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  1. Feb 2026
    1. L'Esprit Critique au Cœur de l'Enquête Privée Spécialisée : Analyse des Pratiques de Benoît Judde

      Ce document de synthèse analyse les interventions de Benoît Judde, détective privé spécialisé, concernant l'évolution de la profession de détective en France, le cadre juridique des dérives sectaires et l'utilisation de l'esprit critique comme outil méthodologique fondamental pour l'administration de la preuve.

      Synthèse

      La profession de détective privé en France, désormais strictement réglementée et contrôlée par le ministère de l'Intérieur (CNAPS), s'est transformée en un auxiliaire de fait pour la défense des intérêts privés et le système judiciaire.

      Benoît Judde, spécialisé dans les faits de manipulation et les dérives sectaires, démontre que l'efficacité de l'enquêteur repose sur une maîtrise rigoureuse du cadre juridique et sur l'application de l'esprit critique.

      Cette approche, adossée aux psychologies cognitive et sociale expérimentales, permet de transformer des phénomènes subjectifs comme la « sujétion psychologique » en éléments de preuve objectifs, circonstanciés et recevables en justice.

      Le passage récent (2024) de la sujétion psychologique au statut d'infraction autonome renforce la nécessité d'une expertise technique capable de caractériser les manœuvres de manipulation sans tomber dans le biais de confirmation.

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      1. Le Cadre Légal et Déontologique de la Profession

      La profession de détective privé, officiellement dénommée « agent de recherche privée », est définie par le Code de la sécurité intérieure (CSI).

      Définition et Prérogatives

      Selon l'article L621-1 du CSI, le détective est un professionnel libéral dont la mission consiste à recueillir des informations ou des renseignements destinés à des tiers, en vue de la défense de leurs intérêts.

      Anonymat d'enquête : C’est la seule profession parajuridique autorisée à enquêter sans révéler sa qualité, son identité réelle ou l’objet de sa mission. Contrairement aux commissaires de justice (huissiers), le détective peut agir sous une identité fictive.

      Recevabilité des preuves : Les rapports de détective doivent être « détaillés, circonstanciés et précis » (DCP) pour être recevables devant les tribunaux, selon une jurisprudence de la Cour de cassation datant de 1962.

      Régulation et Formation

      La profession est passée d'un état de « freestyle » à un encadrement strict :

      Contrôle du CNAPS : Le Conseil national des activités privées de sécurité (sous tutelle du ministère de l'Intérieur) délivre trois agréments distincts (personne physique, structure juridique, carte professionnelle), renouvelables tous les 5 ans après enquête de moralité approfondie.

      Formation obligatoire : Un niveau Bac+3 (licence professionnelle) est requis. Il n'existe que quatre écoles en France (deux universités et deux écoles privées), formant environ 120 nouveaux professionnels par an.

      Déontologie : Les détectives sont soumis au secret professionnel et à une obligation de conseil. Ils doivent notamment vérifier la légitimité de la demande pour éviter de servir des projets de vengeance ou des recherches malveillantes.

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      2. L'Enquête Spécialisée dans les Dérives Sectaires

      Le champ d'action des détectives est vaste (recherche de personnes, contrefaçon, fraude à l'assurance), mais la spécialisation de Benoît Judde porte sur la manipulation mentale.

      Les Critères de la MIVILUDES

      Pour objectiver une dérive sectaire, l'enquêteur s'appuie sur le référentiel de la Mission interministérielle de vigilance et de lutte contre les dérives sectaires (MIVILUDES), qui identifie 10 critères principaux.

      | Catégorie d'atteinte | Exemples de sous-critères | | --- | --- | | Atteintes aux personnes | Rupture avec l'environnement d'origine, perte d'esprit critique, embrigadement des enfants, privation de sommeil ou de nourriture. | | Atteintes aux biens | Exigences financières disproportionnées, endettement, travail dissimulé (ex: détournement du concept de woofing). | | Vie sociale et démocratique | Discours antisocial, trouble à l'ordre public, détournement des circuits économiques. |

      Collaboration Interdisciplinaire

      L'enquêteur travaille en binôme avec un psychologue (spécialisé en psychologie scientifique, cognitive et sociale) pour valider la réalité de l'emprise.

      Cette collaboration permet d'apporter une « parole psychologique » crédible que le juriste ou le détective ne peut formuler seul, notamment pour qualifier le préjudice ou la sujétion devant un juge.

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      3. Évolutions Législatives Récentes (Loi de 2024)

      Le cadre juridique français a récemment évolué pour faciliter la répression des dérives sectaires, rendant le rôle de la preuve plus complexe et crucial.

      Autonomie de la sujétion psychologique : Auparavant liée à l'abus de faiblesse (nécessitant de prouver un état de faiblesse préalable et un préjudice), la « mise en état de sujétion psychologique » est devenue une infraction autonome en 2024.

      Il suffit désormais de prouver l'utilisation de techniques de pression ou de manipulation altérant le jugement.

      Détournement de traitement médical : Une nouvelle infraction punit le fait de provoquer une personne à abandonner un traitement médical thérapeutique ou prophylactique (vaccination) au profit de pratiques pseudo-scientifiques.

      L'Escroquerie et la Cybermalveillance : Dans le domaine numérique, 95 % des arnaques reposent sur l'ingénierie sociale (manipulation humaine) plutôt que sur des failles purement techniques.

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      4. L'Esprit Critique comme Méthodologie d'Enquête

      Pour Benoît Judde, l'esprit critique n'est pas une posture intellectuelle mais un outil de travail permettant d'éviter le biais de confirmation et d'assurer l'objectivité du rapport.

      Les Trois Piliers de la Manipulation

      L'enquêteur analyse les situations à travers trois mécanismes identifiés par la psychologie expérimentale :

      1. L'automanipulation : Utilisation des biais cognitifs naturels des individus.

      2. La soumission librement consentie : Techniques comme le « pied dans la porte » (obtenir un petit engagement pour en obtenir un plus grand) ou la « porte au nez » (demander l'excessif pour obtenir le raisonnable).

      3. La soumission à l'autorité : Référence à l'expérience de Milgram. La manipulation réussit si l'autorité est perçue comme légitime (ex: port d'une blouse, titre de « frère de Jésus », etc.).

      L'Objectivité de la Preuve

      Recours à la technologie : Utilisation de caméras cachées lors d'infiltrations pour fournir une preuve brute et incontestable, évitant ainsi la faillibilité de la mémoire humaine ou les accusations de partialité.

      Nécessité et proportionnalité : L'enquêteur doit justifier que l'atteinte à la vie privée (infiltration, surveillance) était strictement indispensable à la manifestation de la vérité et proportionnée à l'enjeu (droit à la preuve vs droit à la vie privée).

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      5. Conclusion : Vers un Continuum de Sécurité

      Le document souligne que l'État ne peut assurer seul la surveillance de tous les risques, particulièrement dans les domaines complexes des dérives sectaires et thérapeutiques.

      Synergie Public-Privé : Le détective privé intervient là où la police ne peut plus agir (disparitions non inquiétantes, enquêtes pré-pénales pour consolider une plainte).

      Auxiliaire de Justice : En apportant des éléments basés sur un consensus scientifique (psychologie expérimentale), le détective aide le magistrat à fonder sa décision sur des faits plutôt que sur des témoignages contradictoires.

      Complémentarité : L'objectif n'est pas une « américanisation » du système, mais une validation réciproque où le secteur privé complète l'action régalienne en fournissant une expertise technique et de terrain spécifique.

    1. Synthèse Clinique : Comprendre et Accompagner la Cooccurrence TSA-TDAH (ODHD)

      Résumé Exécutif

      Ce document propose une analyse approfondie de la cooccurrence entre le Trouble du Spectre de l'Autisme (TSA) et le Trouble du Déficit de l'Attention avec ou sans Hyperactivité (TDAH), un profil souvent désigné sous l'acronyme anglo-saxon « ODHD ».

      Longtemps ignorée par les classifications officielles (notamment avant le DSM-5 en 2013), cette double problématique est aujourd'hui reconnue comme une entité clinique à part entière, et non une simple addition de symptômes.

      Les points clés de cette analyse incluent :

      Prévalence élevée : Plus de 40 % des individus avec un TSA présentent un TDAH associé.

      Complexité clinique : La combinaison des deux troubles entraîne une sévérité accrue des symptômes, une fatigue majeure (burnout autistique) et des profils sensoriels complexes.

      Prise en charge spécifique : L'approche doit être multidisciplinaire, privilégiant la psychoéducation et une pharmacologie prudente, tout en évitant le recours systématique aux antipsychotiques.

      Changement de paradigme : Il est crucial de passer d'une vision centrée sur le symptôme à une vision axée sur le fonctionnement global et la qualité de l'environnement.

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      1. Analyse du Diagnostic et Prévalence

      1.1 Évolution des Classifications

      Avant 2013, le DSM-5 interdisait formellement le double diagnostic TSA et TDAH. Pourtant, la pratique clinique révélait déjà des patients présentant des caractéristiques marquées des deux troubles. Depuis la levée de cette interdiction, la littérature scientifique et l'expérience de terrain confirment une imbrication fréquente.

      1.2 Statistiques de Cooccurrence

      Les données actuelles mettent en évidence une asymétrie dans la comorbidité :

      TSA avec TDAH : Plus de 40 % des personnes autistes répondent également aux critères du TDAH.

      TDAH avec TSA : Environ 13 % à 20 % des personnes TDAH présentent des traits autistiques associés.

      1.3 L'importance du Diagnostic Différentiel

      Il est impératif de distinguer l'origine des symptômes pour éviter un empilement erroné de diagnostics. Par exemple :

      • Les difficultés sociales du TDAH sont souvent liées à l'impulsivité ou l'inattention, tandis que dans le TSA, elles relèvent de la cognition sociale.

      • Les troubles attentionnels du TSA sont souvent la conséquence d'une hyper-sensorialité ou d'intérêts restreints plutôt que d'un mécanisme TDAH intrinsèque.

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      2. Manifestations Cliniques et Impacts Fonctionnels

      L'association des deux troubles (ODHD) crée un tableau singulier où les symptômes s'influencent mutuellement, augmentant la sévérité globale.

      | Domaine de fonctionnement | Impact de la cooccurrence TSA + TDAH | | --- | --- | | Fonctions Exécutives | Difficultés plus marquées (inhibition, flexibilité, attention) ; profil proche du TDAH isolé mais plus sévère. | | Cognition Sociale | Difficultés sociales accrues, contact visuel moindre et peu d'amélioration spontanée avec le temps. | | Sensorialité | Cumul des hypersensibilités ; profil sensoriel complexe et particulièrement intense. | | Santé Mentale | Risque accru de troubles dépressifs, troubles du sommeil, épuisement majeur et burnout autistique. | | Adaptation | Précarité économique plus importante et difficultés psychosociales majeures. |

      2.1 La Question du "Trouble" vs "Fonctionnement"

      Un point crucial de l'analyse est la distinction entre avoir un fonctionnement neurodivergent et présenter un trouble. Le trouble n'apparaît que lorsqu'il y a une répercussion fonctionnelle négative. Cette répercussion est étroitement liée à la qualité environnementale (par exemple, la personnalité d'un enseignant ou l'adaptation d'un poste de travail).

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      3. Stratégies Thérapeutiques et Accompagnement

      3.1 La Psychoéducation : Le Pilier Central

      La psychoéducation doit être « sextuple » (incluant l'enfant, les parents et la fratrie). Ses objectifs sont de :

      • Donner du sens aux symptômes.

      • Mettre fin aux idées reçues et aux préjugés (notamment ceux des soignants).

      • Réduire l'auto-stigmatisation et la culpabilité.

      • Limiter le "masking" (suradaptation permanente), qui est une cause majeure d'épuisement et de burnout.

      3.2 Approche Médicamenteuse (Méthylphénidate)

      Le recours au méthylphénidate est possible mais nécessite une expertise clinique fine :

      Sensibilité accrue : Les patients TSA sont souvent hyper-sensibles aux substances (perception fine des changements corporels).

      Posologie : Il est recommandé de commencer par des doses très faibles (ex: 5 mg) et d'augmenter de manière très progressive.

      Vigilance : Surveiller l'augmentation potentielle des stéréotypies ou de l'irritabilité.

      Critique des pratiques : Le document dénonce comme une « hérésie » l'usage de première intention des antipsychotiques (type Haldol ou Risperdal) en France, au détriment du méthylphénidate.

      3.3 La "Thérapie de Mamie" et Médiations Corporelles

      L'hygiène de vie et le corps sont des leviers fondamentaux :

      Hygiène de vie : Régime méditerranéen, sommeil de qualité et régulation de l'exposition aux écrans.

      Activité physique : Présente une efficacité majeure prouvée par la littérature pour la régulation du TDAH.

      Régulation émotionnelle : Utilisation d'outils de cohérence cardiaque (ex: RespiRelax) pour agir sur le système nerveux autonome.

      Médiations alternatives : La musicothérapie et la danse-thérapie sont particulièrement efficaces car elles passent par les fréquences et le corps plutôt que par le langage verbal.

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      4. Neurodiversité : Forces et Perspectives Évolutionnistes

      Il est essentiel de ne pas réduire l'individu à ses symptômes mais de reconnaître les forces inhérentes à ces profils.

      Forces du TDAH : Empathie, créativité (issue des stratégies d'adaptation développées), curiosité, enthousiasme, intuition et rapidité.

      Forces du TSA : Précision, sérieux, honnêteté, respect des horaires et sens du détail.

      Lecture évolutionniste : La persistance des troubles du neurodéveloppement (TND) dans l'évolution humaine suggère leur utilité sociale. Par exemple, le TDAH pour l'exploration et la résolution de problèmes rapides, et le TSA pour la vigilance et l'expertise technique au sein d'un groupe.

      Vers des environnements inclusifs

      Le projet « Atipy Friendly » illustre la transition nécessaire vers une société (notamment l'université) capable de s'adapter à la singularité de ces fonctionnements, plutôt que d'exiger une suradaptation systématique des personnes concernées.

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      Conclusion

      Le profil TSA-TDAH (ODHD) nécessite une attention particulière et une coordination accrue entre les professionnels (psychomotriciens, pédopsychiatres, éducateurs).

      L'enjeu n'est pas seulement de traiter des symptômes, mais de répondre aux besoins spécifiques de la personne pour favoriser son autonomie et sa qualité de vie, tout en valorisant les forces liées à sa neurodivergence.

