinical application. Nonetheless, NSG mice remain the gold standard for pre‑clinical in vivo evaluation and their use is generally required by regulatory agencies, such as the FDA, for assessing novel CAR‑T cell therapies; thus these findings are relevant for advancing such treatments.

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good life and eudaimon life relate, so this has to be a must have for finding the good life right? this can lead us to purpose and happiness.

from stanford enc: judging the courageous man and finding the middle ground, no little or too much.

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