    1. THE AMERICAN YAWP Menu Skip to content HomeAbout Barbara Jordan – On the Impeachment of Richard Nixon (1974) Brookes print Casta painting Contributors How the Other Half Lived: Photographs of Jacob Riis Introduction Note on Recommended Readings Press Sample Feedback (@AmericanYawp) Teaching Materials TEST: 11/18/2025 Updates Who Pays for This? 6. A New Nation “The Federal Pillars,” from The Massachusetts Centinel, August 2, 1789. Library of Congress. *The American Yawp is an evolving, collaborative text. Please click here to improve this chapter.* I. IntroductionII. Shays’s RebellionIII. The Constitutional ConventionIV. Ratifying the ConstitutionV. Rights and CompromisesVI. Hamilton’s Financial SystemVII. The Whiskey Rebellion and Jay’s TreatyVIII. The French Revolution and the Limits of LibertyIX. Religious FreedomX. The Election of 1800XI. ConclusionXII. Primary SourcesXIII. Reference Material I. Introduction On July 4, 1788, Philadelphians turned out for a “grand federal procession” in honor of the new national constitution. Workers in various trades and professions demonstrated. Blacksmiths carted around a working forge, on which they symbolically beat swords into farm tools. Potters proudly carried a sign paraphrasing from the Bible, “The potter hath power over his clay,” linking God’s power with an artisan’s work and a citizen’s control over the country. Christian clergymen meanwhile marched arm-in-arm with Jewish leaders. The grand procession represented what many Americans hoped the United States would become: a diverse but cohesive, prosperous nation.1 Over the next few years, Americans would celebrate more of these patriotic holidays. In April 1789, for example, thousands gathered in New York to see George Washington take the presidential oath of office. That November, Washington called his fellow citizens to celebrate with a day of thanksgiving, particularly for “the peaceable and rational manner” in which the government had been established.2 But the new nation was never as cohesive as its champions had hoped. Although the officials of the new federal government—and the people who supported it—placed great emphasis on unity and cooperation, the country was often anything but unified. The Constitution itself had been a controversial document adopted to strengthen the government so that it could withstand internal conflicts. Whatever the later celebrations, the new nation had looked to the future with uncertainty. Less than two years before the national celebrations of 1788 and 1789, the United States had faced the threat of collapse.   II. Shays’s Rebellion Daniel Shays became a divisive figure, to some a violent rebel seeking to upend the new American government, to others an upholder of the true revolutionary virtues Shays and others fought for. This contemporary depiction of Shays and his accomplice Job Shattuck portrays them in the latter light as rising “illustrious from the Jail.” Unidentified artist, Daniel Shays and Job Shattuck, 1787. Wikimedia. In 1786 and 1787, a few years after the Revolution ended, thousands of farmers in western Massachusetts were struggling under a heavy burden of debt. Their problems were made worse by weak local and national economies. Many political leaders saw both the debt and the struggling economy as a consequence of the Articles of Confederation, which provided the federal government with no way to raise revenue and did little to create a cohesive nation out of the various states. The farmers wanted the Massachusetts government to protect them from their creditors, but the state supported the lenders instead. As creditors threatened to foreclose on their property, many of these farmers, including Revolutionary War veterans, took up arms. Led by a fellow veteran named Daniel Shays, these armed men, the “Shaysites,” resorted to tactics like the patriots had used before the Revolution, forming blockades around courthouses to keep judges from issuing foreclosure orders. These protesters saw their cause and their methods as an extension of the “Spirit of 1776”; they were protecting their rights and demanding redress for the people’s grievances. Governor James Bowdoin, however, saw the Shaysites as rebels who wanted to rule the government through mob violence. He called up thousands of militiamen to disperse them. A former Revolutionary general, Benjamin Lincoln, led the state force, insisting that Massachusetts must prevent “a state of anarchy, confusion and slavery.”3 In January 1787, Lincoln’s militia arrested more than one thousand Shaysites and reopened the courts. Daniel Shays and other leaders were indicted for treason, and several were sentenced to death, but eventually Shays and most of his followers received pardons. Their protest, which became known as Shays’s Rebellion, generated intense national debate. While some Americans, like Thomas Jefferson, thought “a little rebellion now and then” helped keep the country free, others feared the nation was sliding toward anarchy and complained that the states could not maintain control. For nationalists like James Madison of Virginia, Shays’s Rebellion was a prime example of why the country needed a strong central government. “Liberty,” Madison warned, “may be endangered by the abuses of liberty as well as the abuses of power.”4   III. The Constitutional Convention The uprising in Massachusetts convinced leaders around the country to act. After years of goading by James Madison and other nationalists, delegates from twelve of the thirteen states met at the Pennsylvania state house in Philadelphia in the summer of 1787. Only Rhode Island declined to send a representative. The delegates arrived at the convention with instructions to revise the Articles of Confederation. The biggest problem the convention needed to solve was the federal government’s inability to levy taxes. That weakness meant that the burden of paying back debt from the Revolutionary War fell on the states. The states, in turn, found themselves beholden to the lenders who had bought up their war bonds. That was part of why Massachusetts had chosen to side with its wealthy bondholders over poor western farmers.5 James Madison, however, had no intention of simply revising the Articles of Confederation. He intended to produce a completely new national constitution. In the preceding year, he had completed two extensive research projects—one on the history of government in the United States, the other on the history of republics around the world. He used this research as the basis for a proposal he brought with him to Philadelphia. It came to be called the Virginia Plan, named after Madison’s home state.6 James Madison was a central figure in the reconfiguration of the national government. Madison’s Virginia Plan was a guiding document in the formation of a new government under the Constitution. John Vanderlyn, Portrait of James Madison, 1816. Wikimedia. The Virginia Plan was daring. Classical learning said that a republican form of government required a small and homogenous state: the Roman republic, or a small country like Denmark, for example. Citizens who were too far apart or too different could not govern themselves successfully. Conventional wisdom said the United States needed to have a very weak central government, which should simply represent the states on certain matters they had in common. Otherwise, power should stay at the state or local level. But Madison’s research had led him in a different direction. He believed it was possible to create “an extended republic” encompassing a diversity of people, climates, and customs. The Virginia Plan, therefore, proposed that the United States should have a strong federal government. It was to have three branches—legislative, executive, and judicial—with power to act on any issues of national concern. The legislature, or Congress, would have two houses, in which every state would be represented according to its population size or tax base. The national legislature would have veto power over state laws.7 Other delegates to the convention generally agreed with Madison that the Articles of Confederation had failed. But they did not agree on what kind of government should replace them. In particular, they disagreed about the best method of representation in the new Congress. Representation was an important issue that influenced a host of other decisions, including deciding how the national executive branch should work, what specific powers the federal government should have, and even what to do about the divisive issue of slavery. For more than a decade, each state had enjoyed a single vote in the Continental Congress. William Patterson’s New Jersey Plan proposed to keep things that way. The Connecticut delegate Roger Sherman, furthermore, argued that members of Congress should be appointed by the state legislatures. Ordinary voters, Sherman said, lacked information, were “constantly liable to be misled” and “should have as little to do as may be” about most national decisions.8 Large states, however, preferred the Virginia Plan, which would give their citizens far more power over the legislative branch. James Wilson of Pennsylvania argued that since the Virginia Plan would vastly increase the powers of the national government, representation should be drawn as directly as possible from the public. No government, he warned, “could long subsist without the confidence of the people.”9) Ultimately, Roger Sherman suggested a compromise. Congress would have a lower house, the House of Representatives, in which members were assigned according to each state’s population, and an upper house, which became the Senate, in which each state would have one vote. This proposal, after months of debate, was adopted in a slightly altered form as the Great Compromise: each state would have two senators, who could vote independently. In addition to establishing both types of representation, this compromise also counted three-fifths of a state’s enslaved population for representation and tax purposes. The delegates took even longer to decide on the form of the national executive branch. Should executive power be in the hands of a committee or a single person? How should its officeholders be chosen? On June 1, James Wilson moved that the national executive power reside in a single person. Coming only four years after the American Revolution, that proposal was extremely contentious; it conjured up images of an elected monarchy.10 The delegates also worried about how to protect the executive branch from corruption or undue control. They endlessly debated these questions, and not until early September did they decide the president would be elected by a special electoral college. In the end, the Constitutional Convention proposed a government unlike any other, combining elements copied from ancient republics and English political tradition but making some limited democratic innovations—all while trying to maintain a delicate balance between national and state sovereignty. It was a complicated and highly controversial scheme.   IV. Ratifying the Constitution Delegates to the Constitutional Convention assembled, argued, and finally agreed in this room, styled in the same manner as during the Convention. Photograph of the Assembly Room, Independence Hall, Philadelphia, Pennsylvania. Wikimedia. Creative Commons Attribution-Share Alike 3.0 Unported. The convention voted to send its proposed Constitution to Congress, which was then sitting in New York, with a cover letter from George Washington. The plan for adopting the new Constitution, however, required approval from special state ratification conventions, not just Congress. During the ratification process, critics of the Constitution organized to persuade voters in the different states to oppose it. Importantly, the Constitutional Convention had voted down a proposal from Virginia’s George Mason, the author of Virginia’s state Declaration of Rights, for a national bill of rights. This omission became a rallying point for opponents of the document. Many of these Anti-Federalists argued that without such a guarantee of specific rights, American citizens risked losing their personal liberty to the powerful federal government. The pro-ratification Federalists, on the other hand, argued that including a bill of rights was not only redundant but dangerous; it could limit future citizens from adding new rights.11 Citizens debated the merits of the Constitution in newspaper articles, letters, sermons, and coffeehouse quarrels across America. Some of the most famous, and most important, arguments came from Alexander Hamilton, John Jay, and James Madison in the Federalist Papers, which were published in various New York newspapers in 1787 and 1788.12 The first crucial vote came at the beginning of 1788 in Massachusetts. At first, the Anti-Federalists at the Massachusetts ratifying convention probably had the upper hand, but after weeks of debate, enough delegates changed their votes to narrowly approve the Constitution. But they also approved a number of proposed amendments, which were to be submitted to the first Congress. This pattern—ratifying the Constitution but attaching proposed amendments—was followed by other state conventions. The most high-profile convention was held in Richmond, Virginia, in June 1788, when Federalists like James Madison, Edmund Randolph, and John Marshall squared off against equally influential Anti-Federalists like Patrick Henry and George Mason. Virginia was America’s most populous state, it had produced some of the country’s highest-profile leaders, and the success of the new government rested upon its cooperation. After nearly a month of debate, Virginia voted 89 to 79 in favor of ratification.13 On July 2, 1788, Congress announced that a majority of states had ratified the Constitution and that the document was now in effect. Yet this did not mean the debates were over. North Carolina, New York, and Rhode Island had not completed their ratification conventions, and Anti-Federalists still argued that the Constitution would lead to tyranny. The New York convention would ratify the Constitution by just three votes, and finally Rhode Island would ratify it by two votes—a full year after George Washington was inaugurated as president.   V. Rights and Compromises Although debates continued, Washington’s election as president cemented the Constitution’s authority. By 1793, the term Anti-Federalist would be essentially meaningless. Yet the debates produced a piece of the Constitution that seems irreplaceable today. Ten amendments were added in 1791. Together, they constitute the Bill of Rights. James Madison, against his original wishes, supported these amendments as an act of political compromise and necessity. He had won election to the House of Representatives only by promising his Virginia constituents such a list of rights. There was much the Bill of Rights did not cover. Women found no special protections or guarantee of a voice in government. Many states continued to restrict voting only to men who owned significant amounts of property. And slavery not only continued to exist; it was condoned and protected by the Constitution. Of all the compromises that formed the Constitution, perhaps none would be more important than the compromise over the slave trade. Americans generally perceived the transatlantic slave trade as more violent and immoral than slavery itself. Many northerners opposed it on moral grounds. But they also understood that letting southern states import more Africans would increase their political power. The Constitution counted each enslaved individual as three fifths of a person for purposes of representation, so in districts with many enslaved people, the white voters had extra influence. On the other hand, the states of the Upper South also welcomed a ban on the Atlantic trade because they already had a surplus of enslaved laborers. Banning importation meant enslavers in Virginia and Maryland could get higher prices when they sold their enslaved laborers to states like South Carolina and Georgia that were dependent on a continued slave trade. New England and the Deep South agreed to what was called a “dirty compromise” at the Constitutional Convention in 1787. New Englanders agreed to include a constitutional provision that protected the foreign slave trade for twenty years; in exchange, South Carolina and Georgia delegates had agreed to support a constitutional clause that made it easier for Congress to pass commercial legislation. As a result, the Atlantic slave trade resumed until 1808 when it was outlawed for three reasons. First, Britain was also in the process of outlawing the slave trade in 1807, and the United States did not want to concede any moral high ground to its rival. Second, the Haitian Revolution (1791–1804), a successful slave revolt against French colonial rule in the West Indies, had changed the stakes in the debate. The image of thousands of armed Black revolutionaries terrified white Americans. Third, the Haitian Revolution had ended France’s plans to expand its presence in the Americas, so in 1803, the United States had purchased the Louisiana Territory from the French at a fire-sale price. This massive new territory, which had doubled the size of the United States, had put the question of slavery’s expansion at the top of the national agenda. Many white Americans, including President Thomas Jefferson, thought that ending the external slave trade and dispersing the domestic slave population would keep the United States a white man’s republic and perhaps even lead to the disappearance of slavery. The ban on the slave trade, however, lacked effective enforcement measures and funding. Moreover, instead of freeing illegally imported Africans, the act left their fate to the individual states, and many of those states simply sold intercepted enslaved people at auction. Thus, the ban preserved the logic of property ownership in human beings. The new federal government protected slavery as much as it expanded democratic rights and privileges for white men.14   VI. Hamilton’s Financial System Alexander Hamilton saw America’s future as a metropolitan, commercial, industrial society, in contrast to Thomas Jefferson’s nation of small farmers. While both men had the ear of President Washington, Hamilton’s vision proved most appealing and enduring. John Trumbull, Portrait of Alexander Hamilton, 1806. Wikimedia. President George Washington’s cabinet choices reflected continuing political tensions over the size and power of the federal government. The vice president was John Adams, and Washington chose Alexander Hamilton to be his secretary of the treasury. Both men wanted an active government that would promote prosperity by supporting American industry. However, Washington chose Thomas Jefferson to be his secretary of state, and Jefferson was committed to restricting federal power and preserving an economy based on agriculture. Almost from the beginning, Washington struggled to reconcile the Federalist and Republican (or Democratic-Republican) factions within his own administration.15 Alexander Hamilton believed that self-interest was the “most powerful incentive of human actions.” Self-interest drove humans to accumulate property, and that effort created commerce and industry. According to Hamilton, government had important roles to play in this process. First, the state should protect private property from theft. Second, according to Hamilton, the state should use human “passions” and “make them subservient to the public good.”16 In other words, a wise government would harness its citizens’ desire for property so that both private individuals and the state would benefit. Hamilton, like many of his contemporary statesmen, did not believe the state should ensure an equal distribution of property. Inequality was understood as “the great & fundamental distinction in Society,” and Hamilton saw no reason why this should change. Instead, Hamilton wanted to tie the economic interests of wealthy Americans, or “monied men,” to the federal government’s financial health. If the rich needed the government, then they would direct their energies to making sure it remained solvent.17 Hamilton, therefore, believed that the federal government must be “a Repository of the Rights of the wealthy.”18 As the nation’s first secretary of the treasury, he proposed an ambitious financial plan to achieve just that. The first part of Hamilton’s plan involved federal “assumption” of state debts, which were mostly left over from the Revolutionary War. The federal government would assume responsibility for the states’ unpaid debts, which totaled about $25 million. Second, Hamilton wanted Congress to create a bank—a Bank of the United States. The goal of these proposals was to link federal power and the country’s economic vitality. Under the assumption proposal, the states’ creditors (people who owned state bonds or promissory notes) would turn their old notes in to the treasury and receive new federal notes of the same face value. Hamilton foresaw that these bonds would circulate like money, acting as “an engine of business, and instrument of industry and commerce.”19 This part of his plan, however, was controversial for two reasons. First, many taxpayers objected to paying the full face value on old notes, which had fallen in market value. Often the current holders had purchased them from the original creditors for pennies on the dollar. To pay them at full face value, therefore, would mean rewarding speculators at taxpayer expense. Hamilton countered that government debts must be honored in full, or else citizens would lose all trust in the government. Second, many southerners objected that they had already paid their outstanding state debts, so federal assumption would mean forcing them to pay again for the debts of New Englanders. Nevertheless, President Washington and Congress both accepted Hamilton’s argument. By the end of 1794, 98 percent of the country’s domestic debt had been converted into new federal bonds.20 Hamilton’s plan for a Bank of the United States, similarly, won congressional approval despite strong opposition. Thomas Jefferson and other Republicans argued that the plan was unconstitutional; the Constitution did not authorize Congress to create a bank. Hamilton, however, argued that the bank was not only constitutional but also important for the country’s prosperity. The Bank of the United States would fulfill several needs. It would act as a convenient depository for federal funds. It would print paper banknotes backed by specie (gold or silver). Its agents would also help control inflation by periodically taking state bank notes to their banks of origin and demanding specie in exchange, limiting the amount of notes the state banks printed. Furthermore, it would give wealthy people a vested interest in the federal government’s finances. The government would control just 20 percent of the bank’s stock; the other eighty percent would be owned by private investors. Thus, an “intimate connexion” between the government and wealthy men would benefit both, and this connection would promote American commerce. In 1791, therefore, Congress approved a twenty-year charter for the Bank of the United States. The bank’s stocks, together with federal bonds, created over $70 million in new financial instruments. These spurred the formation of securities markets, which allowed the federal government to borrow more money and underwrote the rapid spread of state-charted banks and other private business corporations in the 1790s. For Federalists, this was one of the major purposes of the federal government. For opponents who wanted a more limited role for industry, however, or who lived on the frontier and lacked access to capital, Hamilton’s system seemed to reinforce class boundaries and give the rich inordinate power over the federal government. Hamilton’s plan, furthermore, had another highly controversial element. In order to pay what it owed on the new bonds, the federal government needed reliable sources of tax revenue. In 1791, Hamilton proposed a federal excise tax on the production, sale, and consumption of a number of goods, including whiskey.   VII. The Whiskey Rebellion and Jay’s Treaty Grain was the most valuable cash crop for many American farmers. In the West, selling grain to a local distillery for alcohol production was typically more profitable than shipping it over the Appalachians to eastern markets. Hamilton’s whiskey tax thus placed a special burden on western farmers. It seemed to divide the young republic in half—geographically between the East and West, economically between merchants and farmers, and culturally between cities and the countryside. In the fall of 1791, sixteen men in western Pennsylvania, disguised in women’s clothes, assaulted a tax collector named Robert Johnson. They tarred and feathered him, and the local deputy marshals seeking justice met similar fates. They were robbed and beaten, whipped and flogged, tarred and feathered, and tied up and left for dead. The rebel farmers also adopted other protest methods from the Revolution and Shays’s Rebellion, writing local petitions and erecting liberty poles. For the next two years, tax collections in the region dwindled. Then, in July 1794, groups of armed farmers attacked federal marshals and tax collectors, burning down at least two tax collectors’ homes. At the end of the month, an armed force of about seven thousand, led by the radical attorney David Bradford, robbed the U.S. mail and gathered about eight miles east of Pittsburgh. President Washington responded quickly. First, Washington dispatched a committee of three distinguished Pennsylvanians to meet with the rebels and try to bring about a peaceful resolution. Meanwhile, he gathered an army of thirteen thousand militiamen in Carlisle, Pennsylvania. On September 19, Washington became the only sitting president to lead troops in the field, though he quickly turned over the army to the command of Henry Lee, a Revolutionary hero and the current governor of Virginia. As the federal army moved westward, the farmers scattered. Hoping to make a dramatic display of federal authority, Alexander Hamilton oversaw the arrest and trial of a number of rebels. Many were released because of a lack of evidence, and most of those who remained, including two men sentenced to death for treason, were soon pardoned by the president. The Whiskey Rebellion had shown that the federal government was capable of quelling internal unrest. But it also demonstrated that some citizens, especially poor westerners, viewed it as their enemy.21 Around the same time, another national issue also aroused fierce protest. Along with his vision of a strong financial system, Hamilton also had a vision of a nation busily engaged in foreign trade. In his mind, that meant pursuing a friendly relationship with one nation in particular: Great Britain. America’s relationship with Britain since the end of the Revolution had been tense, partly because of warfare between the British and French. Their naval war threatened American shipping, and the impressment of men into Britain’s navy terrorized American sailors. American trade could be risky and expensive, and impressment threatened seafaring families. Nevertheless, President Washington was conscious of American weakness and was determined not to take sides. In April 1793, he officially declared that the United States would remain neutral.22 With his blessing, Hamilton’s political ally John Jay, who was currently serving as chief justice of the Supreme Court, sailed to London to negotiate a treaty that would satisfy both Britain and the United States. Jefferson and Madison strongly opposed these negotiations. They mistrusted Britain and saw the treaty as the American state favoring Britain over France. The French had recently overthrown their own monarchy, and Republicans thought the United States should be glad to have the friendship of a new revolutionary state. They also suspected that a treaty with Britain would favor northern merchants and manufacturers over the agricultural South. In November 1794, despite their misgivings, John Jay signed a “treaty of amity, commerce, and navigation” with the British. Jay’s Treaty, as it was commonly called, required Britain to abandon its military positions in the Northwest Territory (especially Fort Detroit, Fort Mackinac, and Fort Niagara) by 1796. Britain also agreed to compensate American merchants for their losses. The United States, in return, agreed to treat Britain as its most prized trade partner, which meant tacitly supporting Britain in its current conflict with France. Unfortunately, Jay had failed to secure an end to impressment.23 For Federalists, this treaty was a significant accomplishment. Jay’s Treaty gave the United States, a relatively weak power, the ability to stay officially neutral in European wars, and it preserved American prosperity by protecting trade. For Jefferson’s Republicans, however, the treaty was proof of Federalist treachery. The Federalists had sided with a monarchy against a republic, and they had submitted to British influence in American affairs without even ending impressment. In Congress, debate over the treaty transformed the Federalists and Republicans from temporary factions into two distinct (though still loosely organized) political parties.   VIII. The French Revolution and the Limits of Liberty The mounting body count of the French Revolution included that of the queen and king, who were beheaded in a public ceremony in early 1793, as depicted in the engraving. While Americans disdained the concept of monarchy, the execution of King Louis XVI was regarded by many Americans as an abomination, an indication of the chaos and savagery reigning in France at the time. Charles Monnet (artist), Antoine-Jean Duclos and Isidore-Stanislas Helman (engravers), Day of 21 January 1793 the death of Louis Capet on the Place de la Révolution, 1794. Wikimedia. In part, the Federalists were turning toward Britain because they feared the most radical forms of democratic thought. In the wake of Shays’s Rebellion, the Whiskey Rebellion, and other internal protests, Federalists sought to preserve social stability. The course of the French Revolution seemed to justify their concerns. In 1789, news had arrived in America that the French had revolted against their king. Most Americans imagined that liberty was spreading from America to Europe, carried there by the returning French heroes who had taken part in the American Revolution. Initially, nearly all Americans had praised the French Revolution. Towns all over the country hosted speeches and parades on July 14 to commemorate the day it began. Women had worn neoclassical dress to honor republican principles, and men had pinned revolutionary cockades to their hats. John Randolph, a Virginia planter, named two of his favorite horses Jacobin and Sans-Culotte after French revolutionary factions.24 In April 1793, a new French ambassador, “Citizen” Edmond-Charles Genêt, arrived in the United States. During his tour of several cities, Americans greeted him with wild enthusiasm. Citizen Genêt encouraged Americans to act against Spain, a British ally, by attacking its colonies of Florida and Louisiana. When President Washington refused, Genêt threatened to appeal to the American people directly. In response, Washington demanded that France recall its diplomat. In the meantime, however, Genêt’s faction had fallen from power in France. Knowing that a return home might cost him his head, he decided to remain in America. Genêt’s intuition was correct. A radical coalition of revolutionaries had seized power in France. They initiated a bloody purge of their enemies, the Reign of Terror. As Americans learned about Genêt’s impropriety and the mounting body count in France, many began to have second thoughts about the French Revolution. Americans who feared that the French Revolution was spiraling out of control tended to become Federalists. Those who remained hopeful about the revolution tended to become Republicans. Not deterred by the violence, Thomas Jefferson declared that he would rather see “half the earth desolated” than see the French Revolution fail. “Were there but an Adam and an Eve left in every country, and left free,” he wrote, “it would be better than as it now is.”25 Meanwhile, the Federalists sought closer ties with Britain. Despite the political rancor, in late 1796 there came one sign of hope: the United States peacefully elected a new president. For now, as Washington stepped down and executive power changed hands, the country did not descend into the anarchy that many leaders feared. The new president was John Adams, Washington’s vice president. Adams was less beloved than the old general, and he governed a deeply divided nation. The foreign crisis also presented him with a major test. In response to Jay’s Treaty, the French government authorized its vessels to attack American shipping. To resolve this, President Adams sent envoys to France in 1797. The French insulted these diplomats. Some officials, whom the Americans code-named X, Y, and Z in their correspondence, hinted that negotiations could begin only after the Americans offered a bribe. When the story became public, this XYZ Affair infuriated American citizens. Dozens of towns wrote addresses to President Adams, pledging him their support against France. Many people seemed eager for war. “Millions for defense,” toasted South Carolina representative Robert Goodloe Harper, “but not one cent for tribute.”26 By 1798, the people of Charleston watched the ocean’s horizon apprehensively because they feared the arrival of the French navy at any moment. Many people now worried that the same ships that had aided Americans during the Revolutionary War might discharge an invasion force on their shores. Some southerners were sure that this force would consist of Black troops from France’s Caribbean colonies, who would attack the southern states and cause their enslaved laborers to revolt. Many Americans also worried that France had covert agents in the country. In the streets of Charleston, armed bands of young men searched for French disorganizers. Even the little children prepared for the looming conflict by fighting with sticks.27 Meanwhile, during the crisis, New Englanders were some of the most outspoken opponents of France. In 1798, they found a new reason for Francophobia. An influential Massachusetts minister, Jedidiah Morse, announced to his congregation that the French Revolution had been hatched in a conspiracy led by a mysterious anti-Christian organization called the Illuminati. The story was a hoax, but rumors of Illuminati infiltration spread throughout New England like wildfire, adding a new dimension to the foreign threat.28 Against this backdrop of fear, the French Quasi-War, as it would come to be known, was fought on the Atlantic, mostly between French naval vessels and American merchant ships. During this crisis, however, anxiety about foreign agents ran high, and members of Congress took action to prevent internal subversion. The most controversial of these steps were the Alien and Sedition Acts. These two laws, passed in 1798, were intended to prevent French agents and sympathizers from compromising America’s resistance, but they also attacked Americans who criticized the president and the Federalist Party. The Alien Act allowed the federal government to deport foreign nationals, or “aliens,” who seemed to pose a national security threat. Even more dramatically, the Sedition Act allowed the government to prosecute anyone found to be speaking or publishing “false, scandalous, and malicious writing” against the government.29 These laws were not simply brought on by war hysteria. They reflected common assumptions about the nature of the American Revolution and the limits of liberty. In fact, most of the advocates for the Constitution and the First Amendment accepted that free speech simply meant a lack of prior censorship or restraint, not a guarantee against punishment. According to this logic, “licentious” or unruly speech made society less free, not more. James Wilson, one of the principal architects of the Constitution, argued that “every author is responsible when he attacks the security or welfare of the government.”30 In 1798, most Federalists were inclined to agree. Under the terms of the Sedition Act, they indicted and prosecuted several Republican printers—and even a Republican congressman who had criticized President Adams. Meanwhile, although the Adams administration never enforced the Alien Act, its passage was enough to convince some foreign nationals to leave the country. For the president and most other Federalists, the Alien and Sedition Acts represented a continuation of a conservative rather than radical American Revolution. However, the Alien and Sedition Acts caused a backlash in two ways. First, shocked opponents articulated a new and expansive vision for liberty. The New York lawyer Tunis Wortman, for example, demanded an “absolute independence” of the press.31 Likewise, the Virginia judge George Hay called for “any publication whatever criminal” to be exempt from legal punishment.32 Many Americans began to argue that free speech meant the ability to say virtually anything without fear of prosecution. Second, James Madison and Thomas Jefferson helped organize opposition from state governments. Ironically, both of them had expressed support for the principle behind the Sedition Act in previous years. Jefferson, for example, had written to Madison in 1789 that the nation should punish citizens for speaking “false facts” that injured the country.33 Nevertheless, both men now opposed the Alien and Sedition Acts on constitutional grounds. In 1798, Jefferson made this point in a resolution adopted by the Kentucky state legislature. A short time later, the Virginia legislature adopted a similar document written by Madison. The Kentucky and Virginia Resolutions argued that the national government’s authority was limited to the powers expressly granted by the U.S. Constitution. More importantly, they asserted that the states could declare federal laws unconstitutional. For the time being, these resolutions were simply gestures of defiance. Their bold claim, however, would have important effects in later decades. In just a few years, many Americans’ feelings toward France had changed dramatically. Far from rejoicing in the “light of freedom,” many Americans now feared the “contagion” of French-style liberty. Debates over the French Revolution in the 1790s gave Americans some of their earliest opportunities to articulate what it meant to be American. Did American national character rest on a radical and universal vision of human liberty? Or was America supposed to be essentially pious and traditional, an outgrowth of Great Britain? They couldn’t agree. It was on this cracked foundation that many conflicts of the nineteenth century would rest.   IX. Religious Freedom One reason the debates over the French Revolution became so heated was that Americans were unsure about their own religious future. The Illuminati scare of 1798 was just one manifestation of this fear. Across the United States, a slow but profound shift in attitudes toward religion and government began. In 1776, none of the American state governments observed the separation of church and state. On the contrary, all thirteen states either had established, official, and tax-supported state churches, or at least required their officeholders to profess a certain faith. Most officials believed this was necessary to protect morality and social order. Over the next six decades, however, that changed. In 1833, the final state, Massachusetts, stopped supporting an official religious denomination. Historians call that gradual process disestablishment. In many states, the process of disestablishment had started before the creation of the Constitution. South Carolina, for example, had been nominally Anglican before the Revolution, but it had dropped denominational restrictions in its 1778 constitution. Instead, it now allowed any church consisting of at least fifteen adult males to become “incorporated,” or recognized for tax purposes as a state-supported church. Churches needed only to agree to a set of basic Christian theological tenets, which were vague enough that most denominations could support them.34 South Carolina tried to balance religious freedom with the religious practice that was supposed to be necessary for social order. Officeholders were still expected to be Christians; their oaths were witnessed by God, they were compelled by their religious beliefs to tell the truth, and they were called to live according to the Bible. This list of minimal requirements came to define acceptable Christianity in many states. As new Christian denominations proliferated between 1780 and 1840, however, more and more Christians fell outside this definition. South Carolina continued its general establishment law until 1790, when a constitutional revision removed the establishment clause and religious restrictions on officeholders. Many other states, though, continued to support an established church well into the nineteenth century. The federal Constitution did not prevent this. The religious freedom clause in the Bill of Rights, during these decades, limited the federal government but not state governments. It was not until 1833 that a state supreme court decision ended Massachusetts’s support for the Congregational Church. Many political leaders, including Thomas Jefferson and James Madison, favored disestablishment because they saw the relationship between church and state as a tool of oppression. Jefferson proposed a Statute for Religious Freedom in the Virginia state assembly in 1779, but his bill failed in the overwhelmingly Anglican legislature. Madison proposed it again in 1785, and it defeated a rival bill that would have given equal revenue to all Protestant churches. Instead Virginia would not use public money to support religion. “The Religion then of every man,” Jefferson wrote, “must be left to the conviction and conscience of every man; and it is the right of every man to exercise it as these may dictate.”35 At the federal level, the delegates to the Constitutional Convention of 1787 easily agreed that the national government should not have an official religion. This principle was upheld in 1791 when the First Amendment was ratified, with its guarantee of religious liberty. The limits of federal disestablishment, however, required discussion. The federal government, for example, supported Native American missionaries and congressional chaplains. Well into the nineteenth century, debate raged over whether the postal service should operate on Sundays, and whether non-Christians could act as witnesses in federal courts. Americans continued to struggle to understand what it meant for Congress not to “establish” a religion.   X. The Election of 1800 The year 1800 brought about a host of changes in government, in particular the first successful and peaceful transfer of power from one political party to another. But the year was important for another reason: the U.S. Capitol in Washington, D.C. (pictured here in 1800) was finally opened to be occupied by Congress, the Supreme Court, the Library of Congress, and the courts of the District of Columbia. William Russell Birch, A view of the Capitol of Washington before it was burnt down by the British, c. 1800. Wikimedia. Meanwhile, the Sedition and Alien Acts expired in 1800 and 1801. They had been relatively ineffective at suppressing dissent. On the contrary, they were much more important for the loud reactions they had inspired. They had helped many Americans decide what they didn’t want from their national government. By 1800, therefore, President Adams had lost the confidence of many Americans. They had let him know it. In 1798, for instance, he had issued a national thanksgiving proclamation. Instead of enjoying a day of celebration and thankfulness, Adams and his family had been forced by rioters to flee the capital city of Philadelphia until the day was over. Conversely, his prickly independence had also put him at odds with Alexander Hamilton, the leader of his own party, who offered him little support. After four years in office, Adams found himself widely reviled. In the election of 1800, therefore, the Republicans defeated Adams in a bitter and complicated presidential race. During the election, one Federalist newspaper article predicted that a Republican victory would fill America with “murder, robbery, rape, adultery, and incest.”36 A Republican newspaper, on the other hand, flung sexual slurs against President Adams, saying he had “neither the force and firmness of a man, nor the gentleness and sensibility of a woman.” Both sides predicted disaster and possibly war if the other should win.37 In the end, the contest came down to a tie between two Republicans, Thomas Jefferson of Virginia and Aaron Burr of New York, who each had seventy-three electoral votes. (Adams had sixty-five.) Burr was supposed to be a candidate for vice president, not president, but under the Constitution’s original rules, a tie-breaking vote had to take place in the House of Representatives. It was controlled by Federalists bitter at Jefferson. House members voted dozens of times without breaking the tie. On the thirty-sixth ballot, Thomas Jefferson emerged victorious. Republicans believed they had saved the United States from grave danger. An assembly of Republicans in New York City called the election a “bloodless revolution.” They thought of their victory as a revolution in part because the Constitution (and eighteenth-century political theory) made no provision for political parties. The Republicans thought they were fighting to rescue the country from an aristocratic takeover, not just taking part in a normal constitutional process. This image attacks Jefferson’s support of the French Revolution and religious freedom. The letter, “To Mazzei,” refers to a 1796 correspondence that criticized the Federalists and, by association, President Washington. Providential Detection, 1797. Courtesy American Antiquarian Society. Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0. In his first inaugural address, however, Thomas Jefferson offered an olive branch to the Federalists. He pledged to follow the will of the American majority, whom he believed were Republicans, but to respect the rights of the Federalist minority. His election set an important precedent. Adams accepted his electoral defeat and left the White House peacefully. “The revolution of 1800,” Jefferson wrote years later, did for American principles what the Revolution of 1776 had done for its structure. But this time, the revolution was accomplished not “by the sword” but “by the rational and peaceable instrument of reform, the suffrage of the people.”38 Four years later, when the Twelfth Amendment changed the rules for presidential elections to prevent future deadlocks, it was designed to accommodate the way political parties worked. Despite Adams’s and Jefferson’s attempts to tame party politics, though, the tension between federal power and the liberties of states and individuals would exist long into the nineteenth century. And while Jefferson’s administration attempted to decrease federal influence, Chief Justice John Marshall, an Adams appointee, worked to increase the authority of the Supreme Court. These competing agendas clashed most famously in the 1803 case of Marbury v. Madison, which Marshall used to establish a major precedent. The Marbury case seemed insignificant at first. The night before leaving office in early 1801, Adams had appointed several men to serve as justices of the peace in Washington, D.C. By making these “midnight appointments,” Adams had sought to put Federalists into vacant positions at the last minute. On taking office, however, Jefferson and his secretary of state, James Madison, had refused to deliver the federal commissions to the men Adams had appointed. Several of the appointees, including William Marbury, sued the government, and the case was argued before the Supreme Court. Marshall used Marbury’s case to make a clever ruling. On the issue of the commissions, the Supreme Court ruled in favor of the Jefferson administration. But Chief Justice Marshall went further in his decision, ruling that the Supreme Court reserved the right to decide whether an act of Congress violated the Constitution. In other words, the court assumed the power of judicial review. This was a major (and lasting) blow to the Republican agenda, especially after 1810, when the Supreme Court extended judicial review to state laws. Jefferson was particularly frustrated by the decision, arguing that the power of judicial review “would make the Judiciary a despotic branch.”39   XI. Conclusion A grand debate over political power engulfed the young United States. The Constitution ensured that there would be a strong federal government capable of taxing, waging war, and making law, but it could never resolve the young nation’s many conflicting constituencies. The Whiskey Rebellion proved that the nation could stifle internal dissent but exposed a new threat to liberty. Hamilton’s banking system provided the nation with credit but also constrained frontier farmers. The Constitution’s guarantee of religious liberty conflicted with many popular prerogatives. Dissension only deepened, and as the 1790s progressed, Americans became bitterly divided over political parties and foreign war. During the ratification debates, Alexander Hamilton had written of the wonders of the Constitution. “A nation, without a national government,” he wrote, would be “an awful spectacle.” But, he added, “the establishment of a Constitution, in time of profound peace, by the voluntary consent of a whole people, is a prodigy,” a miracle that should be witnessed “with trembling anxiety.”40 Anti-Federalists had grave concerns about the Constitution, but even they could celebrate the idea of national unity. By 1795, even the staunchest critics would have grudgingly agreed with Hamilton’s convictions about the Constitution. Yet these same individuals could also take the cautions in Washington’s 1796 farewell address to heart. “There is an opinion,” Washington wrote, “that parties in free countries are useful checks upon the administration of the government and serve to keep alive the spirit of liberty.” This, he conceded, was probably true, but in a republic, he said, the danger was not too little partisanship, but too much. “A fire not to be quenched,” Washington warned, “it demands a uniform vigilance to prevent its bursting into a flame, lest, instead of warming, it should consume.”41 For every parade, thanksgiving proclamation, or grand procession honoring the unity of the nation, there was also some political controversy reminding American citizens of how fragile their union was. And as party differences and regional quarrels tested the federal government, the new nation increasingly explored the limits of its democracy.   XII. Primary Sources 1. Hector St. Jean de Crèvecœur describes the American people, 1782 Hector St. John de Crèvecœur was born in France, but relocated to the colony of New York and married a local woman named Mehitable Tippet. For a period of several years, de Crèvecœur wrote about the people he encountered in North America. The resulting work was widely successful in Europe. In this passage, Crèvecœur attempts to reflect on the difference between life in Europe and life in North America. 2. A Confederation of Native peoples seek peace with the United States, 1786 In 1786, half a year before the Constitutional Convention, a collection of Native American leaders gathered on the banks of the Detroit River to offer a unified message to the Congress of the United States. Despite this proposal, American surveyors, settlers, and others continued to cross the Ohio River. 3. Mary Smith Cranch comments on politics, 1786-87 In the aftermath of the Revolution, politics became a sport consumed by both men and women. In a series of letters sent to her sister, Mary Smith Cranch comments on a series of political events including the lack of support for diplomats, the circulation of paper or hard currency, legal reform, tariffs against imported tea tables, Shays’s rebellion, and the role of women in supporting the nation’s interests. 4. James Madison, Memorial and Remonstrance Against Religious Assessments, 1785 Before the American Revolution, Virginia supported local Anglican churches through taxes. After the American Revolution, Virginia had to decide what to do with this policy. Some founding fathers, including Patrick Henry, wanted to equally distribute tax dollars to all churches. In this document, James Madison explains why he did not want any government money to support religious causes in Virginia. 5. George Washington, “Farewell Address,” 1796 George Washington used his final public address as president to warn against what he understood as the two greatest dangers to American prosperity: political parties and foreign wars. Washington urged the American people to avoid political partisanship and entanglements with European wars.  6. Venture Smith, A Narrative of the Life and Adventures of Venture Smith, 1798 Venture Smith’s autobiography is one of the earliest slave narratives to circulate in the Atlantic World. Slave narratives grew into the most important genre of antislavery literature and bore testimony to the injustices of the slave system. Smith was unusually lucky in that he was able to purchase his freedom, but his story nonetheless reveals the hardships faced by even the most fortunate enslaved men and women. 7. Susannah Rowson, Charlotte Temple, 1794 In Charlotte Temple, the first novel written in America, Susannah Rowson offered a cautionary tale of a woman deceived and then abandoned by a roguish man. Americans throughout the new nation read the book with rapt attention and many even traveled to New York City to visit the supposed grave of this fictional character. 8. Constitutional ratification cartoon, 1789 The Massachusetts Centinel ran a series of cartoons depicting the ratification of the Constitution.  Each vertical pillar represents a state that has ratified the new government.  In this cartoon, North Carolina’s pillar is being guided into place (it would vote for ratification in November 1789).  Rhode Island’s pillar, however, is crumbling and shows the uncertainty of the vote there.    9. Anti-Thomas Jefferson Cartoon, 1797 This image attacks Jefferson’s support of the French Revolution and religious freedom.  The Altar to “Gallic Despotism” mocks Jefferson’s allegiance to the French. The letter, “To Mazzei,” refers to a 1796 correspondence that criticized the Federalists and, by association, President Washington.    XIII. Reference Material This chapter was edited by Tara Strauch, with content contributions by Marco Basile, Nathaniel C. Green, Brenden Kennedy, Spencer McBride, Andrea Nero, Cara Rogers, Tara Strauch, Michael Harrison Taylor, Jordan Taylor, Kevin Wisniewski, and Ben Wright. Recommended citation: Marco Basile et al., “A New Nation,” Tara Strauch, ed., in The American Yawp, eds. Joseph Locke and Ben Wright (Stanford, CA: Stanford University Press, 2018).   Recommended Reading Allgor, Catherine. Parlor Politics: In which the Ladies of Washington Help Build a City and a Government. Charlottesville: University of Virginia Press, 2000. Appleby, Joyce. Inheriting the Revolution: The First Generation of Americans. Cambridge, Mass.: Belknap Press, 2001. Bartolini-Tuazon, Kathleen. For Fear of an Elective King: George Washington and the Presidential Title Controversy of 1789. Ithaca: Cornell University Press, 2014. Beeman, Richard, Stephen Botein, and Edward C. Carter II eds. Beyond Confederation: Origins of the Constitution and American National Identity. Chapel Hill, N.C.: University of North Carolina Press, 1987. Bilder, Mary Sarah. Madison’s Hand: Revising the Constitutional Convention. Cambridge: Harvard University Press, 2015. Bouton, Terry. “A Road Closed: Rural Insurgency in Post-Independence Pennsylvania,” Journal of American History 87:3 (December 2000): 855-887. Cunningham, Noble E. The Jeffersonian Republicans: The Formation of Party Organization, 1789-1801. Chapel Hill, N.C.: University of North Carolina Press, 1967. Dunn, Susan. Jefferson’s Second Revolution: The Election of 1800 and the Triumph of Republicanism. Boston: Houghton Mifflin, 2004. Edling, Max. A Revolution in Favor of Government: Origins of the U.S. Constitution and the Making of the American State. New York: Oxford University Press, 2003 Gordon-Reed, Annette. The Hemingses of Monticello: An American Family. New York: W. W. Norton, 2008. Halperin, Terri Diane. The Alien and Sedition Acts of 1798: Testing the Constitution. Baltimore: Johns Hopkins University Press, 2016. Holton, Woody. Unruly Americans and the Origins of the Constitution. 1st edition. New York: Hill and Wang, 2007. Kierner, Cynthia A. Martha Jefferson Randolph, Daughter of Monticello: Her Life and Times. Chapel Hill: University of North Carolina Press, 2012. Maier, Pauline. Ratification: The People Debate the Constitution, 1787-1788. New York: Simon & Schuster, 2010. Papenfuse, Eric Robert. “Unleashing the ‘Wildness’: The Mobilization of Grassroots Antifederalism in Maryland,” Journal of the Early Republic 16:1 (Spring 1996): 73-106. Pasley, Jeffrey L. The First Presidential Contest: 1796 and the Founding of American Democracy. Lawrence: The University of Kansas Press, 2013. Smith-Rosenberg, Carroll. “Dis-Covering the Subject of the ‘Great Constitutional Discussion,’ 1786-1789,” Journal of American History 79:3 (December 1992): 841-873 Taylor, Alan. William Cooper’s Town: Power and Persuasion on the Frontier of the Early American Republic. Reprint edition. New York: Vintage, 1996. Rakove, Jack N. Original Meanings: Politics and Ideas in the Making of the Constitution. New York: Vintage Books, 1996. Salmon, Marylynn. Women and the Law of Property in Early America. Chapel Hill, N.C.: University of North Carolina Press, 1989. Sharp, James Roger. American Politics in the Early Republic: The New Nation in Crisis. New Haven: Yale University Press, 1993. Slaughter, Thomas P. The Whiskey Rebellion: Frontier Epilogue to the American Revolution. New York: Oxford University Press, 1988. Waldstreicher, David. In the Midst of Perpetual Fetes : The Making of American Nationalism, 1776-1820. Chapel Hill : Williamsburg, Virginia, by the University of North Carolina Press, 1997. Wood, Gordon. Empire of Liberty: A History of the Early Republic, 1789-1815. Oxford: Oxford University Press, 2011. Zagarri, Rosemarie. Revolutionary Backlash: Women and Politics in the Early American Republic. Philadelphia: University of Pennsylvania Press, 2007. Allgor, Catherine. Parlor Politics: In Which the Ladies of Washington Help Build a City and a Government. Charlottesville: University of Virginia Press, 2000. Appleby, Joyce. Inheriting the Revolution: The First Generation of Americans. Cambridge, MA: Belknap Press, 2001. Bartolini-Tuazon, Kathleen. For Fear of an Elective King: George Washington and the Presidential Title Controversy of 1789. Ithaca, NY: Cornell University Press, 2014. Beeman, Richard, Stephen Botein, and Edward C. Carter II, eds. Beyond Confederation: Origins of the Constitution and American National Identity. Chapel Hill: University of North Carolina Press, 1987. Bilder, Mary Sarah. Madison’s Hand: Revising the Constitutional Convention. Cambridge, MA: Harvard University Press, 2015. Bouton, Terry. “A Road Closed: Rural Insurgency in Post-Independence Pennsylvania.” Journal of American History 87, no. 3 (December 2000): 855–887. Cunningham, Noble E. The Jeffersonian Republicans: The Formation of Party Organization, 1789–1801. Chapel Hill: University of North Carolina Press, 1967. Dunn, Susan. Jefferson’s Second Revolution: The Election of 1800 and the Triumph of Republicanism. Boston: Houghton Mifflin, 2004. Edling, Max. A Revolution in Favor of Government: Origins of the U.S. Constitution and the Making of the American State. New York: Oxford University Press, 2003. Gordon-Reed, Annette. The Hemingses of Monticello: An American Family. New York: Norton, 2008. Halperin, Terri Diane. The Alien and Sedition Acts of 1798: Testing the Constitution. Baltimore: Johns Hopkins University Press, 2016. Holton, Woody. Unruly Americans and the Origins of the Constitution. New York: Hill and Wang, 2007. Kierner, Cynthia A. Martha Jefferson Randolph, Daughter of Monticello: Her Life and Times. Chapel Hill: University of North Carolina Press, 2012. Maier, Pauline. Ratification: The People Debate the Constitution, 1787–1788. New York: Simon and Schuster, 2010. Papenfuse, Eric Robert. “Unleashing the ‘Wildness’: The Mobilization of Grassroots Antifederalism in Maryland.” Journal of the Early Republic 16, no. 1 (Spring 1996): 73–106. Pasley, Jeffrey L. The First Presidential Contest: 1796 and the Founding of American Democracy. Lawrence: University of Kansas Press, 2013. Rakove, Jack N. Original Meanings: Politics and Ideas in the Making of the Constitution. New York: Vintage Books, 1996. Salmon, Marylynn. Women and the Law of Property in Early America. Chapel Hill: University of North Carolina Press, 1989. Sharp, James Roger. American Politics in the Early Republic: The New Nation in Crisis. New Haven, CT: Yale University Press, 1993. Slaughter, Thomas P. The Whiskey Rebellion: Frontier Epilogue to the American Revolution. New York: Oxford University Press, 1986. Smith-Rosenberg, Carroll. “Dis-Covering the Subject of the ‘Great Constitutional Discussion,’ 1786–1789.” Journal of American History 79, no. 3 (December 1992): 841–873. Taylor, Alan. William Cooper’s Town: Power and Persuasion on the Frontier of the Early American Republic. New York: Vintage, 1996. Waldstreicher, David. In the Midst of Perpetual Fetes : The Making of American Nationalism, 1776–1820. Chapel Hill : University of North Carolina Press, 1997. Wood, Gordon. Empire of Liberty: A History of the Early Republic, 1789–1815. Oxford, UK: Oxford University Press, 2011. Zagarri, Rosemarie. Revolutionary Backlash: Women and Politics in the Early American Republic. Philadelphia: University of Pennsylvania Press, 2007   Notes Francis Hopkinson, An Account of the Grand Federal Procession, Philadelphia, July 4, 1788 (Philadelphia: Carey, 1788). []George Washington, Thanksgiving Proclamation, October, 3, 1789; Fed. Reg., Presidential Proclamations, 1791–1991. []Hampshire Gazette (CT), September 13, 1786. []James Madison, The Federalist Papers, (New York: Signet Classics, 2003), no. 63. []Woody Holton, Unruly Americans and the Origins of the Constitution (New York: Hill and Wang, 2007), 8–9. []Madison took an active role during the convention. He also did more than anyone else to shape historians’ understandings of the convention by taking meticulous notes. Many of the quotes included here come from Madison’s notes. To learn more about this important document, read Mary Sarah Bilder, Madison’s Hand: Revising the Constitutional Convention (Cambridge, MA: Harvard University Press, 2015). []Virginia (Randolph) Plan as Amended (National Archives Microfilm Publication M866, 1 roll); The Official Records of the Constitutional Convention; Records of the Continental and Confederation Congresses and the Constitutional Convention, 1774–1789, Record Group 360; National Archives. []Richard Beeman, Plain, Honest Men: The Making of the American Constitution (New York: Random House, 2009), 114. []Herbert J. Storing, What the Anti-Federalists Were For: The Political Thought of the Opponents of the Constitution (Chicago: University of Chicago Press, 1981), 16. []Ray Raphael, Mr. President: How and Why the Founders Created a Chief Executive (New York: Knopf, 2012), 50. See also Kathleen Bartoloni-Tuazon, For Fear of an Elected King: George Washington and the Presidential Title Controversy of 1789 (Ithaca, NY: Cornell University Press, 2014). []David J. Siemers, Ratifying the Republic: Antifederalists and Federalists in Constitutional Time (Stanford, CA: Stanford University Press, 2002). []Alexander Hamilton, James Madison, and John Jay, The Federalist Papers, ed. Ian Shapiro (New Haven, CT: Yale University Press, 2009). []Pauline Maier, Ratification: The People Debate the Constitution, 1787–1788 (New York: Simon and Schuster, 2010), 225–237. []David Waldstreicher, Slavery’s Constitution: From Revolution to Ratification (New York: Hill and Wang, 2009). []Carson Holloway, Hamilton Versus Jefferson in the Washington Administration: Completing the Founding or Betraying the Founding? (New York: Cambridge University Press, 2015). []Alexander Hamilton, The Works of Alexander Hamilton, Volume 1, ed. Henry Cabot Lodge, ed. (New York: Putnam, 1904), 70, 408. []Alexander Hamilton, Report on Manufactures (New York: Childs and Swaine, 1791). []James H. Hutson, ed., Supplement to Max Farrand’s the Records of the Federal Convention of 1787 (New Haven, CT: Yale University Press, 1987), 119. []Hamilton, Report on Manufactures). []Richard Sylla, “National Foundations: Public Credit, the National Bank, and Securities Markets,” in Founding Choices: American Economic Policy in the 1790s, ed. Douglas A. Irwin and Richard Sylla (Chicago: University of Chicago Press, 2011), 68. []Thomas P. Slaughter, The Whiskey Rebellion: Frontier Epilogue to the American Revolution (New York: Oxford University Press, 1986). []“Proclamation of Neutrality, 1793,” in A Compilation of the Messages and Papers of the Presidents Prepared Under the Direction of the Joint Committee on printing, of the House and Senate Pursuant to an Act of the Fifty-Second Congress of the United States (New York: Bureau of National Literature, 1897). []United States, Treaty of Amity, Commerce, and Navigation, signed at London November 19, 1794, Submitted to the Senate June 8, Resolution of Advice and Consent, on condition, June 24, 1795. Ratified by the United States August 14, 1795. Ratified by Great Britain October 28, 1795. Ratifications exchanged at London October 28, 1795. Proclaimed February 29, 1796. []Elizabeth Fox-Genovese and Eugene D. Genovese, The Mind of the Master Class: History and Faith in the Southern Slaveholders Worldview (New York: Cambridge University Press, 2005), 18. []From Thomas Jefferson to William Short, 3 January 1793,” Founders Online, National Archives. http://founders.archives.gov/documents/Jefferson/01-25-02-0016, last modified June 29, 2015; The Papers of Thomas Jefferson, vol. 25, 1 January–10 May 1793, ed. John Catanzariti (Princeton, NJ: Princeton University Press, 1992), 14–17. []Robert Goodloe Harper, June 18, 1798, quoted in American Daily Advertiser (Philadelphia), June 20, 1798. []Robert J. Alderson Jr., This Bright Era of Happy Revolutions: French Consul Michel-Ange-Bernard Mangourit and International Republicanism in Charleston, 1792–1794 (Columbia: University of South Carolina Press, 2008). []Rachel Hope Cleves, The Reign of Terror in America: Visions of Violence from Anti-Jacobinism to Antislavery (New York: Cambridge University Press, 2012), 47. []Alien Act, July 6, 1798, and An Act in Addition to the Act, Entitled “An Act for the Punishment of Certain Crimes Against the United States,” July 14, 1798; Fifth Congress; Enrolled Acts and Resolutions; General Records of the United States Government; Record Group 11; National Archives. []James Wilson, Congressional Debate, December 1, 1787, in Jonathan Elliot, ed., The Debates in the Several State Conventions on the Adoption of the Federal Constitution as Recommended by the General Convention at Philadelphia in 1787, Vol. 2 (New York: s.n., 1888) 448–450. []Tunis Wortman, A Treatise Concerning Political Enquiry, and the Liberty of the Press (New York: Forman, 1800), 181. []George Hay, An Essay on the Liberty of the Press (Philadelphia: s.n., 1799), 43. []Thomas Jefferson to James Madison, August 28, 1789, from The Works of Thomas Jefferson in Twelve Volumes, Federal Edition, ed. Paul Leicester Ford. http://www.loc.gov/resource/mtj1.011_0853_0861 []Francis Newton Thorpe, ed., The Federal and State Constitutions, Colonial Charters, and Other Organic Laws of the States, Territories, and Colonies Now or Heretofore Forming the United States of America Compiled and Edited Under the Act of Congress of June 30, 1906 (Washington, DC: U.S. Government Printing Office, 1909). []Thomas Jefferson, An Act for Establishing Religious Freedom, 16 January 1786, Manuscript, Records of the General Assembly, Enrolled Bills, Record Group 78, Library of Virginia. []Catherine Allgor, Parlor Politics: In Which the Ladies of Washington Help Build a City and a Government (Charlottesville: University of Virginia Press, 2000), 14. []James T. Callender, The Prospect Before Us (Richmond: s.n., 1800). []Letter from Thomas Jefferson to Spencer Roane, September 6, 1819, in The Writings of Thomas Jefferson, 20 vols., ed. Albert Ellery Bergh (Washington, DC: Thomas Jefferson Memorial Association of the United States, 1903), 142. []Harold H. Bruff, Untrodden Ground: How Presidents Interpret the Constitution (Chicago: University of Chicago Press, 2015), 65. []Alexander Hamilton, The Federalist Papers (New York: Signet Classics, 2003), no. 85. []George Washington, Farewell Address, Annals of Congress, 4th Congress, 2869–2870. [] This entry was posted in Uncategorized on June 7, 2013 by All Chapters. Post navigation ← 5. The American Revolution 7. The Early Republic →

      The discussion of Shays’s Rebellion reveals how economic struggles and weak national power under the Articles of Confederation created serious unrest among farmers. While some leaders viewed the rebellion as a dangerous threat to order, others believed it represented the same revolutionary spirit that founded the country.

    1. Puedes publicar lo que quieras, en el formato que quieras, sin que nadie te monitoree. Adicionalmente, compartes enlaces permanentes, que siempre funcionarán, bien sean simples y legibles en tu propio dominio (como ejemplo.com/ideas) o cipherlinks, que funcionan incluso si no tienes un dominio, este cambia o está caído/inaccesible.

      Este sitio web es muy similar Hypothesis, es una herramienta de anotación colaborativa que permite que la lectura sea activa, visible y social; sin que tenga ninguna restricción, que permite tener el control y responsabilidad de lo que publica.

    2. Brea es un generador y gestor de sitios web enfocado en la personalización interactiva y la autonomía,

      Cuando se menciona algún generador de sitios web automáticamente pienso en plataformas como Wix donde se puede hacer una gestión de contenidos de manera sencilla aunque dependa de una suscripción, por lo que una propuesta de este tipo combinando las dinámicas de generación de sitios web y un CMS me parece una forma más libre de interactuar con estas páginas al mencionar a tecnologías como Fossil ya que es una alternativa a Git.

      Al tener más en detalle que es un CMS y un Generador de Sitios Web Estáticos siendo buenos ejemplos wikis o plataformas que usan Markdown es muy interesante ese puente entre dos plataformas que se conforman a partir de la interacción entre usuarios y la colaboración entre ellos dando a relucir el trabajo colaborativo de una manera más fácil y cercana

    3. Brea es un generador y gestor de sitios web enfocado en la personalización interactiva y la autonomía, que permite publicar información integrada desde distintas fuentes, con presentaciones a la medida. Está a medio camino entre un generador de sitios web estáticos y un Sistema Gestor Contenidos (o CMS) desacoplado, debido a la combinación de tecnologías como Fossil y Pharo, que permiten una eficiente gestión, replicación y publicación de archivos estáticos y un entorno de programación en vivo (live coding), para extender y manipular las fuentes de datos, sus presentaciones e interfaces.

      nos muestra con claridad la identidad "híbrida" de Brea, ya que nos dice cómo se posiciona a Brea no solo como una herramienta de publicación al estar entre un sitio estático y un CMS, resuelve el gran dilema de las webs actuales.

      Flexibilidad : Al integrar Fossil, rompe la normatividad de los sistemas cerrados, el uso de "live coding" permite que el usuario no solo llene "cajitas de texto"

    4. Tus datos y contenido son tuyos Cuando publicas algo en la web, debería pertenecerte a ti, no a una empresa. Demasiadas compañías han cerrado y perdido todos los datos de sus usuarios. Otras tienen algoritmos opacos que mercantilizan tu privacidad y condicionan tus hábitos, bajo lógicas extractivistas. Uniéndote a la IndieWeb, tu contenido continúa siendo tuyo y estando bajo tu control.

      Lo que quiero resaltar del texto es lo importante de ser dueño de nuestro dominio y nuestros datos, de esta manera garantizamos que nuestra memoria y nuestra creatividad no dependan del permiso de una corporación.

    5. Está a medio camino entre un generador de sitios web estáticos y un Sistema Gestor Contenidos (o CMS) desacoplado, debido a la combinación de tecnologías como Fossil y Pharo,

      Considero es una mezcla intermedia entre un lenguaje HTML/JS y un CMS lo cual lo hace un poco mas intuitivo mezclando las mejores partes de cada lado

    6. Cuando publicas algo en la web, debería pertenecerte a ti, no a una empresa.

      Me parece muy adecuado el hecho de que en verdad nos pertenece lo que hagamos, pues en paginas como wix, por más que uno pague por el tu dominio y tal, todo esta almacenado en los servidores de ellos, si ellos cierran todo se va, mientras que aca se especifica que me pertenece ese aspecto

    7. permite publicar información integrada desde distintas fuentes

      Este apartado, puede relacionarse con los procesos pertenecientes a la Ciencia de la Información, ya que para la integración de información de diferentes fuentes, es necesario hacer la selección, normalización y organización de la información, lo que favorece a la experiencia del usuario final en IndieWeb.

    8. Cuando publicas algo en la web, debería pertenecerte a ti, no a una empresa.

      Esta idea en particular es la misma que pudimos observar en el video de Hypothesis, una web libre en donde nuestras acciones no están restringidas por las normativas o reglas de una empresa, y que hemos visto en clase, mediante las alternativas al monopolio de los gigantes tecnológicos como Google y su navegador

    9. uando publicas algo en la web, debería pertenecerte a ti, no a una empresa. Demasiadas compañías han cerrado y perdido todos los datos de sus usuarios. Otras tienen algoritmos opacos que mercantilizan tu privacidad y condicionan tus hábitos, bajo lógicas extractivistas. Uniéndote a la IndieWeb, tu contenido continúa siendo tuyo y estando bajo tu control.

      Esto es algo valioso, ya que en esta era de la información desbordada, cualquier cosa que hagamos en la red queda guardada, llega un punto donde en los navegadores que hemos usado aparecen productos o servicios que alguna vez buscamos, por eso ahora el chiste es que "Google lee nuestras mentes", pero en realidad es que vigilan todo lo que hacemos y somos un dato monetizable, por consiguiente me parece excelente una web que sea diferente como lo propone esta página.

    1. Gyuri Lajos Oct 8, 2021The best moat comes from persistence and long term dedication. Engage with something wich is important for the world for which the world i snot ready yet, but you love to do and engages with your whole being. Subsidize "your habit", your "side hustle" with something adjacent, as Frank Herbert would advise you. When the time comes, you will be ready, and it cannot easily be re-engineered. There is no short term fix for levelling up what Long Term Attention can create.It is like the joke, do all that with your lawn, but keep doing it for hundreds of years.

      The best moat

    1. Los fármacos constituyen la piedra angular de la terapéutica moderna; aunque, médicos y legos reconocen que los resultados de la farmacoterapia varían sobremanera con cada persona. Esta variabilidad se ha percibido como un aspecto impredecible y, por lo tanto, inevitable de la farmacoterapia, pero en realidad no es así.

      Importante Para el examen

    1. 2025).

      la cita al Balance Nacional de energía debe ser del 2023, es una mezcla, una cosa es la consulta, otra cosa el año del reporte y otra el año en que se publica la noticia. Debe haber una manera adecuada de poner la cita

    1. De la même manière, la ville peut devenir récit de soi, se mettre en scène comme un jeu, s’éloigner de la simple représentation des choses, des idéologies ou des psychologies sociales.

      Peut-être est ce du à une mauvaise compréhension de ma part, mais je ne parviens pas à saisir à "qui" renvoie ce "soi"

      Une personne ? La ville elle-même devenue quasi personnage ?

    2. le travail de mémoire et le travail du deuil peuvent accompagner la nouveauté

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

    3. La ville, tout comme la maison, nous protège, même sans murs visibles

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

    4. la ville est partagée avec d’autre

      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.

    5. Alors, nous pouvons revenir à la relation initiale entre la ville imaginée — celle des attentes et des désirs — qui restera toujours présente, et peut-être même plus puissante que la ville vécue, bien qu’elle influe à son tour sur ces images.

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

    6. mais cela, sans doute, est une autre histoire

      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 ?

    7. Dans la lecture, le texte déploie tout son potentiel : il éclaire la vie du lecteur, il révèle ce qui restait caché ou encore inquestionné

      Est ce que vous sous entendez qu'il en est de même pour l'architecture ? La proximité que vous défendez au troisième stade du récit pourrait être explicitée.

    8. préfiguration, configuration, refiguration

      J'invite les auteurs à découvrir (ou redécouvrir) les travaux de Jacques Fontanilles sur "les plans d'immanence".

      FONTANILLE, , J. (2015). Formes de vie. In Formes de vie. Presses universitaires de Liège. FONTANILLE, J. (2008). Pratique sémiotique, Presses Universitaires de France. FONTANILLE, J. (2006). Textes, objets, situations et formes de vie. Les niveaux de pertinence du plan de l’expression dans une sémiotique des cultures. In J. Alonso Aldama, D. Bertrand, M. Costantini, & S. Dambrine (éds.), La Transversalité du sens (1 ). Presses universitaires de Vincennes. https://doi.org/10.4000/books.puv.5828 FONTANILLE, J., & ZINNA, A. (2005). Les objets au quotidien. Presses Univ. Limoges.

    9. ma recherche doctorale.

      Il serait utile de mentionner les questionnements et/ou hypothèses de ce travail de thèse, cela pourrait aider à cerner le "pourquoi ces questions sont revenus avec force"

    10. ville, identité, Paris, mémoire, Récit, espace urbain identity, Paris, city, memory, story, narrative, Architecture

      Les mots-clefs ne sont pas les mêmes en Français et en Anglais, pourquoi ?

      Cela sème un trouble déjà bien présent à la double lecture du titre et du résumé : le sujet porte-t-il sur la ville ? sur l'architecture ? la hiérarchisation et le chainage ne permet de trancher . Peut-être faudrait-il clarifier ce point et mettre titre, résumé, mots-clefs et texte au diapason l'un de l'autre !

    11. lieux

      Le lieu est "là où quelque chose se passe" pour reprendre la belle définition d'Augustin Berque (dictionnaire de la géographie et de l'espace des société)

    12. Bibliographie

      C'est surprenant de ne pas trouver un seul ouvrage de Paul Ricoeur dans la bibliographie alors que la réflexion s'appuie en grande partie sur lui.

      Je me permets aussi deux propositions complémentaires de lecture (voir aussi les autres annotations au fil du texte) : Régine Robin "mégapolis ou les derniers pas du flâneur "; Jean-Christophe Bailly "la phrase urbaine",

    1. Dossier de Synthèse : La Psychologie de l'Engagement

      Résumé Exécutif

      Ce document synthétise les concepts clés de la psychologie de l'engagement, tels que présentés par le professeur Fabien Girandola.

      La thèse centrale est que la persuasion traditionnelle, basée sur l'information et l'argumentation, est largement inefficace pour modifier durablement les comportements.

      En opposition, la théorie de l'engagement propose une approche contre-intuitive mais puissante :

      • amener les individus à réaliser un premier acte, peu coûteux et en situation de libre choix, pour les lier à cet acte et
      • les inciter à adopter des comportements plus significatifs par la suite.

      Des techniques comme le "pied-dans-la-porte" et "l'étiquetage", validées par des décennies de recherche expérimentale, démontrent qu'il est possible d'influencer les actions en structurant la situation plutôt qu'en tentant de convaincre les esprits.

      Un effet psychologique majeur de ces techniques est la "naturalisation" : les individus attribuent leur nouveau comportement à leur propre nature ("je suis altruiste") sans avoir conscience de la manipulation situationnelle qui en est la véritable cause.

      La maîtrise de ces techniques soulève des questions éthiques fondamentales, naviguant entre l'influence et la manipulation.

      1. L'Inefficacité de la Persuasion : Le Fossé entre Opinion et Comportement

      La démarche classique pour changer les comportements repose sur la persuasion : l'idée qu'en fournissant des informations et des arguments convaincants, on peut modifier les opinions des individus, ce qui entraînera une modification de leurs actions.

      1.1. Le Postulat de la Persuasion

      L'approche persuasive suppose une chaîne causale directe :

      1. Information : Présenter des faits (ex: "Le tabac tue").

      2. Conviction : L'individu intègre l'information et modifie son opinion.

      3. Action : L'individu ajuste son comportement pour qu'il soit cohérent avec sa nouvelle opinion.

      1.2. La Démonstration de l'Échec

      Des décennies de recherche en psychologie sociale, depuis les années 1960, montrent que ce lien est faible, voire inexistant.

      Savoir quelque chose ne garantit pas de se conformer à cette connaissance.

      Exemples courants :

      ◦ Les fumeurs savent que le tabac est nocif mais continuent de fumer.   

      ◦ La majorité des gens s'accordent sur l'importance de l'écologie mais n'adoptent que peu de comportements pro-environnementaux.

      L'Expérimentation de Bigman (1972) : Cette étude princeps illustre parfaitement le décalage entre l'opinion déclarée et le comportement réel.

      Phase de l'Expérience

      Résultat

      Sondage d'opinion

      95 % des passants déclarent qu'il est important de garder les rues propres.

      Mise en situation

      Confrontés à un papier à ramasser dans la rue, seulement 2 % des mêmes personnes effectuent le geste.

      Cette expérience fondatrice démontre que l'adhésion à une idée (la propreté) ne se traduit pas automatiquement en action.

      2. La Théorie de l'Engagement : Agir d'Abord, Penser Ensuite

      Face aux limites de la persuasion, la théorie de l'engagement, développée notamment par des chercheurs comme Kiesler, Jean-Léon Beauvois et Robert-Vincent Joule, propose de renverser la logique.

      Au lieu de viser les opinions pour changer les actes, elle vise les actes pour, par la suite, influencer les opinions et les comportements futurs.

      2.1. Définition et Principes

      Définition (Kiesler, 1971) : L'engagement est "le lien qui unit l'individu à son acte".

      Principe fondamental : Ce n'est pas l'individu qui s'engage de lui-même, mais la situation qui l'engage.

      L'objectif est d'amener une personne à réaliser de petits actes progressifs qui l'entraîneront vers des comportements plus coûteux qu'elle n'aurait pas réalisés spontanément.

      2.2. Les Facteurs Clés de l'Engagement

      Pour qu'une situation soit engageante, plusieurs facteurs doivent être réunis.

      Facteur

      Description

      Exemple

      Le Sentiment de Liberté

      C'est le facteur le plus crucial. L'individu doit avoir l'impression qu'il a choisi librement de réaliser l'acte.

      Les formules comme "Vous êtes libre d'accepter ou de refuser" ou "Faites comme vous voulez" augmentent considérablement le taux d'acceptation, car elles créent un sentiment de liberté, même si celui-ci est contextuellement contraint.

      Demander de signer une pétition en ajoutant "mais vous êtes libre de refuser" fait passer le taux d'acceptation de 15 % à 45 %.

      Le Caractère Public

      Un acte réalisé publiquement (signer une pétition, prendre la parole) est plus engageant qu'un acte privé.

      Le nom et la signature laissés lient l'individu à son action.

      Signer une pétition avec son nom complet.

      La Répétition de l'Acte

      Répéter un comportement renforce le lien d'engagement.

      Après avoir prêté un objet plusieurs fois, il devient difficile de refuser.

      Prêter un outil à un voisin chaque semaine.

      Le Coût de l'Acte

      Un acte qui demande un effort ou un sacrifice (en temps, en argent, en énergie) est plus engageant.

      Prêter sa voiture est plus engageant que de prêter un stylo.

      L'Étiquetage (Imputation Interne)

      Attribuer une qualité à une personne ("Je sais que vous êtes serviable") l'engage à se comporter conformément à cette étiquette.

      L'acte semble alors "naturel" pour l'individu.

      Dire à quelqu'un "Vous êtes vraiment quelqu'un de bien".

      Note importante : L'engagement ne fonctionne pas en présence de récompenses ou de punitions.

      Si une personne est payée ou menacée pour faire quelque chose, l'acte n'est pas attribué à une décision interne mais à la contrainte externe.

      Il n'y a donc pas d'engagement psychologique.

      3. Les Techniques de Soumission Librement Consentie

      Ces principes théoriques ont été déclinés en techniques d'induction comportementale concrètes, regroupées sous le nom paradoxal de "soumission librement consentie" :

      l'individu se soumet à une demande tout en ayant le sentiment d'avoir agi librement.

      3.1. Le Pied-dans-la-Porte : Demander Peu pour Obtenir Plus

      C'est la technique la plus connue.

      Elle consiste à faire accepter une première requête très peu coûteuse (l'acte préparatoire) pour augmenter significativement les chances que la personne accepte une seconde requête, beaucoup plus coûteuse (le comportement visé).

      Expérimentation de Freedman & Fraser (1966) - Scénario 1 : L'enquête à domicile

      Condition Expérimentale

      Requête

      Taux d'Acceptation

      Contrôle

      Demande directe : Accepter la visite de 2-3h d'une équipe d'enquêteurs pour fouiller la maison.

      22 %

      Pied-dans-la-porte

      1. Acte préparatoire : Répondre à un court questionnaire téléphonique (accepté par tous).<br>

      2. Requête finale (3 jours plus tard) : Accepter la visite de l'équipe d'enquêteurs.

      53 %

      Expérimentation de Freedman & Fraser (1966) - Scénario 2 : Le panneau dans le jardin

      Condition Expérimentale

      Requête

      Taux d'Acceptation

      Contrôle

      Demande directe : Planter un grand panneau de 4x4m pour la sécurité routière dans son jardin.

      17 %

      Pied-dans-la-porte

      1. Acte préparatoire : Apposer un petit autocollant pour la prévention routière sur sa vitre (accepté par tous).<br>

      2. Requête finale (3 jours plus tard) : Accepter de planter le grand panneau.

      76 %

      3.2. L'Étiquetage et le Pied-dans-la-Porte Implicite

      Cette approche combine l'acte préparatoire avec une valorisation de la personne, l'incitant à réaliser d'elle-même un comportement coûteux, sans qu'on le lui demande explicitement.

      Expérimentation de Joule et al. (2002) - Le billet perdu à Aix-en-Provence

      Le comportement visé est l'altruisme : rendre un billet de 10 € tombé de la poche d'un complice.

      L'acte préparatoire consiste à renseigner un "touriste" (un autre complice) sur un plan.

      La variable clé est la manière dont le touriste remercie la personne.

      Condition

      Réponse du "Touriste" après avoir été aidé

      Taux de Restitution du Billet

      Contrôle

      Pas d'interaction préalable avec le touriste.

      30 %

      Pied-dans-la-porte (Remerciement simple)

      "Merci."

      43 %

      Pied-dans-la-porte (Service)

      "Vous m'avez rendu un grand service."

      48 %

      Pied-dans-la-porte + Étiquetage 1

      "Vous êtes serviable."

      70 %

      Pied-dans-la-porte + Étiquetage 2

      "Vous êtes vraiment quelqu'un de bien."

      78 %

      Cette expérience démontre que l'on peut faire varier le taux d'altruisme de 30 % à 78 % uniquement en modifiant une interaction anodine quelques minutes auparavant.

      4. Conséquences Psychologiques et Éthiques

      4.1. La Naturalisation du Comportement

      L'effet le plus remarquable de l'engagement est que les individus n'ont pas conscience d'avoir été influencés. Interrogés sur les raisons de leur acte (ex: rendre le billet), ils répondent systématiquement :

      "C'est normal, je suis quelqu'un d'altruiste/généreux".

      Signification vs. Détermination :

      Signification : L'explication que l'individu donne à son comportement (interne, liée à sa personnalité).  

      Détermination : La cause réelle du comportement (externe, liée à la situation créée par l'expérimentateur).

      Les individus n'ont pas accès à la véritable détermination de leurs actes et la remplacent par une signification qui valorise leur "moi".

      4.2. La Frontière avec la Manipulation

      Le professeur Girandola insiste sur le fait que ces techniques sont puissantes et naviguent à la frontière de la manipulation.

      Leur connaissance est essentielle non seulement pour les utiliser à bon escient (santé publique, éducation) mais aussi pour s'en prémunir.

      Il rappelle que l'usage de ces techniques par les psychologues est encadré par un code de déontologie strict : "il n'y a pas d'action sans éthique".

      5. Lectures et Ressources Recommandées

      Pour approfondir le sujet, plusieurs ouvrages et articles ont été mentionnés :

      Ouvrages de référence :

      Petit traité de manipulation à l'usage des honnêtes gens par R.-V. Joule et J.-L. Beauvois.  

      La soumission librement consentie par les mêmes auteurs.    ◦ Psychologie sociale et Attitude et comportement par F. Girandola.

      Articles en ligne :

      ◦ Des articles de vulgarisation sur la plateforme The Conversation, notamment sur l'application des techniques de manipulation par Donald Trump ou dans le contexte des soldes.

      Vidéo :

      ◦ La reconstitution filmée de l'expérience du "billet perdu" est disponible en ligne.

      6. Conclusion et Perspectives

      La présentation s'est concentrée sur les fondements de la théorie de l'engagement et la technique du pied-dans-la-porte.

      Il a été précisé que d'autres aspects importants n'ont pas été abordés, notamment :

      • Les effets de l'engagement sur les opinions (via la théorie de la dissonance cognitive).

      L'escalade d'engagement, un processus où un individu persévère dans une décision ou un comportement qui s'avère négatif, simplement parce qu'il s'y est initialement engagé.

    1. Daten über den Berliner Baumbestand Bewässerungsdaten des Projekts Gieß den Kiez

      Vielleicht stehe ich auf dem Schlauch: Hier wird mir gesagt, dass ich exakt zwei Datenquellen brauche und dann werden immer die Pumpen als dritte Datenquelle eingebracht. Warum sind die nicht hier erwähnt? Und wie passen die zu meiner Fragestellung? Lautet die: Da, wo es mehr öffentliche Pumpen gibt, gibt es mehr engagierte Gießerinnen?

    1. Zur Reflexion: Am Ende muss da logischerweise herauskommen, dass Daten zum Bewässerungsverhalten nur von den Bürger:innen selbst stammen können, da diese Werte nicht öffentlich erhoben werden können. Auch die “Selbstauskunft” ist keine verlässliche Größe, aber in diesem Fall die einzige, mit der man arbeiten kann. Daher müsste ein Dashboard über die Funktion verfügen, eine selbst gegossene Wassermenge eintragen zu können. Dies ist der Fall bei Gieß den Kiez, ein Projekt des CityLab Berlin, das uns zu unserer Fallstudie inspiriert hat.

      Ist das die abschließende Reflexion oder nur ein Gedanke-Einschub? ... muss da logischerweise herauskommen ... klingt so, als ob wir gleich was hinfälschen wollen.

    1. 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.

    2. 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.

    3. La ciencia de la información[1][2][3] (abreviada como infosci) es un campo académico que se ocupa principalmente del análisis, recopilación, clasificación, manipulación, almacenamiento, recuperación, movimiento, difusión y protección de la información. [4] Los profesionales dentro y fuera del campo participan en el estudio de la aplicación y uso del conocimiento en las organizaciones. Además, examinan la interacción entre personas, organizaciones y cualquier sistema de información existente. El objetivo de este estudio es crear, reemplazar, mejorar o comprender los sistemas 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.

    4. Es una ciencia interdisciplinaria

      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

    5. Social media's power to facilitate topics

      Las conexiones en redes sociales ayudan a esparcir la información a mayor velocidad, pero es claro que también es un medio de desinformación y nosotros como bibliotecarios debemos saber filtrar dicha información cuando se esparce sin control y dar la información real. Aunque es una tarea difícil este tema debe estar de manera principal para poder solucionar la desinformación.

    6. The discipline of documentation science, which marks the earliest theoretical foundations of modern information science, emerged in the late part of the 19th century in Europe together with several more scientific indexes whose purpose was to organize scholarly literature. Many information science historians cite Paul Otlet and Henri La Fontaine as the fathers of information science with the founding of the International Institute of Bibliography (IIB) in 1895.[31] A second generation of European Documentalists emerged after the Second World War, most notably Suzanne Briet.[32] However, "information science" as a term is not popularly used in academia until sometime in the latter part of the 20th century.[33]

      A través de la historia ya habían vestigios de una ciencia para organizar y salvaguardar la información. Solo que no se llamaba como actualmente la conocemos, pero como toda rama comenzó como una necesidad y se fue especializando con el tiempo y dándole forma a lo que actualmente hoy conocemos.

    7. Scope and approach

      Somos una carrera que observa todo a su alrededor y comienza a dar mejoras de todo ello según enfoques que sean necesarios, ya sean desarrollos en tecnología o en el ámbito humano, podria decirse que somos un árbol de vida que mantiene cada rama y la nutre.

    8. transdisciplinary field

      En este aspecto siento que esta palabra es muy fuerte y muchas personas están llevando estos términos muy allá olvidando los campos ya establecidos y que aun falta por mejorar, cuando uno quiere abarcar todo al tiempo al final no hace nada bien.

    9. Information science

      Creo que mas allá del termino lo que significa nuestra carrera, no debemos limitarla a eso, para cada profesional tiene un significado diferente este termino desde las experiencias vividas y desde su labor profesional, para algunos puede ser mas enfocado a lo social y las bibliotecas, para otros mas enfocado a memoria y cultura con el archivo, para otros solo tener un titulo y creerse mas por saber sobre palabras bonitas con significados perfectos. pero en mi humilde opinión la carrera y mas este termino es el sentir de la profesión y como cada uno la aborde desde sus distintos enfoques. Y son igual de validos.

    10. interaction between people, organizations, and any existing information systems.

      Considero que la preocupación por las personas, diferencia a la ciencia de la información de otras disciplinas como la ciencia de datos, la ingeniería de sistemas, etc,

    1. insbesondere mit Variablen, Messkonzepten und dem Umgang mit Datensätzen.

      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.

    2. nteresse an der Interpretation städtischer Strukturdaten

      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)

    3. Grundlegende R-Kenntnisse sind hilfreich, aber nicht zwingend notwendig, da alle Bausteine ausführlich erläutert werden.

      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.

    1. Diese Fallstudie

      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.

    1. 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.

    1. 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.

      --------------------------------------------------------------------------------

      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.

      --------------------------------------------------------------------------------

      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.

      --------------------------------------------------------------------------------

      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.

    1. 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.

    1. 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.

    1. Vous voulez étoffer votre API et permettre aux utilisateurs de passer plusieurs paramètres dans une requête. Quel signe utilisez-vous ?

      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 "&"

    1. 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.

    1. 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.

    1. 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).

      --------------------------------------------------------------------------------

      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. |

      --------------------------------------------------------------------------------

      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.

      --------------------------------------------------------------------------------

      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

    1. 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.

    1. 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.

      --------------------------------------------------------------------------------

      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.

      --------------------------------------------------------------------------------

      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.

      --------------------------------------------------------------------------------

      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.

      --------------------------------------------------------------------------------

      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.

      --------------------------------------------------------------------------------

      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.

      --------------------------------------------------------------------------------

      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.

    1. 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

      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

    1. 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.

    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<sup>L444P/P415R</sup>) and GD2-10-257 (GBA1<sup>L444P/RecNcil</sup>), 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).”

    1. 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.

    1. Soutenir les compétences socio-émotionnelles chez les jeunes enfants : Approches et Dispositifs

      Résumé Exécutif

      Ce document synthétise les interventions de Sylvie Richard (Université de Genève / HP Valais) concernant le soutien aux apprentissages socio-émotionnels durant les premières années de scolarité.

      La recherche scientifique identifie deux leviers complémentaires : l'approche directe (structurée et dirigée par l'enseignant) et l'approche indirecte (développementale, centrée sur le jeu de faire semblant).

      Les données probantes, issues notamment de méta-analyses incluant plus d'un million d'élèves, démontrent que le renforcement des compétences socio-émotionnelles améliore non seulement le bien-être et les comportements sociaux, mais aussi les résultats académiques à long terme.

      La transition vers une pédagogie intégrant le jeu accompagné nécessite toutefois une formation approfondie des enseignants (plus de 20 heures) et un travail réflexif sur leurs propres compétences émotionnelles.

      --------------------------------------------------------------------------------

      1. Cadre Conceptuel des Compétences Socio-Émotionnelles

      Les compétences socio-émotionnelles sont définies selon le modèle de l'organisation Casel, qui regroupe trois grands domaines d'apprentissage :

      Conscience de soi et des autres : Identifier ses propres émotions et comprendre celles d'autrui.

      Gestion des émotions et des relations : Établir et maintenir des relations sociales positives.

      Prise de décision responsable : Apprendre à agir de manière éthique et constructive.

      --------------------------------------------------------------------------------

      2. L'Approche Directe : Programmes Structurés et Dirigés

      L'approche directe repose sur des activités planifiées où l'enseignant cible des savoirs spécifiques via des supports dédiés (jeux de plateau, fiches, lectures).

      Preuves d'Efficacité et Recherche

      La littérature scientifique internationale (méta-analyses de 2022 et 2025) souligne des bénéfices majeurs :

      Impact scolaire : Amélioration significative des résultats académiques comparativement aux élèves ne bénéficiant pas de ces programmes.

      Impact comportemental : Réduction des comportements problématiques et de la détresse émotionnelle.

      Impact à long terme : Diminution de la consommation de drogues à l'entrée de l'âge adulte.

      Programmes en Contexte Francophone

      Il existe un manque de programmes francophones validés par rapport aux modèles anglo-saxons. La simple traduction est jugée insuffisante ; une adaptation socio-culturelle est nécessaire. Deux outils se distinguent :

      | Programme | Origine | Compétences Ciblées | Accessibilité | | --- | --- | --- | --- | | Emotimat | France (Grenoble) | Identification, compréhension et expression des émotions. | Libre d'accès (en ligne). | | Emoti | Suisse (Genève) | Reconnaissance émotionnelle, besoins et régulation. | Payant (coût d'impression des cartes). |

      --------------------------------------------------------------------------------

      3. L'Approche Indirecte : La Pédagogie par le Jeu de Faire Semblant

      Le jeu de faire semblant est une activité où les objets, les paroles et les actions représentent autre chose que leur réalité immédiate. C'est une fonction mentale de haut niveau mobilisant l'imagination.

      Les Composantes du Jeu Mature

      Pour qu'un jeu génère des apprentissages, il doit tendre vers la maturité, caractérisée par plusieurs éléments :

      Substitution d'objets : Utiliser un bâton pour représenter une fusée (inhibition de la fonction réelle de l'objet).

      Attribution de rôles : Endosser une identité (docteur, pirate) et respecter le registre de comportement associé.

      Méta-communication : Planifier et négocier le scénario avec les pairs ("On dirait que tu étais...").

      Raisonnement par hypothèses : Utiliser la logique "Et si..." pour explorer des mondes possibles et des relations de cause à effet.

      Un Laboratoire de Développement

      Le jeu de faire semblant permet à l'enfant :

      1. De s'autoréguler : En s'imposant des règles de comportement liées au rôle choisi.

      2. D'expérimenter sans risque : Tester des situations sociales complexes dans un cadre "pour de faux", sans enjeu de performance.

      3. De traiter le réel : Mettre en scène sa compréhension du monde (ex: jeux liés à la pandémie ou aux soins médicaux) pour réguler ses frustrations ou ses peurs.

      --------------------------------------------------------------------------------

      4. Rôle et Posture de l'Enseignant

      Le passage d'un "jeu libre" à un "jeu accompagné" est crucial. L'enseignant ne doit pas être un simple spectateur, mais un acteur capable d'adopter plusieurs postures :

      Régisseur de scène : Fournir les accessoires et l'espace nécessaires.

      Co-joueur ou Joueur : Entrer dans le scénario pour enrichir le contenu et proposer des défis émotionnels.

      Observateur-Évaluateur : Identifier le niveau de maturité du jeu pour intervenir au bon moment.

      L'Importance de la Formation

      Les recherches indiquent que l'efficacité de ces dispositifs dépend de la préparation de l'adulte :

      Formation technique : Un minimum de 20 heures de formation est recommandé pour maîtriser l'accompagnement du jeu et les concepts socio-émotionnels.

      Dimension réflexive : L'enseignant doit évaluer ses propres compétences émotionnelles et sa capacité à jouer, car il sert de modèle par imitation pour les jeunes enfants.

      --------------------------------------------------------------------------------

      5. Conclusions et Recommandations

      La littérature scientifique actuelle récuse l'idée que le temps alloué au développement socio-émotionnel serait une "perte de temps" au détriment du scolaire. Au contraire :

      Complémentarité : Il est impératif de combiner les séances structurées et les temps de jeu accompagné.

      Enjeu de santé publique : Le déclin de l'engagement des enfants dans le jeu de faire semblant fait de son soutien à l'école une priorité de développement psychologique.

      Apprendre à jouer pour jouer pour apprendre : Le jeu de faire semblant n'est pas inné à un niveau mature ; il doit être enseigné pour devenir un outil d'apprentissage efficace.

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      Nembutal-Natrium-Pentobarbital kaufen Nembutal-Natrium-Pentobarbital kaufen Nembutal-Natrium-Pentobarbital kaufen Nembutal-Natrium-Pentobarbital kaufen Nembutal-Natrium-Pentobarbital kaufen Nembutal-Natrium-Pentobarbital kaufen Nembutal-Natrium-Pentobarbital kaufen Nembutal-Natrium-Pentobarbital kaufen Nembutal-Natrium-Pentobarbital kaufen Nembutal-Natrium-Pentobarbital kaufen Lieferung nur in andere Länder Asiens, Australiens und Afrikas. Innerhalb von 2 Tagen. Alle Sendungen erfolgen per Expressversand, und jeder Kunde ist verpflichtet, die Expressversandkosten zu bezahlen. Ist der Online-Kauf von Nembutal sicher? Vergleich mit anderen Methoden Tödliche Dosis Nembutal-Natrium-Lösung und -Pulver Verfügbarkeit Informationen zu Barbituraten Barbiturate allgemein Wirkmechanismus Toleranz und Entzugserscheinungen Einnahme des Nembutal-Cocktails Zeit bis zum Tod Auswirkungen einer Überdosierung Quellen Die Frage nach der Sicherheit des Online-Kaufs von Nembutal ist ein häufiges Anliegen unserer Kunden. Es ist wichtig zu wissen, dass der Wirkstoff in einigen Ländern kontrolliert wird, z. B. in den USA, Großbritannien und Kanada. In anderen Ländern ist er verboten. In Ländern, in denen der Konsum kontrolliert wird, versenden wir mit einem gültigen ärztlichen Rezept. In Ländern, in denen der Konsum verboten ist, verwenden wir eine besonders sichere Verpackung (Super Secured System Packaging, SSSP), die den Inhalt und die Identität des Empfängers nicht preisgibt. Dies trägt maßgeblich zu einer sicheren Lieferung in jedes Land bei und ist der Grund für unseren langjährigen Erfolg als Lieferant von Nembutal Pentobarbital. Gibt es eine Geld-zurück-Garantie?

      Ja. Nach erfolgreicher Bestellung erhalten Sie eine Sendungsverfolgungsnummer, sobald Ihr Paket bei einem zuverlässigen Kurierdienst (UPS, FedEx, EMS, DHL) registriert wurde. Im Falle eines Paketverlusts erstatten wir Ihnen den Kaufpreis innerhalb von 42 Stunden (2 Werktagen) ohne Zögern. Falls Sie eine erneute Lieferung wünschen, übernehmen wir alle Kosten und versenden die Ware innerhalb von 24 Stunden erneut.

      E-Mail: diewithdignity17@gmail.com

      WhatsApp: +447447025920

    2. HOE KAN IK CONTACT OPNEMEN OM NEMBUTAL TE KOPEN?

      Contact e-mail: diewithdignity17@gmail.com WhatsApp: +447447025920

      We hebben alle nodige maatregelen genomen om de wereld te voorzien van een UITZONDERLIJK medicijn (NEMBUTAL PENTOBARBITAL). Bekend bij velen, maar moeilijk verkrijgbaar. We hebben een duidelijke en transparante leveringsmethode en de Nembutal die elke persoon inneemt, moet de dodelijke dosis zijn.

      Hoe bepaalt u uw dodelijke dosis? NEMBUTAL SOLUTION SUPPLIER GROUP vereist dat iedereen die Nembutal nodig heeft, zijn of haar LEEFTIJD en GEWICHT doorgeeft. Hiermee kunnen onze experts bepalen welke dosis voor u dodelijk is.

      Contact e-mail: diewithdignity17@gmail.com WhatsApp: +447447025920

      Waar leveren we?

      Wij verzenden naar alle landen ter wereld, ongeacht uw locatie. We hebben een wereldwijd geharmoniseerd systeem dat alle continenten omvat en is afgestemd op de verwachtingen van onze klanten: tijdig, te goeder trouw en tegen een gemiddelde prijs.

      In welke landen is levering van Nembutal de volgende dag mogelijk na bestelling en betaling?

      De volgende landen komen in aanmerking voor levering de volgende dag, met een maximale levertijd van 36 uur.

      Nembutal, Nembutal pillen, Nembutal te koop, Wat is Nembutal, Nembutal kopen, Nembutal medicijn, Wat is Nembutal, Nembutal pentobarbital, Bijwerkingen van Nembutal, VERENIGDE STATEN, CANADA, BRAZILIË, VERENIGD KONINKRIJK, ZWEDEN, ZWITSERLAND, FRANKRIJK, DUITSLAND, NOORWEGEN, BELGIË, GRIEKENLAND, POLEN, NEDERLAND, PORTUGAL, ITALIË, DENEMARK, FINLAND HONGARIJE Trefwoord Volume CPC (USD) SERP Fargo forum forum drugs.com Wow forums geneesmiddelinteracties Overwatch forums apotheek bij mij in de buurt het forum Contact e-mail: E-mail: diewithdignity17@gmail.com WhatsApp: +447447025920

      Drinkbare Nembutal Pentobarbital gemengd met zoetstoffen om de bitterheid van de vloeistof te vermijden, Nembutal pentobarbitalnatrium (steriele oplossing) 6,5 gram/100 ml, Nembutal pentobarbitalnatrium (orale oplossing) 10 gram/100 ml, Nembutal Pentobarbitalnatriumcapsules (50 mg & 100 mg), Nembutal Pentobarbital (natriumzout) HOE KAN IK NEMBUTAL KOPEN?

      E-mailadres: diewithdignity17@gmail.com WhatsApp: +447447025920. Nembutal, nembutalnatrium Hoogwaardige nembutalpentobarbital te koop HOOGWAARDIGE NEMBUTALPENTOBARBITAL TE KOOP KOOP ORIGINELE NEMBUTALPENTOBARBITAL IN VLOEIBARE, POEDER- OF PILLENVORM KOOP 100% ZUIVERE NEMBUTALPENTOBARBITALNATRIUM BESTEL NEMBUTALPENTOBARBITAL KOOP TOPKWALITEIT NEMBUTALPENTOBARBITAL ONLINE, NEMBUTALPENTOBARBITALNATRIUM BESCHIKBAAR.

      TOPKWALITEIT NEMBUTALPENTOBARBITAL TE KOOP.

      Originele Nembutal Pentobarbital online verkopen in de VS, het VK, de EU, Canada Originele Nembutal Pentobarbital online verkopen in de Verenigde Staten (VS) Originele Nembutal Pentobarbital online verkopen in het Verenigd Koninkrijk (VK) Originele Nembutal Pentobarbital online verkopen in Londen Originele Nembutal Pentobarbital online verkopen in Italië Originele Nembutal Pentobarbital online verkopen in Australië Originele Nembutal Pentobarbital online verkopen in Rusland Originele Nembutal Pentobarbital online verkopen in Spanje Originele Nembutal Pentobarbital online verkopen in Milaan Koop originele Nembutal Pentobarbital in de Verenigde Arabische Emiraten (VAE), Dubai Koop Nembutal Pentobarbital in de Verenigde Staten (VS) Koop Nembutal Pentobarbital in het Verenigd Koninkrijk Koop Nembutal Pentobarbital in Italië Koop Nembutal Pentobarbital Italië Koop Nembutal Pentobarbital in Spanje Koop Nembutal Pentobarbital in Milaan Koop Nembutal Pentobarbital in Rusland Koop Nembutal Pentobarbital in Australië Contact e-mail: diewithdignity17@gmail.com WhatsApp: +447447025920

      Koop Nembutal Natrium Pentobarbital, koop Nembutal Natrium Pentobarbital Koop Nembutal Natrium Pentobarbital, koop Nembutal Natrium Pentobarbital Koop Nembutal Natrium Pentobarbital, koop Nembutal Natrium Pentobarbital Koop Nembutal Natrium Pentobarbital, koop Nembutal Natrium Pentobarbital Koop Nembutal Natrium Pentobarbital, koop Nembutal Natrium Pentobarbital Koop Nembutal Natrium Pentobarbital, koop Nembutal Natrium Pentobarbital Levering naar andere landen in Azië, Australië en Afrika duurt slechts 2 dagen. Dagen. Alle zendingen zijn expres en elke klant is verplicht te betalen voor expreslevering.

      Is het veilig om Nembutal online te kopen?

      Vergelijking met andere methoden Lethale dosis Nembutal-natriumoplossing en -poeder Beschikbaarheid Informatie over barbituraten Barbituraten in het algemeen Werkingsmechanisme Tolerantie en ontwenningsverschijnselen Inname van de Nembutal-cocktail Tijd tot overlijden Effecten van een overdosis Bronnen De vraag of het veilig is om Nembutal online te kopen, is een veelgestelde vraag onder onze klanten. Het is belangrijk om te weten dat de stof in sommige landen gereguleerd is, zoals de VS, het VK, Canada, enz., terwijl het in andere landen verboden is. In landen waar het gereguleerd is, verzenden we met een geldig medisch recept. Voor landen waar het verboden is, gebruiken we een zogenaamd "super beveiligd systeem voor verpakking (SSSP)" zonder de inhoud te onthullen of de identiteit prijs te geven. Dit draagt ​​in grote mate bij aan een veilige levering naar elk land en is de reden voor ons jarenlange succes als leverancier van NEMBUTAL PENTOBARBITAL.

      Is er een geld-terug-garantie?

      JA. Na een succesvolle bestelling ontvangt u een trackingcode zodra het pakket is geregistreerd bij een gerenommeerde koerier (UPS, FedEx, EMS, DHL). In geval van verlies van het pakket, betalen wij het aankoopbedrag binnen 42 uur (2 werkdagen) zonder aarzeling terug. Indien de klant een nieuwe verzending wenst, dekken wij alle kosten en verzenden wij het pakket binnen 24 uur opnieuw.

      E-mail: diewithdignity17@gmail.com

      WhatsApp: +447447025920

  2. drive.google.com drive.google.com
    1. Las referencias presentan las fuentes de la investigación con el formato requerido por lainstitución para la que se trabaja

      El uso de las Normas APA garantiza formalidad en la presentación de las fuentes.

    2. las características que debe cumplir un objetivo forman el acrónimo SMART:• Específico• Medible• Alcanzable• Relevante• Temporal

      SMART asegura que los objetivos sean claros y alcanzables. Lo que evita plantear metas ambiguas o dificiles de cumplir. La medición y temporalidad permiten evaluar si el estudio logró lo que se propuso.

    3. De la idea de investigación nace la pregunta central de investigación y las preguntasauxiliares de investigación que guiarán el proceso de investigación.

      Las preguntas de investigación son demasiado útiles y sirven como una guia metodológica. Ya que estas orientan todo el proceso y evitan desviaciones.

    4. La descripción del problema:

      El enunciado hace que se pueda contextualizar el problema de una manera profunda. No solo es mencionar el problema, sino que se deben analizar sus causas y efectos. Lo que ayuda a comprender su impacto real.

    5. es importante que se verifique la capacidad de realizar lainvestigación. Para ello se debe tomar en cuenta la disponibilidad de:• Recursos humanos• Recursos materiales• Recursos financieros• Recursos temporales

      La viabilidad garantiza que la investigación pueda realizarse realmente. No solo es tener una buena idea, es necesario contar con los recursos adecuados.

    6. La justificación explica el porqué de la investigación: por qué elproyecto es importante y necesario.

      No se investiga por curiosidad únicamente, sino que también para aportar soluciones, conocimiento o beneficios prácticos. Evaluar lo que es su conveniencia y relevancia fortalece el valor académico del proyecto.

    7. Un problema planteado de forma correcta está parcialmente resuelto, ya que, a mayorexactitud, hay más posibilidades de obtener una solución satisfactoria.

      Esta idea es clave porque nos muestra que la claridad en la formulación del problema es muy importante y determina la calidad de toda la investigación. Si el problema no es exacto, el estudio perderá dirección. Por lo que hay que definirlo correctamente para aumentar las probabilidades de obtener resultados válidos y útiles.

  3. ushift.tecnico.ulisboa.pt ushift.tecnico.ulisboa.pt
    1. slide 6, dos "shapes don't match OSM", - acrescentar que o próprio standatr não define como devem ser desenhadas as rotas - o que gera um problema gigante de consistência, até dentro do mesmo operador e da mesma rota para serviços diferentes! (isso é algo que terá de ser ultrapassado no futuro pelo standart, mas que conseguimos ultrapassar aqui)

    2. ah já entendi. mas então suguro ou no primeiro mapa mudar a escala de cores, ou no segundo meter o lwd também relativo à frequência (mais largas e mais claras -> mais frequentes)

    1. Reviewer #2 (Public review):

      Summary:

      In this manuscript, Thompson et al. investigate the impact of prior ATP exposure on later macrophage functions as a mechanism of immune training. They describe that ATP training enhances bactericidal functions which they connect to the P2x7 ATP receptor, Nlrp3 inflammasome activation, and TWIK2 K+ movement at the cell surface and subsequently at phagosomes during bacterial engulfment. This is an incremental addition to existing literature, which has previously explored how ATP alters TWIK2 and K+, and linked it to Nlrp3 activation. The novelty here is in discovering the persistence of TWIK2 change and exploring the impact this biology may have on bacterial clearance. Additional experiments could strengthen their hypothesis that the in vivo protective effect of ATP-training on bacterial clearance is mediated by alveolar macrophages.

      Strengths:

      The authors demonstrate three novel findings: 1) prolonged persistence of TWIK2 at the macrophage plasma membrane following ATP that can translocate to the phagosome during particle engulfment, 2) a persistent impact of ATP exposure on remodeling chromatin around nlrp3, and 3) administering mice intra-nasal ATP to 'train' lungs protects mice from otherwise fatal bacterial infection.

      Weaknesses:

      (1) Some methods remain unclear including the timing and method by which lung cellularity was assessed in Figure 2. It is also difficult to understand how many mice were used in experiments 1, 2 and 6 and thus how rigorous the design was. A specific number is only provided for 1D and the number of mice stated in legend and methods do not match.

      (2) The study design is not entirely ideal for the authors' in vivo question. Overall, the discussion would benefit from a clear summary of study caveats, which are primarily that that 1) in vitro studies attributing ATP training-mediated bacterial killing to persistent TWIK2 relocation, K+ influx, a glycolytic metabolic shift , and epigenetic nlrp3 reprogramming were performed in BMDM or RAW cells and not primary AMs, 2) data does not eliminate the possibility that non-AM immune or non-immune cells in the lung are "trained" and responsible for ATP-mediated protection in vivo; flow data examined total lung digest which may obscure important changes in alveolar recruitment, and 3) in vivo work shows data on acute bacterial clearance but does not explore potential risks that "training" for a more responsive inflammasome may have for the severity of lung injury during infection.

      (3) The is some lack of transparency on data and rigor of methods. Clear data is not provided regarding the RNA-sequencing results. Specific identities of DEGs is not provided, only one high-level pathway enrichment figure. It would also be ideal if controls were included for subcellular fractionating to confirm pure fractions and for dye microscopy to show negative background.

      (4) In results describing 5A, the text states that "ATP-induced macrophage training effects, as measured by augmented bactericidal activity, were diminished in macrophages treated with protease inhibitors". However, these data are not identified significant in the figure; protease dependence can be described as a trend that supports the authors' hypothesis but should not be stated as significant data in text.

      In summary, this work contains some useful data showing how ATP can train macrophages via TWIK2/Nlrp3. Revisions have significantly improved methods reporting, added some data to strengthen the conclusions, and toned down on overstatements to bring conclusions more in line with data presented. The title still overstates what the authors have actually tested, since no macrophage-specific targeting in vivo (no conditional gene deletion, macrophage depletion etc) was performed in infection studies. However, in vitro data provide clear evidence that macrophages can be trained by ATP, and through caveats remain, it is plausible that macrophage training is a key mechanism for the protection observed here in the lung.

    2. Author response:

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

      Public Reviews:

      Reviewer #1 (Public review):

      (1) First, the concept of training or trained immunity refers to long-term epigenetic reprogramming in innate immune cells, resulting in a modified response upon exposure to a heterologous challenge. The investigations presented demonstrate phenotypic alterations in AMs seven days after ATP exposure; however, they do not assess whether persistent epigenetic remodeling occurs with lasting functional consequences. Therefore, a more cautious and semantically precise interpretation of the findings would be appropriate.

      In response, we have performed epigenetic analysis (ATAC seq analysis) as requested (Supp Fig. 1).

      (2) Furthermore, the in vivo data should be strengthened by additional analyses to support the authors' conclusions. The authors claim that susceptibility to Pseudomonas aeruginosa infection differs depending on the ATP-induced training effect. Statistical analyses should be provided for the survival curves, as well as additional weight curves or clinical assessments. Moreover, it would be appropriate to complement this clinical characterization with additional measurements, such as immune cell infiltration analysis (by flow cytometry), and quantification of pro-inflammatory cytokines in bronchoalveolar lavage fluid and/or lung homogenates.

      We have added the statistical analyses provided for the survival curves (new Fig. 1D), immune cell infiltration analysis, and quantification of pro-inflammatory cytokines in the lung (new Figs. 1, 2).

      (3) Moreover, the authors attribute the differences in resistance to P. aeruginosa infection to the ATP-induced training effect on AMs, based on a correlation between in vivo survival curves and differences in bacterial killing capacity measured in vitro. These are correlative findings that do not establish a causal role for AMs in the in vivo phenotype. ATP-mediated effects on other (i.e., non-AM) cell populations are omitted, and the possibility that other cells could be affected should be, at least, discussed. Adoptive transfer experiments using AMs would be a suitable approach to directly address this question.

      We have performed additional experiments and found that the numbers of lung macrophages were not significantly altered before and after ATP training (new Fig. 2), indicating the training effects are focused on lung resident macrophages.

      Reviewer #2 (Public review):

      (1) Missing details from methods/reported data: Substantial sections of key methods have not been disclosed (including anything about animal infection models, RNA-sequencing, and western blotting), and the statistical methods, as written, only address two-way comparisons, which would mean analysis was improperly performed. In addition, there is a general lack of transparency - the methods state that only representative data is included in the manuscript, and individual data points are not shown for assays.

      We have revised the methods and statistical analysis.

      (2) Poor experimental design including missing controls: Particularly problematic are the Seahorse assay data (requires normalization to cell numbers to interpret this bulk assay - differences in cell growth/loss between conditions would confound data interpretation) and bacterial killing assays (as written, this method would be heavily biased by bacterial initial binding/phagocytosis which would confound assessment of killing). Controls need to be included for subcellular fractionating to confirm pure fractions and for dye microscopy to show a negative background. Conclusions from these assays may be incorrect, and in some cases, the whole experiment may be uninterpretable.

      Seahorse assay methodology was updated to confirm the order of cell counting, time at seeding and cell counts. Methods were also updated to address the distinction between bacterial killing (Fig. 1B) and overall decrease in bacterial load.

      (3) The conclusions overstate what was tested in the experiments: Conceptually, there are multiple places where the authors draw conclusions or frame arguments in ways that do not match the experiments used. Particularly:

      (a) The authors discuss their findings in the context of importance for AM biology during respiratory infection but in vitro work uses cells that are well-established to be poor mimics of resident AMs (BMDM, RAW), particularly in terms of glycolytic metabolism.

      We have adjusted the text to reflect that the metabolic assay was performed on BMDMs. AMs are fragile for certain manipulations in vitro. We expect that the metabolic change is similar across several macrophage systems as well as the bacterial load reduction.

      (b) In vivo work does not address whether immune cell recruitment is triggered during training.

      We have performed immune cell infiltration analysis (new Fig. 2).

      (c) Figure 3 is used to draw conclusions about K+ in response to bacterial engulfment, but actually assesses fungal zymosan particles.

      We have corrected this in the manuscript.

      (d) Figure 5 is framed in bacterial susceptibility post-viral infection, but the model used is bacterial post-bacterial.

      We have corrected this in the manuscript.

      (e) In their discussion, the authors propose to have shown TWIK2-mediated inflammasome activation. They link these separately to ATP, but their studies do not test if loss of TWIK2 prevents inflammasome activation in response to ATP (Figure 4E does not use TWIK2 KO).

      We have now added the TWIK2 KO results (new Fig. 5E).

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      As noted in the public review, it would be advisable to further characterize the in vivo phenotype in order to strengthen the conclusions. Specifically, it would be useful to quantify the bacterial load in the bronchoalveolar lavage fluid and lung homogenates, as well as to measure cytokine levels both in the respiratory compartment and systemically. Additionally, a broader characterization of the immune response in the presence or absence of ATP-induced training would be valuable. In the absence of direct evidence demonstrating that trained AMs mediate the observed phenotype, the authors should adopt a more cautious interpretation of their results. Moreover, careful attention to semantic accuracy is recommended. The concept of trained immunity refers specifically to long-term epigenetic reprogramming that leads to an altered response of target cells upon a secondary challenge, distant from the initial stress. The data presented do not fully demonstrate this phenomenon, and the interpretations should remain aligned with the evidence provided.

      Bacterial load has been quantified (see more details in the Methods part). And we also measured immune cell infiltration, quantification of pro-inflammatory cytokines in the lung (new Figs. 1, 2), and epigenetic evaluation of vehicle- and ATP-treated cells (Supp. Fig. 1).

      Reviewer #2 (Recommendations for the authors):

      (1) It cannot be overstated how lacking the methods are. This includes no discussion of IACUC approval for animal procedures, which must be included as part of research ethics. It also needs to be made clear where raw data is being archived. This notably includes an accession for deposited RNA-sequencing data, although unmanipulated microscopy and western blot images should also be shown. Methods should discuss any pre-processing that occurred with images.

      We have revised the methods in the manuscript.

      (2) Per statistics, in addition to generally providing more detail and adjusting analyses if they have not been correctly performed, please disclose if SD or SEM is shown. Reporting aggregate data versus representative data would provide more rigor. Perhaps replicate experiments could be included in the supplemental if they cannot, for some reason, be aggregated. Detailed statistical methods for RNA-seq analysis also need to be included.

      More details have been provided in the methods section.

      (3) It is unclear whether bacterial killing assays were correctly designed and can be interpreted. What does cells collected mean? If the assay was focused on intracellular macrophage bacterial load, it is critical to assess and report phagocytosis since different input loads would confound the assessment of killing. A rigorous wash or an antibiotic to eliminate extracellular bacteria should also have been performed and be described in this case. If the total bacterial burden was assessed, that would use cells+media and also needs to be clear and described. With the information provided, it is unclear whether the assays performed are sufficiently rigorous to assess bacterial killing. In addition, Figure 1B reports using an MOI of 50-100, but all data is compiled in one graph - data from different levels of infection should be separated. Figure 5A shows a model with E.coli followed by PA, but that does not appear to be how the assay was structured in B or C. This also does not match how the experiment is written in the results section, which references S. aureus. It is unclear what tissue (or cells) were assessed in Figure 5. Whole lung? BAL? As written, no data provided regarding bacterial killing is of sufficient quality to be considered valid.

      We have re-written the bacterial killing assay in the manuscript. The methodology was corrected to distinguish bacterial killing vs load decrease and generally accurate methodology.

      (4) The in vitro data provide reasonable evidence that BMDM/RAW macrophage training can occur in response to ATP exposure. However, it is unclear whether training is an important mechanism for resident AM in vivo, or whether, in vivo, a broader inflammatory response is generated, recruiting additional immune cells that persist and change infection susceptibility. The authors argue for resident AM immune training, but do not provide sufficient evidence to counter the latter possibility (resident AM are never themselves directly assessed, and the presence of other immune cells in vivo is not excluded). See Iliakis et al 2023 (PMID 37640788) for discussion of how this issue continues to drive uncertainty in the field. For this study, at least providing flow cytometry data quantifying myeloid and lymphoid immune populations in BALF before and after various treatments would help address this caveat. Without knowing this, it also confounds the interpretation of Figure 1B; if BAL is not pure AM after training, perhaps 1B could be repeated with ex vivo training or resident AM could be purified?

      We have performed immune cell infiltration analysis in the lung (both to BALF and in-tissue, new Fig. 2).

      (5) Figure 3A appears to show that fewer than 50% of cells express GFP. Is it expected that only a fraction of RAW cells express TWIK2-GFP? How was this addressed in the analyses for Figure 3? Were cells not appearing to express any significant GFP, included in phagosomal-negative or excluded from analysis? Please include in the methods.

      The RAW cells were transfected with TWIK2-GFP and variable GFP expression was expected. These cells were expressing a non-integrated transgene, which has been added to the methods as well as the consideration of cells for the analysis. Cells without visible GFP expression were excluded.

      (6) Why are many data points in Figure 3D negative? This suggests that settings were not optimized for microscopy - perhaps there is a very high background signal and the ION stain is barely above it. This is concerning for the quality of data. Further, is it expected that only some cells are positive for ION K+? The images shown clearly differentiate phagosomal K with ATP versus the absence of K without, but it is surprising that some cells appear not to contain any ION K+ signal (not completely clear given lack of brightfield or other cell staining) - this may again point to issues with imaging settings that confound data interpretation. This analysis should be carefully assessed.

      This has been updated in the methodology. In old Fig. 3D (new Fig. 4D), the presented data is the net intensity of the phagosome, subtracting the average cytoplasmic MFI from that of the area corresponding to an engulfed zymosan-af594 bead. Thus, a negative value has higher cytoplasmic IonK signal than that of the phagosome.

      (7) The Discussion states that it will be interesting to test whether ATP-TWIK2 is a common mechanism of training and specifically references LPS as an ATP-generating signal. However, Figure 2D data show that LPS induces only transient TWIK2 translocation; the authors have data suggesting that, in the context of LPS, TWIK2 'training' will not be engaged. This line of discussion shows incomplete consideration of the data.

      We have further limited this language in the text such that this may require differential sensitivity/damage sustained by macrophages as compared to that of epi/endothelial cells in response to bacterial endotoxin.

      (8) For RNA-sequencing, plots of the actual genes changed for the mitochondrial pathways of interest would be helpful information for readers, as would a heat map showing sample purity between groups for macrophage markers versus possible contaminant cells, which can also be generated from precursors in BMDM cultures. In general, information in Methods regarding how the analyses in Figure 4B were run is necessary, per cutoffs used to determine DEGs, number of samples in each group, sex of samples used, etc. Greater transparency of data would be appreciated, so plots that show variation between replicates, such as heat maps, would be ideal. Supplemental tables would also be nice.

      We have added to the methodology of the RNA sequencing analysis

      (9) The use of alternate DAMPs is a positive addition to the experimental design, but no data is given regarding the concentrations used. Ideally, positive controls showing histones/NAD are used at acutely activating concentrations could be included but at least references supporting the doses chosen or information about how doses were selected should be given. It is easy to find substantial literature on histones as a DAMP, but it was unclear why/how NAD was selected.

      We have added these concentrations and corresponding references.

      (10) The E.coli CFU reported in Figure 5B are extraordinarily low. In addition, CFU are generally shown on a log scale, but this appears to be linear. Please confirm that these data are correct. Perhaps improved methods might explain why? Is the second hit a low dose?

      These have been corrected in the new Fig. 6B.

      (11) Given that loss of either TWIK2 or Nlrp3 ablates bacterial protection, a link should be tested - experiments should test whether loss of TWIK2 prevents inflammasome activation in response to ATP (TWIK2 KO in 4E) and if loss of Nlrp3 changes TWIK2 translocation (Nlrp3 KO in at least some experiments of Figures 2/3).

      We have now added the TWIK KO results (new Fig. 5E).

      (12) One of the most striking data pieces is Figure 1D. It would, therefore, strengthen the paper to repeat those experiments (even just with the high-dose ATP) using TEIK2/P2X7/NLRP3 KO mice and really show the importance of these pathways in vivo. This is conceptually Figure 5, but the survival data of Figure 1 is far more convincing than the relatively weak bacterial load data of Figure 5.

      Unfortunately, our previous laboratory has been closed and we have trouble acquiring enough mice for additional survival data during the transition period. However, the bacterial load data has been adjusted to the same bacterial counts per 5 mg lung tissue instead of per individual sampling, giving a more contextual interpretation of the data.

    1. Author response:

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

      Public Reviews:

      Reviewer #1 (Public reviews):

      (1) The absence of replicate paired-end datasets limits confidence in peak localization.

      The reviewer was under the impression that that we did not perform biological replicates of our ChIP-seq experiments. All ChIP-seq (and ATAC-seq) experiments were performed with biological replicates and the Pearson’s correlations (all >0.9) between replicates were provided in Supplementary Table 1. We had indicated this in the text and methods but will try to make this even clearer.

      (2) The analyses are primarily correlative, making it difficult to fully assess robustness or to support strong mechanistic conclusions.

      Histone modifications are difficult to alter genetically because of the high copy number of histone genes and inhibition of HATs/HDACs in general leads to alterations in other histone modifications. It is an inherent challenge in establishing causality of histone modifications, especially histone acetylation marks.

      (3) Some claims (e.g., specificity for CpG islands, "dynamic" regulation during differentiation) are not fully supported by the analyses as presented.

      We have modified the text in response to this point. The new text reads: “Non-CGI promoters have lower overall levels of transcription compared to CGI promoters, and for this promoter class H3K115ac enrichment detected by ChIP is only really seen for the highest quartile of transcription (4SU) quartile of expression (Figure 1G). CGI promoters on the other hand, exhibit significant levels of detected H3K115ac even for the lowest quartile of expression. These results suggest a special link between CGI promoters and H3K115ac”.

      (4) Overall, the study introduces an intriguing new angle on globular PTMs, but additional rigor and mechanistic evidence are needed to substantiate the conclusions.

      We agree that the paper does not provide mechanistic details or solid causality of H3K115ac. We have only emphasized the potential role of H3K115ac in nucleosome fragility based on our in vivo data and previously published in-vitro experiments (Manohar et.al., 2009, Chatterjee et. al., 2015). We do provide the evidence that H3K115ac is enriched on subnucleosomal particles via sucrose gradient sedimentation of MNase-digested chromatin (Figure 3C-D).

      Reviewer #2 (Public review):

      (1) I am not fully convinced about the specificity of the antibody. Although the experiment in Figure S1A shows a specific binding to H3K115ac-modified peptides compared to unmodified peptides, the authors do not show any experiment that shows that the antibody does not bind to unrelated proteins. Thus, a Western of a nuclear extract or the chromatin fraction would be critical to show. Also, peptide competition using the H3K115ac peptide to block the antibody may be good to further support the specificity of the antibody. Also, I don't understand the experiment in Figure S1B. What does it tell us when the H3K115ac histone mark itself is missing? The KLF4 promoter does not appear to be a suitable positive control, given that hundreds of proteins/histone modifications are likely present at this region. It is important to clearly demonstrate that the antibody exclusively recognizes H3K115ac, given that the conclusion of the manuscript strongly depends on the reliability of the obtained ChIP-Seq data.

      ChIP-qPCR in S1B includes competition from native chromatin and shows high specificity to its target. We have provided antibody validation in three ways:

      - Western blot with dot-blot of synthetic peptides (Figure S1A).

      - Western blots with Whole cell extracts (Figure 4D).

      - ChIP-qPCR on native chromatin spiked with a cocktail of synthetic mono-nucleosomes, each carrying a single acetylation and a specific barcode (SNAP-ChIP K-AcylStat Panel).

      We could not include H3K115ac marked nucleosomes as they are not available in the panel. Figure S1B shows that the H3K115ac antibody exhibits negligible binding to known K-acyl marks, comparable to an unmodified nucleosome. Because of the absence of a H3K115ac modified barcoded nucleosome, we used the KLF4 promoter from mESCs as a positive control, in agreement with ChIP-seq signal shown in the genome browser profile (Figure 1E), the KLF4 promoter shows a significantly higher signal than the gene body.

      (2) The association of H3K115ac with fragile nucleosomes is based on MNase-sensitivity and fragment length, which are indirect methods and can have technical bias. Experiments that support that the H3K115ac modified nucleosomes are indeed more fragile are missing.

      We have performed ChIP-seq on MNase digested mESC chromatin fractionated on sucrose gradients and this shows that H3K115ac is enriched in fractions containing sub-nucleosomal and fragile nucleosomes but depleted in fractions containing stable nucleosomes (Figure 3D).

      (3) The comparison of H3K115ac with H3K122ac and H3K64ac relies on publicly available datasets. Since the authors argue that these marks are distinct, data generated under identical experimental conditions would be more convincing. At a minimum, the limitations of using external datasets should be discussed.

      H3K64ac and H3K122ac datasets were generated by us in a previous publication (Pradeepa et. al., 2016) using same native MNase ChIP protocol as used here. The ChIP-seq datasets for H3K122ac and H3K27ac are processed in an identical manner, with the same computational pipelines, to the H3K115ac data sets generated in this paper.

      (4) The enrichment of H3K115ac at enhancers and CTCF binding sites is notable but remains descriptive. It would be interesting to clarify whether H3K115ac actively influences transcription factor/CTCF binding or is a downstream correlate.

      We agree with the reviewer’s comment, but we have not claimed causality.

      (5) No information is provided about how H3K115ac may be deposited/removed. Without this information, it is difficult to place this modification into established chromatin regulatory pathways.

      Due to broad target specificity, redundancies and crosstalk among different classes of HATs and HDACs, it is not tractable to answer this question in the current manuscript.

      Reviewer #3 (Public reviews):

      Reviewer 3 is mistaken in thinking our ChIP experiments are performed under cross-linked conditions. As clearly stated in the main text and methods, all our ChIP-seq for histone modifications is done on native MNase-digested chromatin – with no cross-linking. This includes the spike-in experiment shown in Fig S1B to test H3K115ac antibody specificity against the bar-coded SNAP-ChIP® K-AcylStat Panel from Epicypher. We could not include H3K115ac bar-coded nucleosomes in that experiment since they are not available in the panel.

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      (1) I have two primary concerns that resound through the entire paper:

      (a) Overall, the manuscript is making strong claims based on entirely correlative datasets. No quantitative analyses are performed to demonstrate co-occupancy/localization. Please see more detailed descriptions below.

      Our responses to specific points are provided against each comment below.

      (b) Lack of paired-end replicates for H3K115ac ChIP-seq. While the reviewer token for the deposited data was not made accessible to me, looking at Supplementary Table 1, it appears there are two H3K115ac ChIP-seq datasets. One is paired-end and is single-read. So are peaks called with only one replicate of PE? Or are inaccurate peaks called with SR datasets? Either way, this is not a rigorous way to evaluate H3K115ac localization.

      We are sorry that this reviewer was not able to access the data – the token for the GEO accession was provided for reviewers at the journal’s request. All ChIP-seq (and ATAC-seq) experiments (paired and single-end) were performed with two biological replicates and the Pearson’s correlations (all >0.9) between replicates were provided in Supplementary Table 1. This was indicated in both the main text and in the methods. In the revised manuscript we have tried to make this even clearer and have put the relevant Pearsons coefficient (r) into the text at the appropriate places. For the reviewer’s information, here is the complete list of data samples in the GEO Accession:

      Author response image 1.

      While I agree that H3K115ac occupancy is high at +CGIs, the authors downplay that H3K122ac and H3K27ac is also more highly enriched at these locations (page 7, last sentence of first paragraph). I imagine this is all due to the more highly transcribed nature of these genes. Sub-stratifying the K27ac and K122ac by transcription (as in Figure 1G) would help to demonstrate a unique nature of H3K115ac. But even better would be to do an analysis that plots H3K115ac enrichment vs transcription for every individual gene rather than aggregate analyses that are biased by single locations. For example, make an XY scatterplot of RNAPII occupancy or 4SU-seq signal vs H3K115ac level, where each point represents a single gene. Because the interpretation that it is CGI-based and not transcription is confounded with the fact that -CGI are more lowly transcribed. So, looking at Figure 1G, even the -CGI occupancy of H3K115ac is correlated with transcription, but it is just more lowly transcribed.

      We thank the reviewer for these suggestions but point out that Figure 1G shows H3K115ac signal for CGI+ and CGI– TSS that are matched for expressions levels (quartiles of 4SU-seq). Fig 1F shows that H3k115ac is much more of a discriminator between CGI+ and – than H3K27ac or H3K122ac.

      (2) H3K115ac, H3K27ac, and H3K122ac are all more enriched (in aggregate) at +CGI locations (Fig 1F); so do these locations just have more positioned nucleosomes? More H3.3? So that these PTMs are just more enriched due to the opportunity?

      Positioned nucleosomes are generally found downstream of the TSS of active CpG island promoters, so what the reviewer suggests may well account for the relative enrichment of H327ac and H3K122ac at CGI+ vs CGI- promoters in Fig.1F. But H3K115ac localisation is distinct, with the peak at the nucleosome-depleted region not the +1 nucleosome. This is also confirmed by the contour plots in Fig 3. Our observation is also not explained by an enrichment of H3.3 at CGI promoters, since we show that H3K115ac is not specific to H3.3 (Fig 4D).

      (3) The authors note in paragraph 2 of page 7 that "H3K115ac does not scale linearly with gene expression..." but the authors never show a quantification of this; stratification in four clusters is not able to make a linear correlation. Furthermore, in the second line of page 7, the authors state that the levels do generally correlate with transcription. To claim it is a specific CGI link and not transcription is tricky, but I encourage the authors to consider more quantifiable ways, rather than correlations, to demonstrate this point, if it is observed.

      We thank the reviewer for this comment, and taking it into consideration, we have decided to re-phrase this paragraph. The new text reads: “Non-CGI promoters have lower overall levels of transcription compared to CGI promoters, and for this promoter class H3K115ac enrichment detected by ChIP is only really seen for the highest quartile of transcription (4SU) quartile of expression (Figure 1G). CGI promoters on the other hand, exhibit significant levels of detected H3K115ac even for the lowest quartile of expression. These results suggest a special link between CGI promoters and H3K115ac”.

      (4) The authors claim on page 7 that "on average, transcription increased from TSS that also gained H3K115ac but to a modest extent, compared with the more substantial loss of H3K115ac from downregulated TSS". However, both upregulated and downregulated are significant; the difference in magnitude could simply be due to more highly or more lowly transcribed locations, meaning that fold change could be more robustly detected. I caution the authors to substantiate claims like this rather than stating a correlation.

      We thank the reviewer for this comment which relates to the data in Fig 2A. It is Fig. 2B shows that the association of H3K115ac loss with downregulation is statistically stronger than H3K115ac gain with upregulation, but only for CGI promoters. With regard to the text on the original pg 7 that is referred to, we have now reworded this to read “Average levels of transcription increased from TSS that also gained H3K115ac, and there was loss of H3K115ac from downregulated TSS (Figure 2A).”

      (5) For Figure 2C, the authors argue that H3K115ac correlate with bivalent locations. So this is all qualitative and aggregate localization; please quantitatively demonstrate this claim.

      Figure S2D provides statistics for this (observed/expected and Fishers exact test).

      (6) The authors claim in Figure 2 that H3115ac is dynamic during differentiation (title of Figure 2). However, there are locations that gain and lose, or maintain H3K115ac. In fact, the most discussed locations are H3K115ac with no change (2C); which means it is NOT dynamic during differentiation. So what is the message for the role during differentiation? From Supplemental Table 1, it appears there is a single ChIP experiment for H3K115ac in NPC, and it is a single read. So this is also a difficult claim with one replicate. Related to this, in S2A, the authors show K115ac where there is no change in transcription; so what is the role of H3K115ac at TSSs relevant to differentiation - it is at both locations changed and unchanged in transcription, but H3K115ac levels itself do not change at these subsets. So, how is this dynamic? This is very confusing, and clearer analyses and descriptions are necessary to deconvolute these data.

      We apologise for the misleading title for Figure 2. This has now been amended to “Changes in H3K115ac during differentiation”. The message of this figure is that whilst changes in H3K115ac at TSS are small (panels A-C), at enhancers the changes are much more dramatic (panel D). The reviewer is incorrect about the number of replicates for NPCs – there are two biological replicates (see response to point 1b).

      (7) The authors go on to examine H3K115ac enrichment on fragile nucleosomes through sucrose gradient sedimentation. A control for H3K27ac or H3K122ac would be nice for comparison.

      We do not have the material available to perform these experiments

      (8) When discussing Figures 3 and SF3, the authors mention performing a different MNase for a second ChIP. Showing the MNase distribution for both the more highly digested and the lowly digested would be nice. a) Related to the above, the authors show input in SF3E to argue that the difference in H3K115ac vs H3K27ac is not due to the library, but they do not show the MNase digestion patterns, which is more important for this argument.

      Input libraries (first two graphs of FigS3E) are the MNase-digested chromatin. Comparison of nucleotide frequencies from millions of reads is more robust method than the fragment length patterns.

      (9) The authors move on to examine H3K115ac at enhancers. Just out of curiosity, given what was found at promoters, is H3K115ac enriched at +CGI enhancers? And what is the correlation with enhancer transcription?

      This is an interesting point, but the number of enhancers associated with CGI is not very high and so we did not focus on this. We have not analysed a correlation with eRNAs in this paper.

      (10) The authors state on page 14 that the most frequent changes in H3K115ac during differentiation are at these enhancers. So do these changes connect with differentiation-specific genes, and/or genes that have altered transcription during differentiation? Just trying to understand the functional role.

      Given the challenges of connecting enhancers with target genes, we have not addressed this question quantitatively. However, we draw the reviewer’s attention to the Genome Browser shots in Figures 2D and S2C, which show clear gain of H3K115ac (and ATAC-seq peaks) at intra and intergenic regions close to genes whose transcription is activated during the differentiation to NPCs.

      (11) Related, at the end of page 14, the authors state that the changes in H3K115ac correlate with changes in ATAC-seq; I imagine this dynamic is not unique for H3K115ac and this is observed for other PTMs (H3K27ac), so assessing and clarifying this, to again get to the specific interest of H3K115ac, would be ideal.

      We have not claimed that chromatin accessibility is unique to H3K115ac. It is the location of H3K115ac which is found inside the ATAC-seq peak region while H3K27ac is found only upstream/downstream of the ATAC peak that is so striking. This is apparent in Fig 4C.

      (12) The authors examine levels of H3K115ac in H3.3 KO cell lines via western blot (Figure 4D), but no replicates and/or quantification are shown.

      We now provide a biological replicate for the Western Blot (new FigS4H) together with an image of the whole gel for the data in Fig 4D

      (13) In Figure S4 and at the end of page 17, the authors are arguing that there is a link to pioneer TF complexes, based on Oct4 binding. First, while Oct4 has pioneering activity, not all Oct4 sites (or motifs) are pioneering; this has been established. So if you want to use Oct4, substratifying by pioneer vs no pioneer is necessary. Second, demonstrating this is unique to pioneer and not to non-pioneer TFs would be an important control.

      In response to the reviewer’s comment, we have removed the term “pioneer” from the manuscript.

      (14) Minor point: Figure 4 A and B, there are some formatting issues with the scale bars.

      We thank the reviewer for pointing this out, and the errors have been corrected in the revised figure.

      (15) Minor point is that it should be clear when single replicates of data are used and when PE/SR sequences are combined or which one is used in each analysis, as this was hard to discern when reading the paper and figure legends.

      We have clearly stated in the text that, after Figure2, we repeated all experiments in paired-end mode. All processing steps are defined separately for single end and paired end datasets in the method section. Details of biological replicates are provided in Sup. Table 1. These concerns are also addressed in our response to Reviewer’s public comment-1.

      (16) Minor point: it is surprising that different MNase and different units were used in the ChIP vs sucrose sedimentation. Could the authors clarify why?

      Chromatin prep for sucrose gradients were done on a much larger scale than for ChIP-seq and required different setups to obtain the right level of MNase digestion.

      (17) The authors note that fragile nucleosomes contain H2A.Z and H3.3, but they never perform an analysis of available data to demonstrate a correlation (or better a quantifiable correlation) between H3K115ac occupancy and these marks at the locations they identify H3K115ac.

      Since have shown (Fig. 4) that depletion of H3.3 does not affect overall levels of H3K115ac, we do not think there is value in further quantitative correlative analyses of H3K115ac and variant histones.

      (18) Minor point: What is the overlap in peaks for H3K115ac, H3K122ac, and H3K27ac (Figure 1C)?

      Nearly all H3K115ac peaks overlap with H3K122ac and/or H3K27ac. Its most distinct properties are its association with CGI promoters, fragile nucleosomes and its unique localisation within the NDRs, three points that the manuscript is focussed on.

      Reviewer #3 (Recommendations for the authors):

      (1) The western blot results in Figure 4D probing for H3, H3.3, and H3K115ac use Ponceau S staining, presumably of an area of the membrane where histones might be expected to migrate, as a measure of loading. However, the Ponceau S bands appear uniformly weaker in the H3.3KO lanes, yet despite this, blotting with H3.3 antibody detects a band in H3.3 knockout ESCs, suggesting that the antibody does not have a high degree of specificity. Again, a blocking experiment with appropriate peptides would instill more confidence in the specificity of these reagents, and/or the authors could provide independent validation of the knockout model to differentiate between a partial knockout or antibody cross-reactivity (e.g., by Sanger sequencing).

      In a revised Fig. S4H we now show the whole gel corresponding to this blot but including co-staining with an antibody for H4 to provide a better loading control. We also provide a biological replicate of this Western blot in the lower panel of Fig. S4H.

      (2) The manuscript would benefit from in vitro follow-up and validation, but if the authors intend to keep the manuscript primarily in silico, I suggest dedicating a few lines in each section to explain the plots, their axes, and their purpose, as well as to assist with interpretation, rather than directly discussing the results. This would make the manuscript more accessible and understandable for a broader audience in the field of epigenetics.

      In the revised version, we have tried to improve the text to make the data more accessible to a broad audience.

    1. Reviewer #1 (Public Review):

      The authors tested the hypothesis that at high elevations avian eggs will be adapted to prevent desiccation that might arise from loss of water to surrounding drier air. They used a combination of gas diffusion experiments and scanning electron microscopy to examine water vapour conductance rates and eggshell structure, including thickness, pore size, and pore density among 197 bird species distributed along an elevational gradient in the Andes. While there was a correlation between water vapour conductance and elevation among species, a decrease in water vapour conductance with elevation was not associated with eggshell thickness, pore size, and pore density, suggesting the variation in the structure of the eggshells is unlikely to do with among species differences in water loss along elevational gradients. This study is very interesting and timely, especially with increasing water vapour pressure due to climate warming. It is a very well-written study and easy to read. However, I have some concerns about the conclusions drawn from the results.

      There are more than twice as many species in low and medium-elevation sites compared to high-elevation sites, so the amount of variation in low and medium-elevation should be expected to be higher by default. The argument for a wider range of variation in low-elevation species will be stronger if the comparison was a similar sample size. Moreover, the pattern clearly breaks down within families. Note also that for Low and medium elevation there is no difference in the amount of variation in conductance residuals possibly because the sample sizes are similar. The seemingly strong positive correlation between eggshell conductance and egg mass may be driven by the five high and two medium-elevation species with large eggs. There seem to be hardly any high-elevation species with egg mass greater than 12g whereas species in low elevation egg size seem to be as high as 80g (Figure 2a). Since larger eggs (and thus eggs of larger birds) lose more water compared to smaller eggs, the correlation between water vapour conductance and elevation may be more strongly associated with body size distribution along elevational gradients rather than egg structure and function.

      Authors argue that the observed variation in the relationship between water vapour conductance and elevation among and within bird families suggests potential differences in the adaptive response to common selective pressures in terms of eggshell thickness and pore density, and size. The evidence for this is generally weak from the data analyses because the decrease in water vapour conductance with elevation was not consistent across taxonomic groups nor were differences associated with specific patterns in eggshell thickness and pore density, and size.

      It is not clear how the authors expected the relationship between water vapour conductance and elevation to differ among taxonomic groups and there was no attempt to explain the biological implication of these differences among taxonomic groups based on the specific traits of the species or their families. This missing piece of information is crucial to justify the argument that differences among taxonomic groups may be due to differences in adaptive response.

    2. Author response:

      Reviewer #1 (Public Review):

      The authors tested the hypothesis that at high elevations avian eggs will be adapted to prevent desiccation that might arise from loss of water to surrounding drier air. They used a combination of gas diffusion experiments and scanning electron microscopy to examine water vapour conductance rates and eggshell structure, including thickness, pore size, and pore density among 197 bird species distributed along an elevational gradient in the Andes. While there was a correlation between water vapour conductance and elevation among species, a decrease in water vapour conductance with elevation was not associated with eggshell thickness, pore size, and pore density, suggesting the variation in the structure of the eggshells is unlikely to do with among species differences in water loss along elevational gradients. This study is very interesting and timely, especially with increasing water vapour pressure due to climate warming. It is a very well-written study and easy to read. However, I have some concerns about the conclusions drawn from the results.

      There are more than twice as many species in low and medium-elevation sites compared to high-elevation sites, so the amount of variation in low and medium-elevation should be expected to be higher by default. The argument for a wider range of variation in lowelevation species will be stronger if the comparison was a similar sample size. Moreover, the pattern clearly breaks down within families. Note also that for Low and medium elevation there is no difference in the amount of variation in conductance residuals possibly because the sample sizes are similar. The seemingly strong positive correlation between eggshell conductance and egg mass may be driven by the five high and two medium-elevation species with large eggs. There seem to be hardly any high-elevation species with egg mass greater than 12g whereas species in low elevation egg size seem to be as high as 80g (Figure 2a). Since larger eggs (and thus eggs of larger birds) lose more water compared to smaller eggs, the correlation between water vapour conductance and elevation may be more strongly associated with body size distribution along elevational gradients rather than egg structure and function.

      We thank the reviewer for this thoughtful observation. As noted in our response to comment 3, we recognize that the higher number of species at low and mid-elevations reflects the natural turnover in species richness along elevational gradients, and we are transparent about this caveat in our revised Discussion section. Nevertheless, to address this specific concern, we conducted additional analyses excluding the species with large eggs (i.e., egg mass >12g, which are only present at low and mid-elevations in our dataset). These analyses are now included in the Supplementary Figure 1, and the main pattern of lower water vapor conductance at high elevations holds even when larger eggs are excluded.

      We agree that the well-known scaling relationship between egg mass and conductance (recognized since the 1970s) may partially explain the observed trends across the elevational gradient. Our aim was to explore whether the known relationship between egg size and conductance varies when incorporating environmental variables such as elevation, which brings with it changes in humidity and oxygen availability. While we acknowledge the possible confounding effect of body size distributions along the gradient, our results, even after controlling for egg size (residual analysis), still suggest a decrease in conductance at higher elevations, consistent with predictions based on environmental conditions.

      We have clarified these points in the revised Discussion, including the acknowledgment that disentangling the relative contributions of body size and elevation to conductance patterns remains challenging and warrants further study.

      Authors argue that the observed variation in the relationship between water vapour conductance and elevation among and within bird families suggests potential differences in the adaptive response to common selective pressures in terms of eggshell thickness and pore density, and size. The evidence for this is generally weak from the data analyses because the decrease in water vapour conductance with elevation was not consistent across taxonomic groups nor were differences associated with specific patterns in eggshell thickness and pore density, and size.

      We appreciate the reviewer’s comments on the observed variation in water vapor conductance across taxonomic groups. As mentioned in response to comment 7, we have removed the explicit analyses and figures showing within-family comparisons, as these were exploratory and not directly tied to a specific hypothesis. We have also toned down our speculations regarding the potential adaptive drivers of the observed variation. In the revised Discussion, we emphasize the need for further research to explore these patterns and acknowledge the limitations of our current dataset in making strong conclusions about the adaptive responses to selective pressures.

      It is not clear how the authors expected the relationship between water vapour conductance and elevation to differ among taxonomic groups and there was no attempt to explain the biological implication of these differences among taxonomic groups based on the specific traits of the species or their families. This missing piece of information is crucial to justify the argument that differences among taxonomic groups may be due to differences in adaptive response.

      We appreciate the reviewer’s point. To clarify, we were not expecting the relationship between water vapor conductance and elevation to differ among taxonomic groups. Rather, our primary hypothesis was that water vapor conductance would decrease with elevation due to the drier conditions in highland habitats, and we sought to link this pattern with structural characteristics of the eggshell. The suggestion of potential differences among taxonomic groups arose from the lack of a consistent pattern across families, which prompted us to consider possible adaptive variation. We now address this more clearly in the Discussion section, acknowledging the need for further exploration into the potential selective pressures driving this variation among taxonomic groups.

      Reviewer #2 (Public Review):

      This paper represents a strong advance for two main reasons. First, it provides evidence that egg physiology varies with elevation as predicted by the hypothesis that eggs are physiologically adapted to certain climatic conditions. This means egg physiological adaptation is a factor that could influence species' elevational ranges. Second, it is a proof-of-concept study that shows it is possible to measure eggshell physiology for a large number of species in the field in order to test hypotheses. As such, it should inspire many further tests that examine adaptation in egg physiology in the context of species' distributions along environmental gradients.

      There are two caveats that readers should be aware of. First, measuring these traits is difficult, and there remain questions about the efficacy of different methods. For example, the authors note that quantifying eggshell structures is very difficult, with several unresolved questions about their method of using scanning electron microscopy images to measure eggshell pores. Similarly, the authors mention that temperature variation may partially influence their main result that high-elevation eggs lose water at slower rates than low-elevation eggs (temperatures were colder for experiments at high elevations than for low elevations). Second, I regard the analyses of eggshell traits for specific families as exploratory. There are no a priori expectations for how different families might be expected to differ in their patterns. These analyses are fruitful in that they generate additional hypotheses that future work can test. However, it does mean that the statistical significance of eggshell trait relationships with elevation for specific families should be interpreted with caution.

      We thank Reviewer 2 for these insightful comments. As mentioned earlier, measuring these traits is indeed very challenging, and we acknowledge the limitations of our methods, particularly when it comes to using scanning electron microscopy to quantify eggshell structures. We are aware of the unresolved questions around these techniques, and we plan to continue refining these methods in future studies. Regarding the influence of temperature variation on water loss, we recognize that colder temperatures at high elevations may have influenced our results, and we address this potential confounding factor in the Discussion section, Line 257.

      We also agree with the reviewer’s point regarding the exploratory nature of the family-specific analyses. These analyses were not guided by specific hypotheses, other than the expectation of replicating the overall pattern, and we recognize that they should be interpreted with caution. They serve primarily to generate additional hypotheses for future studies. In the revised manuscript, we have toned down the emphasis on the statistical significance of eggshell trait relationships with elevation for specific families, and we emphasize the need for further research to confirm these patterns.

    1. Reviewer #1 (Public Review):

      This study aims to develop a new system to analyze genetic determinants of neutrophil function by large-scale genetic screens. For that, the authors use a genetically-engineered ER-Hoxb8 neutrophil progenitor line that expresses Cas9 to perform CRISPR screens and to identify genes required for neutrophil survival and differentiation.

      A main strength of this study is that the authors develop a pooled CRISPR sgRNA library applicable to neutrophils and show potential determinants of neutrophil differentiation in vitro using this screening methodology.

      A main weakness of this study is that identified candidates associated with neutrophil differentiation, as those indicated in Fig. 4A, were not validated in vivo using neutrophil-specific K.O. models or further characterized in vitro (e.g. transcriptional or epigenetic changes during maturation when compared to non-targeting sgRNA controls).

      As secondary strengths, the authors provide evidence of efficient gene editing in Cas9+ER-Hoxb8 neutrophils both in vivo and in vitro and provide evidence of the specificity of this methodology using a Cas9+ER-Hoxb8 immortalized cell line that differentiates into macrophages.

      In terms of methodology, this study provides a useful tool to explore gene regulatory networks in neutrophils in large-scale genetic screens. However, it falls short in identifying and validating the true potential of this kind of methodology in the biology of the neutrophil.

      Moreover, the technical advances in the field are only incremental as several studies, including those using CRISPR/Cas9 technology in Hoxb-8 immortalized neutrophil progenitor cell lines have been already performed.

    2. Reviewer #2 (Public Review):

      In this manuscript, Jong et al. provide and validate a very useful resource for performing CRISPR screenings to study neutrophil differentiation and function. The major strength of the paper lies in its careful validation of many aspects of the Hoxb8-immortalized progenitor cells, including their differentiation capacity, their ability to clear bacteria, and their capacity to differentiate in vivo. The authors succeed at this, with results correctly supporting their conclusions. The major weaknesses are its presentation and writing, some of which are poorly organized. Finally, while the potential impact of this resource in the field could be very large, the CRISPR screening results appear half-baked, almost preliminary, and could be better validated, or at least presented. A few other points that warrant revision are included below:

      • The introduction should be better constructed and organized. It should be written with more connectors to present facts in a stream that flows naturally, from the broad general facts to the experimental details implemented in the manuscript. It should also discuss other similar approaches used in the literature, such as LaFleur et al. 2019, and relate in which ways these presented methods could be better.

      • The scheme in Figure 4A should more clearly indicate the timings, doublings, numbers of cells, and other aspects of the experimental design.

      • The volcano plot in Figure 4B is poorly informative and almost redundant. What does one make of it?

      • The representation (normalized reads) of each sgRNA in the library and across multiple experiments, including their correlation, should be checked and plotted, to visualize how robust these replicates are.

      • In Figure 4E, the distribution of the hit sgRNAs should be compared to all other targeting guides (instead of just to non-targeting controls). Linear density distribution plots or scatter plots of all guides are usually the best way, but there are others (for example, see Figure 4 of LaFleur et al. 2019). Ideally, each independent sgRNA for each gene in the library, as well as biological replicates, should be separately shown, with hits clearly highlighted.

      • While in vivo differentiation is shown as possible with these cell lines, it is unclear whether CRISPR screenings could be performed in vivo too. Would sgRNA representation suffice for genome-wide? At least some of the new hits could be validated by testing differentiation in vivo (i.e. WASH complex).

      • In the methods section, the RNA-seq analysis pipeline details are missing (versions, software for alignment, quantification, differential gene expression, and enrichment). Also, parameters for MAGeCK and MAGeCKFlute should be explicit and detailed.

      • The discussion is mostly a summary of the results. It is lacking in detail and thoughtful discussion regarding novelty and impact beyond the validation of the cell line. What about potential applications? What about extending screenings to test bacterial-killing, as suggested in Figure 2? What about limitations compared to other similar methods out there? There is little discussion of such important potential matters. Also, a large part of the discussion is dedicated to discussing details about Cebpe that are all well known in the literature and add little value.

      • Figure legends are typically too succinct and hard to interpret, especially for non-experts. The text should enable the figure reader to correctly interpret what is shown in each panel.

    1. Author response:

      The following is the authors’ response to the previous reviews

      Public Reviews:

      Reviewer #1 (Public review):

      The authors assess the effectiveness of electroporating mRNA into male germ cells to rescue the expression of proteins required for spermatogenesis progression in individuals where these proteins are mutated or depleted. To set up the methodology, they first evaluated the expression of reporter proteins in wild-type mice, which showed expression in germ cells for over two weeks. Then, they attempted to recover fertility in a model of late spermatogenesis arrest that produces immotile sperm. By electroporating the mutated protein, the authors recovered the motility of ~5% of the sperm; although the sperm regenerated was not able to produce offspring using IVF, the embryos reached the 2-cell state (in contrast to controls that did not progress past the zygote state).

      This is a comprehensive evaluation of the mRNA methodology with multiple strengths. First, the authors show that naked synthetic RNA, purchased from a commercial source or generated in the laboratory with simple methods, is enough to express exogenous proteins in testicular germ cells. The authors compared RNA to DNA electroporation and found that germ cells are efficiently electroporated with RNA, but not DNA. The differences between these constructs were evaluated using in vivo imaging to track the reporter signal in individual animals through time. To understand how the reporter proteins affect the results of the experiments, the authors used different reporters: two fluorescent (eGFP and mCherry) and one bioluminescent (Luciferase). Although they observed differences among reporters, in every case expression lasted for at least two weeks. The authors used a relevant system to study the therapeutic potential of RNA electroporation. The ARMC2-deficient animals have impaired sperm motility phenotype that affects only the later stages of spermatogenesis. The authors showed that sperm motility was recovered to ~5%, which is remarkable due to the small fraction of germ cells electroporated with RNA with the current protocol. The sperm motility parameters were thoroughly assessed by CASA. The 3D reconstruction of an electroporated testis using state-of-the-art methods to show the electroporated regions is compelling.

      The main weakness of the manuscript is that although the authors manage to recover motility in a small fraction of the sperm population, it is unclear whether the increased sperm quality is substantial to improve assisted reproduction outcomes. The authors found that the rescued sperm could be used to obtain 2-cell embryos via IVF, but no evidence for more advanced stages of embryo differentiation was provided. The motile rescued sperm was also successfully used to generate blastocyst by ICSI, but the statistical significance of the rate of blastocyst production compared to non-rescued sperm remains unclear. The title is thus an overstatement since fertility was never restored for IVF, and the mutant sperm was already able to produce blastocysts without the electroporation intervention.

      Overall, the authors clearly show that electroporating mRNA can improve spermatogenesis as demonstrated by the generation of motile sperm in the ARMC2 KO mouse model.

      We thank the reviewer for this thoughtful and constructive comment. We agree that our study demonstrates a partial functional recovery of spermatogenesis rather than a complete restoration of fertility. Our main objective was to establish and validate a proof-of-concept approach showing that mRNA electroporation can rescue the expression of a missing or mutated protein in post-meiotic germ cells and result in the production of motile sperm.

      To address the reviewer’s concern, we have the title and discussion to more accurately reflect the scope of our findings. The new title reads:

      “Sperm motility in mice with oligo-astheno-teratozoospermia restored by in vivo injection and electroporation of naked mRNA”

      In the manuscript, we now emphasize that while motility recovery was significant, complete fertility restoration was not achieved. We have also clarified that:

      The 5% recovery in motile sperm represents a substantial improvement considering the small population of germ cells reached by the current electroporation method.

      The 2-cell embryo formation observed after IVF serves as a strong indication of partial functional recovery

      Finally, we now explicitly state in the Discussion that this approach should be considered a therapeutic proof-of-concept, demonstrating feasibility and potential, rather than a fully curative intervention.

      Reviewer #2 (Public review):

      The authors inject, into the rete testes, mRNA and plasmids encoding mRNAs for GFP and then ARMC2 (into infertile Armc2 KO mice) in a gene therapy approach to express exogenous proteins in male germ cells. They do show GFP epifluorescence and ARMC2 protein in KO tissues, although the evidence presented is weak. Overall, the data do not necessarily make sense given the biology of spermatogenesis and more rigorous testing of this model is required to fully support the conclusions, that gene therapy can be used to rescue male infertility.

      In this revision, the authors attempt to respond to the critiques from the first round of reviews. While they did address many of the minor concerns, there are still a number to be addressed. With that said, the data still do not support the conclusions of the manuscript.

      We thank the reviewer for their careful and detailed assessment of our manuscript. We appreciate the concerns raised regarding mRNA stability, GFP localization, and the interpretation of spermatogenesis stages, and we have addressed these points in the manuscript and in the responses below.

      (1) The authors have not satisfactorily provided an explanation for how a naked mRNA can persist and direct expression of GFP or luciferase for ~3 weeks. The most stable mRNAs in mammalian cells have half-lives of ~24-60 hours. The stability of the injected mRNAs should be evaluated and reported using cell lines. GFP protein's half-life is ~26 hours, and luciferase protein's half-life is ~2 hours.

      We thank the reviewer for this important comment. The stability of mRNA-GFP was assessed by RT-QPCR in HEK cells and seminiferous tubule cells (Fig. 5). mRNA-GFP was detected for up to 60 hours in HEK cells and for up to two weeks in seminiferous tubule cells (Fig. 5A). Together, these results suggest that the long-lasting fluorescence observed in our experiments reflects a combination of transcript stability, efficient translation within germ cells and the slow protein turnover that is typical of the spermatogenic lineage.

      (2) There is no convincing data shown in Figs. 1-8 that the GFP is even expressed in germ cells, which is obviously a prerequisite for the Armc2 KO rescue experiment shown in the later figures! In fact, to this reviewer the GFP appears to be in Sertoli cell cytoplasm, which spans the epithelium and surrounds germ cells - thus, it can be oft-confused with germ cells. In addition, if it is in germ cells, then the authors should be able to show, on subsequent days, that it is present in clones of germ cells that are maturing. Due to intracellular bridges, a molecule like GFP has been shown to diffuse readily and rapidly (in a matter of minutes) between adjacent germ cells. To clarify, the authors must generate single cell suspensions and immunostain for GFP using any of a number of excellent commercially-available antibodies to verify it is present in germ cells. It should also be present in sperm, if it is indeed in the germline.

      We thank the reviewer for this insightful comment. To directly address the concern, we performed additional experiments to assess GFP expression in germ cells following in vivo mRNA delivery. GFP-encoding mRNA was injected and electroporated into the testes on day 0. On day 1, testes were collected, enzymatically dissociated, and the resulting seminiferous tubule cell suspensions were cultured for 12 hours. Live cells were then analyzed by fluorescence microscopy (Fig. 10).

      We observed GFP expression in various germ cell types, including pachytene spermatocytes (53,4 %) (Fig 10 A-), round spermatids (25 %) (Fig 10B-E) and in elongated spermatids (11,4%) (Fig 10 C-E). The identification of these cell types was based on DAPI nuclear staining patterns, cell size fig 10 F, non-adherent characteristics, and the use of an enzymatic dissociation protocol.

      Fluorescence imaging revealed strong cytoplasmic GFP signals in each of these populations, confirming efficient transfection and translation of the delivered mRNA. These results demonstrate that the in vivo injection and electroporation protocol enables effective mRNA transfection across multiple stages of spermatogenesis. These results confirm that the injected mRNA is efficiently translated in germ cells at various stages of spermatogenesis. Together, these data validate the germ cell-specific nature of the GFP signal, supporting the Armc2 KO rescue experiments.

      As mentioned previously, we assessed the stability of mRNA-GFP using RT-QPCR in HEK cells and seminiferous tubule cells (see Fig. 5). mRNA-GFP was detected for up to 60 hours in HEK cells and for up to two weeks in seminiferous tubule cells. Together, these results suggest that the long-lasting fluorescence observed in our experiments reflects a combination of transcript stability and local translation within germ cells, as well as the slow protein turnover typical of the spermatogenic lineage.

      Other comments:

      70-1 This is an incorrect interpretation of the findings from Ref 5 - that review stated there were ~2,000 testis-enriched genes, but that does not mean "the whole process involves around two thousand of genes"

      We thank the reviewer for this helpful comment. We agree that our previous phrasing was imprecise. We have revised the sentence to clarify that approximately 2,000 genes show testis-enriched expression, rather than implying that the entire spermatogenic process is limited to these genes. The corrected sentence now reads:

      “Spermatogenesis involves the coordinated expression of a large number of genes, with approximately 2,000 showing testis-enriched expression, about 60% of which are expressed exclusively in the testes”

      74 would specify 'male':

      we have now specified it as you suggested.

      79-84 Are the concerns with ICSI due to the procedure itself, or the fact that it's often used when there is likely to be a genetic issue with the male whose sperm was used? This should be clarified if possible, using references from the literature, as this reviewer imagines this could be a rather contentious issue with clinicians who routinely use this procedure, even in cases where IVF would very likely have worked:

      We thank the reviewer for this important comment. Concerns about ICSI outcomes indeed reflect two partly overlapping causes: the procedure itself (direct sperm injection and associated laboratory manipulations) and the clinical/genetic background of couples undergoing ICSI (especially men with severe male-factor infertility). Large reviews and meta-analyses report a small increase in some perinatal and congenital risks after ART/ICSI, but these studies conclude that it is difficult to fully disentangle procedural effects from parental factors. Importantly, genetic or epigenetic abnormalities in the male (which motivate use of ICSI) likely contribute to adverse outcomes in offspring, while some studies also suggest that ICSI-specific manipulations may alter epigenetic marks in embryos. For these reasons professional bodies recommend reserving ICSI for appropriate male-factor indications rather than as routine insemination for non-male-factor cases

      We have revised the text accordingly to clarify this distinction:

      “ICSI can efficiently overcome the problems faced.  Nevertheless, concerns persist regarding the potential risks associated with this technique, including blastogenesis defect, cardiovascular defect, gastrointestinal defect, musculoskeletal defect, orofacial defect, leukemia, central nervous system tumors, and solid tumors [1-4]. Statistical analyses of birth records have demonstrated an elevated risk of birth defects, with a 30-40 % increased  likelihood in cases involving ICSI [1-4], and a prevalence of birth defects between 1 % and 4 % [3]. It is important to note, however, that the origin of these risks remains debated. Several large epidemiological and mechanistic studies indicate that both the procedure itself (direct microinjection and in vitro manipulation) and the underlying genetic or epigenetic abnormalities often present in men requiring ICSI contribute to the observed outcomes [1, 3] [5, 6] . To overcome these drawbacks, a number of experimental strategies have been proposed to bypass ARTs and restore spermatogenesis and fertility, including gene therapy [7-10].”

      199 Codon optimization improvement of mRNA stability needs a reference;

      We have added the references accordingly: [11-15]

      In one study using yeast transcripts, optimization improved RNA stability on the order of minutes (e.g., from ~5 minutes to ~17 minutes); is there some evidence that it could be increased dramatically to days or weeks?

      We agree with the reviewer that codon optimization can enhance mRNA stability, but available evidence indicates that this effect is moderate. In Saccharomyces cerevisiae, Presnyak et al. (2015) [16] showed that codon optimization increased mRNA half-life from approximately 5 minutes to ~17 minutes, representing a several-fold improvement rather than a shift to days or weeks. Similar codon-dependent stabilization has been observed in mammalian systems, where transcripts enriched in optimal codons exhibit longer half-lives and enhanced translation efficiency [11]; [17]). However, these studies consistently report effects on the scale of minutes to hours. In mammalian cells, the prolonged stability of therapeutic or vaccine mRNAs—lasting for days—is primarily achieved through additional features such as optimized untranslated regions, chemical nucleotide modifications (e.g., N¹-methylpseudouridine), and protective delivery systems, rather than codon usage alone ([18]; [19]).

      Other molecular optimizations that improve in vivo mRNA stability and translation include a poly(A) tail, which binds poly(A)-binding proteins to protect the transcript from 3′ exonuclease degradation and promotes ribosome recycling, and a CleanCap structure at the 5′ end, which mimics the natural Cap 1 configuration, protects against 5′ exonuclease attack, and enhances translational initiation [11-15]. Together, these modifications act synergistically to stabilize the transcript and support efficient translation.

      472-3 The reported half-life of EGFP is ~36 hours - so, if the mRNA is unstable (and not measured, but certainly could be estimated by qRT-PCR detection of the transcript on subsequent days after injection) and EGFP is comparatively more stable (but still hours), how does EGFP persist for 21 days after injection of naked mRNA??

      We thank the reviewer for this important comment. The stability of mRNA-GFP was assessed by RT-QPCR in HEK cells and seminiferous tubule cells (Fig. 5). mRNA-GFP was detected for up to 60 hours in HEK cells and for up to two weeks in seminiferous tubule cells (Fig. 5). Together, these results suggest that the long-lasting fluorescence observed in our experiments reflects a combination of transcript stability, efficient translation within germ cells and the slow protein turnover that is typical of the spermatogenic lineage.

      Curious why the authors were unable to get anti-GFP to work in immunostaining?

      We appreciate the reviewer’s question. We attempted to detect GFP using several commercially available anti-GFP antibodies under various standard immunostaining conditions. However, in our hands, these antibodies consistently produced either no signal or high background staining, making the results unreliable. We therefore relied on direct detection of GFP fluorescence, which provides a more accurate and specific readout of protein expression in our system.

      In Fig. 3-4, the GFP signals are unremarkable, in that they cannot be fairly attributed to any structure or cell type - they just look like blobs; and why, in Fig. 4D-E, why does the GFP signal appear stronger at 21 days than 15 days? And why is it completely gone by 28 days? This data is unconvincing.

      We would like to thank the reviewer for their comments. Figure 3–4 provides a global overview of GFP expression on the surface of the testis. The entire testis was imaged using an inverted epifluorescence microscope, and the GFP signal represents a composite of multiple seminiferous tubules across the tissue surface. Due to this whole-organ imaging approach, it is not possible to resolve individual structures such as the basement membrane or lumen, which is why the signals may appear as diffuse “blobs.”

      Regarding the time-course in Figure 4D–E, the apparent increase in GFP signal at 21 days compared with 15 days likely reflects accumulation and translation of the delivered mRNA in germ cells over time, whereas the absence of signal at 28 days corresponds to the natural turnover and degradation of GFP protein and mRNA in the tissue. We hope this explanation clarifies the observed patterns of fluorescence.

      If the authors did a single cell suspension, what types or percentage of cells would be GFP+? Since germ cells are not adherent in culture, a simple experiment could be done whereby a single cell suspension could be made, cultured for 4-6 hours, and non-adherent cells "shaken off" and imaged vs adherent cells. Cells could also be fixed and immunostained for GFP, which has worked in many other labs using anti-GFP.

      We thank the reviewer for this insightful comment. To directly address the concern, we performed additional experiments to assess GFP expression in germ cells following in vivo mRNA delivery. GFP-encoding mRNA was injected and electroporated into the testes on day 0. On day 1, testes were collected, enzymatically dissociated, and the resulting seminiferous tubule cell suspensions were cultured for 12 hours. Live cells were then analyzed by fluorescence microscopy (Fig. 10).

      We observed GFP expression in various germ cell types, including pachytene spermatocytes (53,4 %) (Fig 10 A-), round spermatids (25 %) (Fig 10B-E) and in elongated spermatids (11,4%) (Fig 10 C-E). The identification of these cell types was based on DAPI nuclear staining patterns, cell size fig 10 F, non-adherent characteristics, and the use of an enzymatic dissociation protocol.

      Fluorescence imaging revealed strong cytoplasmic GFP signals in each of these populations, confirming efficient transfection and translation of the delivered mRNA. These results demonstrate that the in vivo injection and electroporation protocol enables effective mRNA transfection across multiple stages of spermatogenesis.

      These results confirm that the injected mRNA is efficiently translated in germ cells at various stages of spermatogenesis. Together, these data validate the germ cell-specific nature of the GFP signal, supporting the Armc2 KO rescue experiments.

      As mentioned previously, we assessed the stability of mRNA-GFP using RT-QPCR in HEK cells and seminiferous tubule cells (see Fig. 5). mRNA-GFP was detected for up to 60 hours in HEK cells and for up to two weeks in seminiferous tubule cells. Together, these results suggest that the long-lasting fluorescence observed in our experiments reflects a combination of transcript stability and local translation within germ cells, as well as the slow protein turnover typical of the spermatogenic lineage.

      In Fig. 5, what is the half-life of luciferase? From this reviewer's search of the literature, it appears to be ~2-3 h in mammalian cells. With this said, how do the authors envision detectable protein for up to 20 days from a naked mRNA? The stability of the injected mRNAs should be shown in a mammalian cell line - perhaps this mRNA has an incredibly long half-life, which might help explain these results. However, even the most stable endogenous mRNAs (e.g., globin) are ~24-60 hrs.

      We did not directly assess the stability of luciferase mRNA, but we evaluated the persistence of GFP mRNA, which was synthesized and optimized using the same sequence optimization and chemical modification strategy as the luciferase mRNA. In these experiments, mRNA-GFP was detectable in seminiferous tubule cells for up to two weeks after injection. We therefore expect a similar stability profile for the luciferase mRNA. These findings suggest that the prolonged fluorescence or bioluminescence observed in our study likely reflects a combination of factors, including enhanced transcript stability, local translation within germ cells, and the inherently slow protein turnover characteristic of the spermatogenic lineage.

      527-8 The Sertoli cell cytoplasm is not just present along the basement membrane as stated, but also projects all the way to the lumina:

      we clarified the sentence " Sertoli cells have an oval to elongated nucleus and the cytoplasm presents a complex shape (“tombstone” pattern) along the basement membrane, with long projections that extend toward the lumen."

      529-30 This is incorrect, as round spermatids are never "localized between the spermatocytes and elongated spermatids" - if elongated spermatids are present, rounds are not - they are never coincident in the same testis section:

      We thank the reviewer for this important comment and for drawing attention to the detailed staging of the seminiferous epithelium. We agree that the spatial organization of germ cells varies depending on the stage of spermatogenesis. While round spermatids (steps 1–8) and elongated spermatids (steps 9–16) are typically associated with distinct stages, transitional stages of the seminiferous epithelium can contain both cell types in close proximity, reflecting the continuous and overlapping nature of spermatid differentiation (Meistrich, 2013, Methods Mol. Biol. 927:299–307). We have revised the text to clarify this point, indicating that the relative positioning of germ cell types depends on the stage of the seminiferous cycle rather than implying their constant coexistence within the same tubule section.

      Fig. 7. To this reviewer, all of the GFP appears to be in Sertoli cell cytoplasm In Figs 1-8 there is no convincing evidence presented that GFP is expressed in germ cells! In fact, it appears to be in Sertoli cells.

      We thank the reviewer for their observation. As previously mentioned, we have included an additional experiment specifically demonstrating GFP expression in germ cells (fig 10). This new data provides clear evidence that the GFP signal is not restricted to Sertoli cells and confirms successful uptake and translation of GFP mRNA in germ cells.

      Fig. 9 - alpha-tubuline?

      We corrected the figure.

      Fig. 11 - how was sperm morphology/motility not rescued on "days 3, 6, 10, 15, or 28 after surgery", but it was in some at 21 and 35? How does this make sense, given the known kinetics of male germ cell development??

      We note the reviewer’s concern regarding the timing of motile sperm appearance. Variability among treated mice is expected because transfection efficiency differed between spermatogonia and spermatids. Full spermiogenesis requires ~15 days, and epididymal transit adds ~8 days, consistent with motile sperm appearing around 21 days post-injection in some mice.

      And at least one of the sperm in the KO in Fig. B5 looks relatively normal, and the flagellum may be out-of-focus in the image? With only a few sperm for reviewers to see, how can we know these represent the population?

      We thank the reviewer for their comment. Upon closer examination of the image, the flagellum of the spermatozoon in question is clearly abnormally short and this is not due to being out of focus. Furthermore, the supplementary figure shows that the KO consistently lacks normal spermatozoa. These defects are consistent with previous findings from our laboratory [22], confirming that the observed phenotype is representative of the KO population rather than an isolated occurrence.

      Reviewer #3 (Public review):

      Summary:

      The authors used a novel technique to treat male infertility. In a proof-of-concept study, the authors were able to rescue the phenotype of a knockout mouse model with immotile sperm using this technique. This could also be a promising treatment option for infertile men.

      Strengths:

      In their proof-of-concept study, the authors were able to show that the novel technique rescues the infertility phenotype of Armc2 knockout spermatozoa. In the current version of the manuscript, the authors have added data on in vitro fertilisation experiments with Armc2 mRNA-rescued sperm. The authors show that Armc2 mRNA-rescued sperm can successfully fertilise oocytes that develop to the blastocyst stage. This adds another level of reliability to the data.

      Weaknesses:

      Some minor weaknesses identified in my previous report have already been fixed. The technique is new and may not yet be fully established for all issues. Nevertheless, the data presented in this manuscript opens the way for several approaches to immotile spermatozoa to ensure successful fertilisation of oocytes and subsequent appropriate embryo development.

      [Editors' note: The images in Figure 12 do not support the authors' interpretation that 2-cell embryos resulted from in vitro fertilization. Instead, the cells shown appear to be fragmented, unfertilized eggs. Combined with the lack of further development, it seems highly unlikely that fertilization was successful.]

      We thank the reviewer for their careful evaluation and constructive feedback. We appreciate the acknowledgment of the strengths of our study, particularly the proof-of-concept demonstration that Armc2-mRNA electroporation can rescue sperm motility in Armc2 knockout mice.

      Regarding the concern raised by the editor about Figure 12, we would like to clarify two technical points. First, the IVF experiments were performed using CD1 oocytes and B6D2 sperm. Due to strain-specific incompatibilities, fertilization of CD1 oocytes by B6D2 sperm typically does not progress beyond the two-cell stage (Fernández-González [23] et al., 2008, Biology of Reproduction). Therefore, the observation of two-cell embryos represents the expected limit of development in this cross and serves as a strong indication of successful fertilization, even though further development is not possible. Second, the oocytes used in these experiments were treated with collagenase to remove cumulus cells. This enzymatic treatment can sometimes affect the morphology of early embryos, which may explain why the two-cell embryos in Figure 12 appear less regular or somewhat fragmented. We also included a control showing embryos from B6D2 sperm with the same collagenase treatment on CD1 oocytes, which yielded similar appearances (Fig14 A4).

      To provide additional functional evidence, we complemented the IVF experiments with ICSI using rescued Armc2<sup>–/–</sup> sperm and B6D2 oocytes, which allowed embryos to develop to the blastocyst stage. In these experiments, 25% of injected oocytes reached the blastocyst stage with rescued sperm compared to 13% for untreated Armc2–/– sperm (Supplementary Fig. 9) These results support the functional competence of rescued sperm and demonstrate partial recovery of fertilization ability following Armc2 mRNA electroporation.

      We have clarified these points in the revised Results and Discussion sections to emphasize that the IVF data indicate partial functional recovery of rescued sperm rather than full fertility restoration. These clarifications address the editor’s concern while accurately representing the technical limitations of the strain combination used in our experiments.

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      Fig 12 and Supplementary Fig 9 are mislabeled in the text and rebuttal.

      We thank the reviewer for pointing this out. We have carefully checked the manuscript and the rebuttal text, and corrected all references to Figure 12 and Supplementary Figure 9 to ensure they are accurately labeled and consistent throughout the text.

      Reviewer #3 (Recommendations for the authors):

      The contribution of the newly added authors should be clarified. All other aspects of inadequacy raised in my previous report have been adequately addressed.

      No further comments.

      We thank the reviewer for noting this. The contributions of the newly added authors have been clarified in the Author Contributions section of the revised manuscript. All other points raised in the previous review have been addressed as indicated.

      References

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      (2) Halliday, J.L., et al., Increased risk of blastogenesis birth defects, arising in the first 4 weeks of pregnancy, after assisted reproductive technologies. Hum Reprod, 2010. 25(1): p. 59-65.

      (3) Davies, M.J., et al., Reproductive technologies and the risk of birth defects. N Engl J Med, 2012. 366(19): p. 1803-13.

      (4) Kurinczuk, J.J., M. Hansen, and C. Bower, The risk of birth defects in children born after assisted reproductive technologies. Curr Opin Obstet Gynecol, 2004. 16(3): p. 201-9.

      (5) Graham, M.E., et al., Assisted reproductive technology: Short- and long-term outcomes. Dev Med Child Neurol, 2023. 65(1): p. 38-49.

      (6) Palermo, G.D., et al., Intracytoplasmic sperm injection: state of the art in humans. Reproduction, 2017. 154(6): p. F93-f110.

      (7) Usmani, A., et al., A non-surgical approach for male germ cell mediated gene transmission through transgenesis. Sci Rep, 2013. 3: p. 3430.

      (8) Raina, A., et al., Testis mediated gene transfer: in vitro transfection in goat testis by electroporation. Gene, 2015. 554(1): p. 96-100.

      (9) Michaelis, M., A. Sobczak, and J.M. Weitzel, In vivo microinjection and electroporation of mouse testis. J Vis Exp, 2014(90).

      (10) Wang, L., et al., Testis electroporation coupled with autophagy inhibitor to treat non-obstructive azoospermia. Mol Ther Nucleic Acids, 2022. 30: p. 451-464.

      (11) Wu, Q., et al., Translation affects mRNA stability in a codon-dependent manner in human cells. eLife, 2019. 8: p. e45396.

      (12) Gallie, D.R., The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency. Genes & Development, 1991. 5(11): p. 2108-2116.

      (13) Henderson, J.M., et al., Cap 1 messenger RNA synthesis with co-transcriptional CleanCap® analog improves protein expression in mammalian cells. Nucleic Acids Research, 2021. 49(8): p. e42.

      (14) Stepinski, J., et al., Synthesis and properties of mRNAs containing novel “anti-reverse” cap analogs. RNA, 2001. 7(10): p. 1486-1495.

      (15) Sachs, A.B., P. Sarnow, and M.W. Hentze, Starting at the beginning, middle, and end: translation initiation in eukaryotes. Cell, 1997. 89(6): p. 831-838.

      (16) Presnyak, V., et al., Codon optimality is a major determinant of mRNA stability. Cell, 2015. 160(6): p. 1111-24.

      (17) Cao, D., et al., Unlock the sustained therapeutic efficacy of mRNA. J Control Release, 2025. 383: p. 113837.

      (18) Karikó, K., et al., Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther, 2008. 16(11): p. 1833-40.

      (19) Pardi, N., et al., mRNA vaccines — a new era in vaccinology. Nature Reviews Drug Discovery, 2018. 17(4): p. 261-279.

      (20) Meistrich, M.L. and R.A. Hess, Assessment of Spermatogenesis Through Staging of Seminiferous Tubules, in Spermatogenesis: Methods and Protocols, D.T. Carrell and K.I. Aston, Editors. 2013, Humana Press: Totowa, NJ. p. 299-307.

      (21) Au - Mäkelä, J.-A., et al., JoVE, 2020(164): p. e61800.

      (22) Coutton, C., et al., Bi-allelic Mutations in ARMC2 Lead to Severe Astheno-Teratozoospermia Due to Sperm Flagellum Malformations in Humans and Mice. Am J Hum Genet, 2019. 104(2): p. 331-340.

      (23) Fernández-Gonzalez, R., et al., Long-term effects of mouse intracytoplasmic sperm injection with DNA-fragmented sperm on health and behavior of adult offspring. Biol Reprod, 2008. 78(4): p. 761-72.

    1. Author response:

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

      Reviewer #1:

      Summary:

      In their study, the authors investigated the F. graminearum homologue of the Drosophila Misato-Like Protein DML1 for a function in secondary metabolism and sensitivity to fungicides.

      Strengths:

      Generally, the topic of the study is interesting and timely, and the manuscript is well written, albeit in some cases, details on methods or controls are missing.

      Weaknesses:

      However, a major problem I see is with the core result of the study, the decrease in the DON content associated with the deletion of FgDML1. Although some growth data are shown in Figure 6, indicating a severe growth defect, the DON production presented in Figure 3 is not related to biomass. Also, the method and conditions for measuring DON are not described. Consequently, it could well be concluded that the decreased amount of DON detected is simply due to decreased growth, and the specific DON production of the mutant remains more or less the same.

      To alleviate this concern, it is crucial to show the details on the DON measurement and growth conditions and to relate the biomass formation under the same conditions to the DON amount detected. Only then can a conclusion as to an altered production in the mutant strains be drawn.

      We appreciate it very much that you spent much time on my paper and give me good suggestions, we tried our best to revise the manuscript. I have revised my manuscript according to your suggestions. The point to point responds to the reviewer’s comments are listed as following. Our method for DON quantification was based on the amount per unit of mycelium. After obtaining the absorbance value from the ELISA reaction, the concentration of DON was calculated according to a standard curve and a formula, then divided by the dry weight of the mycelium to obtain the DON content per unit of mycelium, with the results finally expressed in µg/g.

      (1) Line 139f

      ... FgDML1 is a critical positive regulator of virulence ....

      Clearly, the deletion of FgDML1 impacts virulence, but it is too much of a general effect to say it is a regulator. DML1 acts high up in the cascade, impacting numerous processes, one of which is virulence. Generally, it has to be considered that deletion of DML1 causes a severe growth defect, which in turn is likely to lead to a plethora of effects. Besides discussing this fact, please also revise the manuscript to avoid references to "direct effects" or "regulator".

      Thank you very much for your advice. Our method for determining the amount of DON is based on the amount of mycelium per unit. After obtaining the absorbance value through Elisa reaction, we calculate the concentration of DON toxin according to the established standard curve and formula. Then, we divide it by the dry weight of mycelium to obtain the DON toxin content per unit mycelium, and finally present the results in µg/g. In summary, we conclude that the decrease in DON production by ΔFgDML is not due to slower hyphal growth, but rather a decrease in the ability of unit hyphae to produce DON toxins compared to the wild type. Given the decrease in DON toxin synthesis caused by FgDML1 deficiency, we believe that using a regulator is reasonable.

      (2) Line 143

      Please define "toxin-producing conditions".

      Thank you very much for your advice. We have accurately defined the conditions for toxin-producing conditions in the manuscript' toxin-inducing conditions '(28°C, 145 ×g, 7 days incubation)' (in L163-164)

      (3) Line 149

      A brief intro on toxisomes should be provided in the introduction to better integrate this into the manuscript's results.

      Thank you very much for your advice. We have added corresponding content about toxin producing bodies in the introduction section 'The biosynthesis of DON entails a reorganization of the endoplasmic reticulum into a specialized compartment termed the "toxisome" (Tang et al., 2018). The assembly of the toxisome coincides with the aggregation of key biosynthetic enzymes, which in turn enhances the efficiency of DON production. Concurrently, this compartmentalization serves as a self-defense mechanism, protecting the fungus from the autotoxicity of TRI pathway intermediates (Boenisch et al., 2017). The proteins TRI1, TRI4, TRI14, and Hmr1 are confirmed constituents of this structure(Kistler and Broz, 2015; Menke et al., 2013).' (in L86-93)

      (4) Line 153

      DON production decreases by about 80 %, but not to 0. Consequently, DML1 is important, but NOT essential for DON production.

      Thank you very much for your advice. We have made changes to the wording of the corresponding sections based on your suggestions. 'FgDML1 is essential for the biosynthesis of the DON toxin. '(in L161)

      (5) Line 168ff

      Please provide a reference for FgDnm1 being critical for mitochondrial fission and state whether such an interaction has been shown in other organisms.

      Thank you very much for your advice. We have made changes to the wording of the corresponding sections based on your suggestions. 'FgDnm1 is a key dynamin-related protein mediating mitochondrial fission(Griffin et al., 2005; Kang et al., 2023), suggesting that FgDML1 may form a complex with FgDnm1 to regulate mitochondrial fission and fusion processes. To our knowledge, this is the first report documenting an interaction between DML1 and Dnm in any fungal species, including model organisms such as S. cerevisiae. This novel finding provides new insights into the molecular mechanisms underlying mitochondrial dynamics in filamentous fungi. '(in L277-283)

      (6) Line 178

      Please specify whether Complex III activity was related to biomass and provide a p-value or standard deviation for the value.

      Thank you very much for your question. The activity determination of complex III was completed using a complex III enzyme activity kit (Solarbio, Beijing, China) (Li, et al 2022; Wang, et al 2022). Take 0.1 g of standardized mycelium as the sample for the experiment. Given that the mycelium has been homogenized, we believe that there is no necessary correlation between the activity and biomass of complex III. And we also refined the specific measurement steps in the article. ' Briefly, 0.1 g of mycelia was homogenized with 1 mL of extraction buffer in an ice bath. The homogenate was centrifuged at 600 ×g for 10 min at 4°C. The resulting supernatant was then subjected to a second centrifugation at 11,100 ×g for 10 min at 4°C. The pellet was resuspended in 200 μL of extraction buffer and disrupted by ultrasonication (200 W, 5 s pulses with 10 s intervals, 15 cycles). Complex III enzyme activity was finally measured by adding the working solution as per the manufacturer's protocol. Each treatment group contains three biological replicates and three technical replicates. '(in L511-517)

      Li C, et al. Amino acid catabolism regulates hematopoietic stem cell proteostasis via a GCN2-eIF2 axis. Cell Stem Cell. 2022 Jul 7; 29(7):1119-1134.e7. doi: 10.1016/j.stem.2022.06.004. PMID: 35803229.

      Wang K, et al. Locally organised and activated Fth1hi neutrophils aggravate inflammation of acute lung injury in an IL-10-dependent manner. Nat Commun. 2022 Dec 13;13(1):7703. doi: 10.1038/s41467-022-35492-y. PMID: 36513690; PMCID: PMC9745290

      (7) Line 185

      Albeit this headline is a reasonable hypothesis, you actually did not show that the conformation is altered. Please reword accordingly.

      Please also add references for cyazofamid acting on the QI site versus other fungicides acting on the QO site.

      Thank you very much for your advice. We have made changes to the wording of the corresponding sections based on your suggestions. 'Overexpression of FgQCR2, FgQCR8, and FgQCR9 may alters the conformation of the QI site, resulting in reduced sensitivity to cyazofamid. '(in L212-213). For fungicides targeting Qi and QO sites, we have added corresponding descriptions in the respective sections 'Numerous fungicides have been developed to inhibit the Qo site (e.g., pyraclostrobin, azoxystrobin)(Nuwamanya et al., 2022; Peng et al., 2022) and the Qi site (e.g., cyazofamid)(Mitani et al., 2001) of the cytochrome bc1 complex. '(in L327-329)

      (8) Line 200

      This section on growth should be moved up right after introducing the mutant strain.

      Thank you very much for your advice. We have advanced the part of nutritional growth and sexual asexual development before DON toxin to promote better reading and understanding. We arranged the sequence in the previous way to emphasize the new discovery between mitochondria and DON toxin. We found a significant decrease in DON toxin in ΔFgDML1, defects in the formation of toxin producing bodies, and downregulation of FgTRis at both the gene and protein levels. In summary, we believe that the absence of FgDML1 does indeed lead to a decrease in the content of DON toxin, and FgDML1 plays a regulatory role in the synthesis of DON toxin. In addition, our measurements of DON toxin, acetyl CoA, ATP and other indicators are all based on the amount per unit hyphae, excluding differences caused by hyphal biomass or growth. We have further refined the materials and methods to facilitate better reading and understanding.

      (9) Line 203

      "... significantly reduced growth rates ..."

      This is not what was measured here. Figure 6A shows a plate assay that can be used to assess hyphal extension. In the figure, it is also visible that the mycelium of the deletion mutant is much denser, maybe due to increased hyphal branching. Please reword.

      Additionally, it is important to include a biomass measurement here under the conditions used for DON assessment. Hyphal extension measurements cannot be used instead of biomass.

      Thank you very much for your advice. We have made changes to the wording of the corresponding sections based on your suggestions. 'The ΔFgDML1 strain displayed a distinct growth phenotype characterized by retardation in radial growth and the formation of more compact, denser hyphal networks on all tested media compared to the PH-1 and ΔFgDML-C strains. '(in L136-138).

      (10) Line 217

      Please include information on how long the cultures were monitored. Given the very slow growth of the mutant, perithecia formation may be considerably delayed beyond 14 days.

      Thank you very much for your advice. Based on your suggestion, we have extended the incubation time for sexual reproduction to 21 days to more accurately evaluate its sexual reproduction ability. Our results show that even after 21 days, Δ FgDML1 still cannot produce ascospores and ascospores, which proves that the absence of FgDML1 does indeed cause sexual reproduction defects in F. graminearum.

      Author response image 1.

      Discussion

      (11) Please mention your summary Figure 8 early on in the discussion, and explain conclusions with this figure in mind. Please avoid repetition of the results section as much as possible.

      Also, please state clearly what was already known from previous research and is in agreement with your results, and what is new (in fungi or generally).

      Thank you very much for your advice. Based on your suggestion, we mentioned Fig8 earlier in the first half of the discussion and provided guidance for the following text. We also conducted a more comprehensive discussion by analyzing our research results and comparing them with previous studies. 'Our study defines a novel mechanism through which FgDML1 governs mitochondrial homeostasis. We demonstrate that FgDML1 directly interacts with the key mitochondrial fission regulator FgDnm1 and positively modulates cellular bioenergetic metabolism, as evidenced by elevated ATP and acetyl-CoA levels (Fig. 8). '(in L250-253). 'The Misato/DML1 protein family is evolutionarily conserved from yeast to humans and plays a critical role in mitochondrial regulation. In S. cerevisiae, DML1 is an essential gene; its deletion is lethal, while its overexpression results in fragmented mitochondrial networks and aberrant cellular morphology, underscoring its necessity for normal mitochondrial function (Gurvitz et al., 2002). Similarly, in Homo sapiens, the homolog Misato localizes to the mitochondrial outer membrane, and both its depletion and overexpression are sufficient to disrupt mitochondrial morphology and distribution (Kimura and Okano, 2007). '(in L241-244).

      (12) Line 262ff

      Please specify if this interaction was shown previously in other organisms and provide references.

      Thank you very much for your advice. We have clearly stated in the corresponding section that the interaction between FgDML and FgDnm is the first reported, and to our knowledge, no relevant reports have been found in other species so far. ' Notably, FgDML1 was found to interact with FgDnm1 (Fig. 5E), FgDnm1 is a key dynamin-related protein mediating mitochondrial fission(Griffin et al., 2005; Kang et al., 2023), suggesting that FgDML1 may form a complex with FgDnm1 to regulate mitochondrial fission and fusion processes. To our knowledge, this is the first report documenting an interaction between DML1 and Dnm in any fungal species, including model organisms such as S. cerevisiae. This novel finding provides new insights into the molecular mechanisms underlying mitochondrial dynamics in filamentous fungi. '(in L276-283)

      (13) Line 287ff

      There is no result that would justify this speculation. Please remove.

      Thank you very much for your advice. We have modified the corresponding wording in the corresponding section. 'In conclusion, our findings suggest that the overexpression of assembly factors FgQCR2, FgQCR7, and FgQCR8 in ΔFgDML1 potentially modifies the conformation of the Qi site, which specifically modulates the sensitivity of F. graminearum to cyazofamid. '(in L352-355)

      Materials and methods

      (14) A table with all primer sequences used in the study and their purpose is missing. For every experiment, the number of technical and biological replicates needs to be stated.

      Thank you very much for your advice. We have presented all the primers used in this study in Supplementary Table 1 (in Table S1) .We added the number of technical and biological replicates in the material and method descriptions for each experiment. 'For each sample, a total of 200 conidia were counted. The experiment included three biological replicates with three technical replicates each.'(in L434-436). 'Each treatment group contains three biological replicates. '(in L444-445). 'Each treatment group contains three biological replicates and three technical replicates. ' (in L463-464). 'Each treatment group contains three biological replicates and three technical replicates. '(in L474-475). 'Each treatment group contains three biological replicates. '(in L483). 'Each treatment group contains three biological replicates and three technical replicates.'(in L501-502). 'Each treatment group contains three biological replicates and three technical replicates. '(in L516-517). 'The experiment was independently repeated three times. '(in L533-534).

      (15) Line 369ff

      Please provide final concentrations used for assays here.

      Thank you very much for your advice. The final concentration has been displayed in the Figure (in Fig6. A, B) (in Fig. S3). And we have provided supplementary Table 2 to reflect the concentration in a more intuitive way.(in Table. S2)

      (16) Line 383

      Please provide a reference or data on the use of F2du for transformant selection and explain the abbreviation.

      Thank you very much for your advice. Based on your suggestion, we have provided the full name and references of F2du. 'Transformants were selected on PDA plates containing either 100 μg/mL Hygromycin B (Yeasen, Shanghai, China) or 0.2 μmol/mL 5-Fluorouracil 2'-deoxyriboside (F2du) (Solarbio, Beijing, China)(Zhao et al., 2022). '(in L405-407).

      (17) Line 407

      Please provide a reference for the method and at least a brief description.

      Thank you very much for your advice. Based on your suggestion, we have added references and provided a brief introduction to the method. 'As previously described (Tang et al., 2020; Wang et al., 2025), Specifically, coleoptiles were inoculated with conidial suspensions and incubated for 14 days, while leaves were inoculated with fresh mycelial plugs and incubated for 5 days, followed by observation and quantification of disease symptoms. DON toxin was measured using a Wise Science ELISA-based kit (Wise Science, Jiangsu, China) (Li et al., 2019; Zheng et al., 2018). '(in L466-471)

      (18) Line 414ff

      Also, here, the amount of biomass has to be considered for the measurement to be able to distinguish if actually less of the compounds were produced or if the effect seen was merely due to an altered amount of biomass present.

      Thank you very much for your advice. We believe that biomass is not within the scope of our measurement indicators, as we have measured and calculated based on unit hyphae. Therefore, we have ruled out experimental bias caused by a decrease in biomass.

      RNA and RT-qPCR

      (19) Line 461

      When the strains were transferred to AEA medium, was the biomass measured, at least wet weight, and in which culture volume was it done? It makes a big difference if the amount of (wet) biomass dilutes a small amount of fungicide-containing culture or if biomass is added in at least roughly equal amounts in sufficient growth medium to ensure equal conditions.

      Thank you very much for your question. Our sample processing controlled the wet weight of the samples before dosing, ensuring that the wet weight of the mycelium obtained from each sample before dosing was 0.2g. The mycelium was obtained through AEA with a volume of 100mL. This ensured consistency in the initial biomass between groups before dosing, and also ensured the accuracy of the drug concentration.

      (20) Line 466

      Please provide the name and supplier of the kit.

      Thank you very much for your advice. We have added corresponding content in the corresponding location. 'Mycelium was collected and total RNA was extracted following the instructions provided by the Total RNA Extraction Kit (Tiangen, Beijing, China).' (in L523-524).

      (21) All primer sequences must be provided in a table.

      Thank you very much for your advice. We have presented all the primers used in this study in Supplementary Table 1. (in Table S1).

      (22) For RT qPCR it is essential to check the RNA quality to be sure that the obtained results are not artifacts due to varying quality, which may exceed differences. Please state how quality control was done and which threshold was applied for high-quality RNA to be used in RTqPCR (like RIN factor, etc).

      Thank you very much for your question. We performed stringent quality control on the extracted total RNA. First, a micro-spectrophotometer was used to measure RNA concentration and purity, confirming that the A260/A280 ratio was between 1.8 and 2.0 and the A260/A230 ratio was greater than 2.0, indicating good RNA purity without significant protein or organic solvent contamination.Subsequently, verification by agarose gel electrophoresis revealed distinct 28S and 18S rRNA bands, demonstrating good RNA integrity and absence of degradation.

      Author response image 2.

      (B): Minor Comments:

      (1) Please increase the font size of the labels and annotations of the figures; it is hard to read as it is now.

      Thank you very much for your advice. We have increased the size of annotations or numerical labels in the corresponding images for better reading.

      (2) Throughout the manuscript: Please check that all abbreviations are explained at first use.

      Thank you very much for your advice. We have checked the entire text to ensure that abbreviations have their full names when they first appear.

      (3) I do hope that the authors can clarify all concerns and provide an amended manuscript of this interesting story.

      Thank you very much for your advice. Sincerely thank you for your suggestions and questions, which have been very helpful to us.

      Reviewer #2:

      The manuscript entitled "Mitochondrial Protein FgDML1 Regulates DON Toxin Biosynthesis and Cyazofamid Sensitivity in Fusarium graminearum by affecting mitochondrial homeostasis" identified the regulatory effect of FgDML1 in DON toxin biosynthesis and sensitivity of Fusarium graminearum to cyazofamid. The manuscript provides a theoretical framework for understanding the regulatory mechanisms of DON toxin biosynthesis in F. graminearum and identifies potential molecular targets for Fusarium head blight control. The paper is innovative, but there are issues in the writing that need to be addressed and corrected.

      We appreciate it very much that you spent much time on my paper and give me good suggestions, we tried our best to revise the manuscript. I have revised my manuscript according to your suggestions with red words. In the response comments, to highlight the specific positions of the revised parts in the manuscript with red line number. The point to point responds to the reviewer’s comments are listed as following.

      Weaknesses:

      (1) The authors speculate that cyazofamid treatment caused upregulation of the assembly factors, leading to a change in the conformation of the Qi protein, thus restoring the enzyme activity of complex III. But no speculation was given in the discussion as to why this would lead to the upregulation of assembly factors, and how the upregulation of assembly factors would change the protein conformation, and is there any literature reporting a similar phenomenon? I would suggest adding this to the discussion.

      Thank you very much for your advice. Based on your suggestion, we have added content related to the assembly factor of complex III in the discussion section and made modifications to the corresponding wording. 'Previous studies have reported that mutations in the Complex III assembly factors TTC19, UQCC2, and UQCC3 impair the assembly and activity of Complex III (Feichtinger et al., 2017; Wanschers et al., 2014). '(in L345-347). 'In conclusion, our findings suggest that the overexpression of assembly factors FgQCR2, FgQCR7, and FgQCR8 in ΔFgDML1 potentially modifies the conformation of the Qi site, which specifically modulates the sensitivity of F. graminearum to cyazofamid. '(in L352-355).

      (2) Would increased sensitivity of the mutant to cell wall stress be responsible for the excessive curvature of the mycelium?

      Thank you very much for your question. We believe that the sensitivity of ΔFgDML1 to osmotic stress is reduced, which may not be related to hyphal bending, as shown in the Author response image 3. During the conidia stage, ΔFgDML1 cannot germinate in YEPD, while the application of 1M Sorbitol promotes its germination. But it is caused by internal unknown mechanisms, which is also the focus of our future research.

      Author response image 3.

      (3) The vertical coordinates of Figure 7B need to be modified with positive inhibition rates for the mutants.

      Thank you very much for your advice. The display in Figure 7B truly reflects its inhibition rate. In the Δ FgDML1 mutant, when subjected to osmotic stress treatment, the inhibition rate becomes negative, indicating that the colony growth is greater than that of the CK. Therefore, the negative inhibition rate is shown in Figure 7B.

      (1) In Figure 1B, Figure 3C, and Figure 6C, the scale below the picture is not clear. In Figure 5D, the histogram is unclear, and it is recommended to redraw the graph.

      Thank you very much for your advice. The issue with the above images may be due to Word compression. We have changed the settings and enlarged the images as much as possible to better display them.

      (2) The full Latin name of the strain should be used in the title of figures and tables.

      Thank you very much for your advice. Based on your suggestion, we have used the full names of the strains appearing in the title of figures and tables.

      (3) Proteins in line 117 should be abbreviated.

      Thank you very much for your advice. Based on your suggestion, we have abbreviated the corresponding positions. 'The DML1 protein from S. cerevisiae was used as a query for a BLAST search against the Fusarium genome database, resulting in the identification of the putative DML1 gene FgDML1 (FGSG_05390) in F. graminearum. '(in L118-120).

      (4) The sentence in lines 187-189, which is supposed to introduce why the test is sensitive to the three drugs, is currently illogical.

      Thank you very much for your advice. Based on your suggestion, we have made modifications to the corresponding sections. 'Since Complex III is involved in the action of both cyazofamid (targeting the QI site) and pyraclostrobin (targeting the QO site), the sensitivity of ΔFgDML1 to cyazofamid and pyraclostrobin was investigated. ' (in L214-216).

      (5) The expression of FgQCR2, FgQCR7, and FgQCR8 was significantly upregulated in ΔFgDML1 at transcription levels. Do FgQCR2, FgQCR8, and FgQCR9 show upregulated expression at the protein level?

      Thank you very much for your question. Based on your suggestion, we evaluated the protein expression levels of FgQCR2, FgQCR7, and FgQCR8 in PH-1 and ΔFgDML1, and we found that the protein expression levels of FgQCR2, FgQCR7, and FgQCR8 in ΔFgDML1 were higher than those in PH-1. (in Fig. 6F).

      (6) In Figure 7B, it is recommended to adjust the position of the horizontal axis labels in the histogram.

      Thank you very much for your advice. Based on your suggestion, we have made modifications to the corresponding sections.(in Fig. 7B)

      (7) There are numerous errors in the writing of gene names in the text. Please check the full text and change the writing of gene names and mutant names to italic.

      Thank you very much for your advice. We have checked the entire text to ensure that all genes have been italicized.

      (8) All acronyms should be spelled out in figure and table captions. e.g., F. graminearum.

      Thank you very much for your advice. Based on your suggestion, we have used the full names of the strains appearing in the title of figures and tables.

      (9) In line 492, P should be lowercase and italic.

      Thank you very much for your advice. Based on your suggestion, we have made adjustments to the corresponding content.

      Reviewer #3:

      Summary:

      The manuscript "Mitochondrial 1 protein FgDML1 regulates DON toxin biosynthesis and cyazofamid sensitivity in Fusarium graminearum by affecting mitochondrial homeostasis" describes the construction of a null mutant for the FgDML1 gene in F. graminearum and assays characterising the effects of this mutation on the pathogen's infection process and lifecycle. While FgDML1 remains underexplored with an unclear role in the biology of filamentous fungi, and although the authors performed several experiments, there are fundamental issues with the experimental design and execution, and interpretation of the results.

      Strengths:

      FgDML1 is an interesting target, and there are novel aspects in this manuscript. Studies in other organisms have shown that this protein plays important roles in mitochondrial DNA (mtDNA) inheritance, mitochondrial compartmentalisation, chromosome segregation, mitochondrial distribution, mitochondrial fusion, and overall mitochondrial dynamics. Indeed, in Saccharomyces cerevisiae, the mutation is lethal. The authors have carried out multi-faceted experiments to characterise the mutants.

      Weaknesses:

      However, I have concerns about how the study was conceived. Given the fundamental importance of mitochondrial function in eukaryotic cells and how the absence of this protein impacts these processes, it is unsurprising that deletion of this gene in F. graminearum profoundly affects fungal biology. Therefore, it is misleading to claim a direct link between FgDML1 and DON toxin biosynthesis (and virulence), as the observed effects are likely indirect consequences of compromised mitochondrial function. In fact, it is reasonable to assume that the production of all secondary metabolites is affected to some extent in the mutant strains and that such a strain would not be competitive at all under non-laboratory conditions. The order in which the authors present the results can be misleading, too. The results on vegetative growth rate appeared much later in the manuscript, which should have come first, as the FgDML1 mutant exhibited significant growth defects, and subsequent results should be discussed in that context. Moreover, the methodologies are not described properly, making the manuscript hard to follow and difficult to replicate.

      We appreciate it very much that you spent much time on my paper and give me good suggestions, we tried our best to revise the manuscript. I have revised my manuscript according to your suggestions with red words. In the response comments, to highlight the specific positions of the revised parts in the manuscript with red line number. The point to point responds to the reviewer’s comments are listed as following.

      For weaknesses,we arranged the sequence in this way to emphasize the novel discovery between mitochondria and DON toxin. We found a significant decrease in DON toxin in Δ FgDML1, defects in the formation of toxin producing bodies, and downregulation of FgTRis at both the gene and protein levels. In summary, we believe that the absence of FgDML1 does indeed lead to a decrease in the content of DON toxin, and FgDML1 plays a regulatory role in the synthesis of DON toxin. In addition, our measurements of DON toxin, acetyl CoA, ATP and other indicators are all based on the amount per unit hyphae, excluding differences caused by hyphal biomass or growth. We have further refined the materials and methods to facilitate better reading and understanding.

      (1) Lines 37-39: The disease itself does not produce toxins; it is the fungus that causes the disease that produces toxins. Moreover, the disease symptoms observed are likely caused by the toxins produced by the fungus.

      Thank you very much for your advice. We have made modifications to the wording of the corresponding sections. 'Studies have shown that increased DON levels are positively correlated with the pathogenicity rate of F. graminearum.'(in L36-37).

      (2) Lines 82-87: While it is challenging to summarise the role of ATP in just a few words, this section needs improvement for clarity and accuracy. Additionally, I do not believe that drawing a direct link between mitochondrial defects and toxin production is an appropriate strategy in this case.

      Thank you very much for your advice. Based on your suggestion, we have added corresponding descriptions in the corresponding positions to provide more information on the relationship between ATP and toxins, in order to better prepare for the following text. 'Pathogen-intrinsic ATP homeostasis is recognized as a critical, rate-limiting determinant for toxin biosynthesis. Previous studies indicate that dual-target inhibition of ATP synthase (AtpA) and adenine deaminase (Ade) by a specific small-molecule probe effectively depletes intracellular ATP, consequently suppressing the synthesis of key virulence factors TcdA and TcdB transcriptionally and translationally(Marreddy et al., 2024). The systemic toxicity of Anthrax Edema Toxin (ET) is primarily attributed to its catalytic activity, which depletes the host cell's ATP reservoir, thereby triggering a bioenergetic collapse that culminates in cell lysis and death(Liu et al., 2025). '(in L78-86).

      (3) Lines 125-126: The manuscript does not clearly describe how subcellular localisation was determined. This methodology needs to be properly detailed.

      Thank you very much for your advice. The subcellular localization was validated through co-localization analysis with MitoTracker Red CMXRos, a mitochondrial-specific dye. The observed overlap between the FgDML1-GFP signal and the mitochondrial marker confirmed mitochondrial localization. Based on these results, we determined that FgDML1 is definitively localized to the mitochondria.We have incorporated this description in the appropriate section of the manuscript. 'Furthermore, subcellular localization studies confirmed that FgDML1 localizes to mitochondria, as demonstrated by colocalization with a mitochondria-specific dye MitoTracker Red CMXRos (Fig. 1B). '(in L125-127).

      (4) Regarding the organisation of the Results section, it needs to be revised. While I understand the authors' intention to emphasise the impact on virulence, the results showing how FgDML1 deletion affects vegetative growth, asexual and sexual reproduction, and sensitivity to stressors should be presented before the virulence assays and effects on DON production. Additionally, the authors do not provide any clear evidence that FgDML1 directly interacts with proteins involved in asexual or sexual reproduction, stress responses, or virulence. Therefore, it is misleading to suggest that FgDML1 directly regulates these processes. The observed phenotypes are, rather, a consequence of severely impaired mitochondrial function. Without functional mitochondria, the cell cannot operate properly, leading to widespread physiological defects. In this regard, statements such as those in lines 139-140 and 343-344 are misleading.

      Thank you very much for your advice. We have adjusted the order of the images based on your suggestion, placing the characterization of ΔFgDML1 in nutritional growth, sexual reproduction, and other aspects before DON toxin. And we have made adjustments to the corresponding statements. 'These findings demonstrate that FgDML1 is a positive regulator of virulence in F. graminearum. '(in L140-141).

      (5) Lines 185-186: The authors do not provide sufficient evidence to support the claim that FgQCR2, FgQCR8, and FgQCR9 overexpression is the main cause of reduced cyazofamid sensitivity. Although expression of these genes is altered, reduced sensitivity may result from changes in other proteins or pathways. To strengthen this claim, overexpression of FgQCR2, 8, and 9 in the wild-type background, followed by assessment of cyazofamid resistance, would be necessary. As it stands, there is no support for the claim presented in lines 329-332.

      Thank you very much for your advice. To establish a causal link between the overexpression of FgQCR2, FgQCR7, and FgQCR8 and the observed reduction in cyazofamid sensitivity, we first quantified the protein levels of these assembly factor. Western blot analysis confirmed their elevated expression in the ΔFgDML1 mutant compared to the wild-type PH-1. We further generated individual overexpression strains for FgQCR2, FgQCR7, and FgQCR8 in the wild-type PH-1 background. Fungicide sensitivity assays revealed that all three overexpression mutants displayed significantly reduced sensitivity to cyazofamid compared to the parental strain. These genetic complementation experiments confirm that upregulation of FgQCR2, FgQCR7, and FgQCR8 is sufficient to confer reduced cyazofamid sensitivity.We have incorporated these explanations and provided supporting images in the appropriate section of the manuscript. 'To further clarify whether the upregulated expression of FgQCR2, FgQCR7, and FgQCR8 genes affects their protein expression levels, we measured the protein levels. The results showed that the protein expression levels of FgQCR2, FgQCR7, and FgQCR8 in ΔFgDML1 were higher than those in PH-1(Fig. 6F). Subsequently, we overexpressed FgQCR2, FgQCR7, and FgQCR8 in the wild-type background, and the corresponding overexpression mutants exhibited reduced sensitivity to cyazofamid(Fig. 6E). '(in L205-211)(in Fig. 6E, F)

      (6) Lines 187-190: This segment is confusing and difficult to follow. It requires rewriting for clarity.

      Thank you very much for your advice. Based on your suggestion, we have made corresponding modifications in the corresponding locations. 'Since Complex III is involved in the action of both cyazofamid (targeting the QI site) and pyraclostrobin (targeting the QO site), the sensitivity of ΔFgDML1 to cyazofamid and pyraclostrobin was investigated. ''(in L214-216)

      (7) Lines 345-346: The authors state that in this study, FgDML1 is localised in mitochondria, which implies that in other studies, its localisation was different. Is this accurate? Clarification is needed.

      Thank you very much for your question. In previous studies, the localization of this protein was not clearly defined, and its function was only emphasized to be related to mitochondria. Whether in yeast or in Drosophila melanogaster. (Miklos et al., 1997; Gurvitz et al., 2002)

      Miklos GLG, Yamamoto M-T, Burns RG, Maleszka R. 1997. An essential cell division gene of drosophila, absent from saccharomyces, encodes an unusual protein with  tubulin-like and myosin-like peptide motifs. Proc Natl Acad Sci 94:5189–5194. doi:10.1073/pnas.94.10.5189

      Gurvitz A, Hartig A, Ruis H, Hamilton B, de Couet HG. 2002. Preliminary characterisation of DML1, an essential saccharomyces cerevisiae gene related to misato of drosophila melanogaster. FEMS Yeast Res 2:123–135. doi:10.1016/S1567-1356(02)00083-1

      Material and Methods Section

      (8) In general, the methods require more detailed descriptions, including the brands and catalog numbers of reagents and kits used. Simply stating that procedures were performed according to manufacturers' instructions is insufficient, particularly when the specific brand or kit is not identified.

      Thank you very much for your advice. We have added corresponding content based on your suggestion to more comprehensively display the reagent brand and complete product name. 'Transformants were selected on PDA plates containing either 100 μg/mL Hygromycin B (Yeasen, Shanghai, China) or 0.2 μmol/mL 5-Fluorouracil 2'-deoxyriboside (F2du) (Solarbio, Beijing, China)(Zhao et al., 2022). ' (in L405-407). 'DON toxin was measured using a Wise Science ELISA-based kit (Wise Science, Jiangsu, China) (Li et al., 2019; Zheng et al., 2018) '. (in L469-471)

      (9) Line 364: What do CM and MM stand for? Please define.

      Thank you very much for your advice. Based on your suggestion, we have made modifications in the corresponding locations. 'To evaluate vegetative growth, complete medium (CM), minimal medium (MM), and V8 Juice Agar (V8) media were prepared as described previously(Tang et al., 2020). '(in L385-387)

      Generation of Deletion and Complemented Mutants:

      (10) This section lacks detail. For example, were PCR products used directly for PEG-mediated transformation, or were the fragments cloned into a plasmid?

      Thank you very much for your question. We directly use the fused fragments for protoplast transformation after sequencing confirmation. We have clearly defined the fragment form used for transformation at the corresponding location. 'The resulting fusion fragment was transformed into the wild-type F. graminearum PH-1 strain via polyethylene glycol (PEG)-mediated protoplast transformation. '(in L403-405).

      (11) PCR and Southern blot validation results should be included as supplementary material, along with clear interpretations of these results.

      Thank you very much for your advice. In the supplementary material we submitted, Supplementary Figure 2 already includes the results of PCR and Southern blot validation.(in Fig. S2)

      (12) There is almost no description of how the mutants mentioned in lines 388-390 were generated.

      Thank you very much for your advice. Based on your suggestions, we have added relevant content in the appropriate sections to more comprehensively and clearly reflect the experimental process. 'Specifically, FgDML1, including its native promoter region and open reading frame (ORF) (excluding the stop codon), was amplified.The PCR product was then fused with the XhoI -digested pYF11 vector. After transformation into E. coli and sequence verification, the plasmid was extracted and subsequently introduced into PH-1 protoplasts. For FgDnm1-3×Flag, the 3×Flag tag was added to the C-terminus of FgDnm1 by PCR, fused with the hygromycin resistance gene and the FgDnm1 downstream arm, and then introduced into PH-1 protoplasts. The overexpression mutant was constructed according to a previously described method. Specifically, the ORF of FgDML1 was amplified and the PCR product was ligated into the SacII-digested pSXS overexpression vector. The resulting plasmid was then transformed into PH-1 protoplasts (Shi et al., 2023). For the construction of PH-1::FgTri1+GFP and ΔFgDML1::FgTri1+GFP, the ORF of FgTri1 was amplified and ligated into the XhoI-digested pYF11 vector as described above. The resulting vectors were then transformed into protoplasts of PH-1 or ΔFgDML1, respectively.'(in L413-426).

      Vegetative Growth and Conidiation Assays:

      (13) There is no information about how long the plates were incubated before photos were taken. Judging by the images, it appears that different incubation times may have been used.

      Thank you very much for your advice. Due to the slower growth of ΔFgDML1, we adopted different incubation periods and have supplemented the relevant content in the corresponding section. 'All strains were incubated at 25°C in darkness; however, due to ΔFgDML1 slower growth, the ΔFgDML1 mutant required a 5-day incubation period compared to the 3 days used for PH-1 and ΔFgDML1-C. '(in L490-493).

      (14) There is no description of the MBL medium.

      Thank you very much for your advice. Based on your suggestion, we have supplemented the corresponding content in the corresponding positions. 'Mung bean liquid (MBL) medium was used for conidial production, while carrot agar (CA) medium was utilized to assess sexual reproduction(Wang et al., 2011). '(in L387-389).

      DON Production and Pathogenicity Assays:

      (15) Were DON levels normalised to mycelial biomass? The vegetative growth assays show that FgDML1 null mutants exhibit reduced growth on all tested media. If mutant and wild-type strains were incubated for the same period under the same conditions, it is reasonable to assume that the mutants accumulated significantly less biomass. Therefore, results related to DON production, as well as acetyl-CoA and ATP levels, must be normalised to biomass.

      Thank you very much for your question. We have taken into account the differences in mycelial biomass. Therefore, when measuring DON, acetyl-CoA, and ATP levels, all data were normalized to mycelial mass and calculated as amounts per unit of mycelium, thereby avoiding discrepancies arising from variations in biomass.

      Sensitivity Assays:

      (16) While the authors mention that gradient concentrations were used, the specific concentrations and ranges are not provided. Importantly, have the plates shown in Figure 5 been grown for different periods or lengths? Given the significantly reduced growth rate shown in Figure 6A, the mutants should not have grown to the same size as the WT (PH-1) as shown in Figures 5A and 5B unless the pictures have been taken on different days. This needs to be explained.

      Thank you very much for your question. Due to the slower growth of ΔFgDML1, we adopted different incubation periods and have supplemented the relevant content in the corresponding section. 'All strains were incubated at 25°C in darkness; however, due to ΔFgDML1 slower growth, the ΔFgDML1 mutant required a 5-day incubation period compared to the 3 days used for PH-1 and ΔFgDML1-C. '(in L490-493).

      (17) Additionally, was inhibition measured similarly for both stress agents and fungicides? This should be clarified.

      Thank you very much for your question. We have supplemented the specific concentration gradient of fungicides. 'The concentration gradients for each fungicide in the sensitivity assays were set up according to Supplementary Table S2. '(in L493-494)(in Table. S2).

      Complex III Enzyme Activity:

      (18) A more detailed description of how this assay was performed is needed.

      Thank you very much for your advice. We have provided further detailed descriptions of the corresponding sections. 'Briefly, 0.1 g of mycelia was homogenized with 1 mL of extraction buffer in an ice bath. The homogenate was centrifuged at 600 ×g for 10 min at 4°C. The resulting supernatant was then subjected to a second centrifugation at 11,000 ×g for 10 min at 4°C. The pellet was resuspended in 200 μL of extraction buffer and disrupted by ultrasonication (200 W, 5 s pulses with 10 s intervals, 15 cycles). Complex III enzyme activity was finally measured by adding the working solution as per the manufacturer's protocol. '(in L511-517)

      (19) Were protein concentrations standardised prior to the assay?

      Thank you very much for your question. Protein concentrations for all Western blot samples were quantified using a BCA assay kit to ensure equal loading.

      (20) Line 448: Are ΔFgDML1::Tri1+GFP and ΔFgDML1+GFP the same strain? ΔFgDML1::Tri1+GFP has not been previously described.

      Thank you very much for your question. These two strains are not the same strain, and we have supplemented their construction process in the corresponding section. 'For the construction of PH-1::FgTri1+GFP and ΔFgDML1::FgTri1+GFP, the ORF of FgTri1 was amplified and ligated into the XhoI-digested pYF11 vector as described above. The resulting vectors were then transformed into protoplasts of PH-1 or ΔFgDML1, respectively. '(in L423-426)

      (21) Lines 460 and 468: Please adopt a consistent nomenclature, either RT-qPCR or qRT-PCR.

      Thank you very much for your advice. We have unified it and modified the corresponding content in the corresponding sections. 'Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) was carried out using the QuantStudio 6 Flex real-time PCR system (Thermo, Fisher Scientific, USA) to assess the relative expression of three subunits of Complex III (FgCytb, FgCytc1, FgISP), five assembly factors (FgQCR2, FgQCR6, FgQCR7, FgQCR8, FgQCR9), and DON biosynthesis-related genes (FgTri5 and FgTri6). '(in L526-531)

      (22) Lines 472-473: Why was FgCox1 used as a reference for FgCytb? Clarification is needed.

      Thank you very much for your question. FgCytb (cytochrome b) and FgCOX1 (cytochrome c oxidase subunit I) are both encoded by the mitochondrial genome and serve as core components of the oxidative phosphorylation system (Complex III and Complex IV, respectively). Their transcription is co-regulated by mitochondrial-specific mechanisms in response to cellular energy status. Consequently, under experimental conditions that perturb energy homeostasis, FgCOX1 expression exhibits relative, context-dependent stability with FgCytb, or at least co-varies directionally, making it a superior reference for normalizing target gene expression. In contrast, FgGapdh operates within a distinct genetic and regulatory system. Using FgCOX1 ensures that both reference and target genes reside within the same mitochondrial compartment and functional module, thereby preventing normalization artifacts arising from independent variation across disparate pathways.

      (23) Lines 476-477: This step requires a clearer and more detailed explanation.

      Thank you very much for your advice. We provided detailed descriptions of them in their respective positions. 'For FgDnm1-3×Flag, the 3×Flag tag was added to the C-terminus of FgDnm1 by PCR, fused with the hygromycin resistance gene and the FgDnm1 downstream arm, and then introduced into PH-1 protoplasts. '(in L417-419). 'The FgDnm1-3×Flag fragment was introduced into PH-1 and FgDML1+GFP protoplasts, respectively, to obtain single-tagged and double-tagged strains. '(in L541-543)

      Western blotting:

      (24) Uncropped Western blot images should be provided as supplementary material.

      Thank you very much for your advice. All Western blot images will be submitted to the supplementary material package.

      (25) Lines 485-489: A more thorough description of the antibodies used (including source, catalogue number, and dilution) is necessary.

      Thank you very much for your advice. The antibodies used are clearly stated in terms of brand, catalog number, and dilution. We have added the dilution ratio. 'All antibodies were diluted as follows: primary antibodies at 1:1000 and secondary antibodies at 1:10000. '(in L550-551)

      (26) The Western blot shown in Figure 3D appears problematic, particularly the anti-GAPDH band for FgDML1::FgTri1+GFP. Are both anti-GAPDH bands derived from the same gel?

      Thank you very much for your advice. We are unequivocally certain that these data derive from the same gel. Therefore, we are providing the original image for your inspection.

      Author response image 4.

    1. Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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      Reply to the reviewers

      Reviewer #1 (Evidence, reproducibility and clarity (Required)):

      Summary: This manuscript reports the identification of putative orthologues of mitochondrial contact site and cristae organizing system (MICOS) proteins in Plasmodium falciparum - an organism that unusually shows an acristate mitochondrion during the asexual part of its life cycle and then this develops cristae as it enters the sexual stage of its life cycle and beyond into the mosquito. The authors identify PfMIC60 and PfMIC19 as putative members and study these in detail. The authors at HA tags to both proteins and look for timing of expression during the parasite life cycle and attempt (unsuccessfully) to localise them within the parasite. They also genetically deleted both gene singly and in parallel and phenotyped the effect on parasite development. They show that both proteins are expressed in gametocytes and not asexuals, suggesting they are present at the same time as cristae development. They also show that the proteins are dispensible for the entire parasite life cycle investigated (asexuals through to sporozoites), however there is some reduction in mosquito transmission. Using EM techniques they show that the morphology of gametocyte mitochondria is abnormal in the knock out lines, although there is great variation.

      Major comments: The manuscript is interesting and is an intriguing use of a well studied organism of medical importance to answer fundamental biological questions. My main comments are that there should be greater detail in areas around methodology and statistical tests used. Also, the mosquito transmission assays (which are notoriously difficult to perform) show substantial variation between replicates and the statistical tests and data presentation are not clear enough to conclude the reduction in transmission that is claimed. Perhaps this could be improved with clearer text?

      We would like to thank the reviewer for taking the time to review our manuscript. We are happy to hear the reviewer thinks the manuscript is interesting and thank the reviewer for their constructive feedback.

      To clarify the statistical analyses used, we included a new supplementary dataset with all statistical analyses and p-values indicated per graph. Furthermore, figure legends now include the information on the exact statistical test used in each case.

      Regarding mosquito experiments, while we indeed reported a reduction in transmission and oocysts numbers we are aware that this effect might be due to the high variability in mosquito feeding assays. To highlight this point, we deleted the sentence "with the transmission reduction of [numbers]...." and we included the sentence "The high variability encountered in the standard membrane feeding assays, though, partially obstructs a clear conclusion on the biological relevance of the observed reduction in oocyst numbers"

      More specific comments to address: Line 101/Fig1E (and figure legend) - What is this heatmap showing. It would be helpful to have a sentence or two linking it to a specific methodology. I could not find details in the M+M section and "specialized, high molecular mass gels" does not adequately explain what experiments were performed. The reference to Supplementary Information 1 also did not provide information.

      We added the information "high molecular mass gels with lower acrylamide percentage" to clarify methodology in the text. Furthermore, we extended the figure legend to include all relevant information. Further experimental details can be found in the study cited in this context, where the dataset originates from (Evers et al., 2021).

      Line 115 and Supplementary Figure 2C + D - The main text says that the transgenic parasites contained a mitochondrially localized mScarlet for visualization and localization, but in the supplementary figure 2 it shows mitotracker labelling rather than mScarlet. This is very confusing. The figure legend also mentions both mScarlet and MitoTracker. I assume that mScarlet was used to view in regular IFAs (Fig S2C) and the MitoTracker was used for the expansion microscopy (Fig S2D)? Please clarify.

      We thank the reviewer for pointing this out - this was indeed incorrectly annotated. We used the endogenous mito-mScarlet signal in IFA and mitoTracker in U-ExM. The figure annotation has now been corrected.

      Figure 2C - what is the statistical test being used (the methods say "Mean oocysts per midgut and statistical significance were calculated using a generalized linear mixed effect model with a random experiment effect under a negative binomial distribution." but what test is this?)?

      The statistic test is now included in the material and method section with the sentence "The fitted model was used to obtain estimated means and contrasts and were evaluated using Wald Statistics". The test is now also mentioned in the figure legend.

      Also the choice of a log10 scale for oocyst intensity is an unusual choice - how are the mosquitoes with 0 oocysts being represented on this graph? It looks like they are being plotted at 10^-1 (which would be 0.1 oocysts in a mosquito which would be impossible).

      As the data spans three orders of magnitude with low values being biologically meaningful, we decided that a log scale would best facilitate readability of the graph. As the 0 values are also important to show, we went with a standard approach to handle 0s in log transformed data and substituted the 0s with a small value (0.001). We apologize for not mentioning this transformation in the manuscript. To make this transformation transparent, we added a break at the lower end of the log‑scaled y‑axis and relabelled the lowest tick as '0'. This ensures that mosquitoes with zero oocysts are shown along the x‑axis without being assigned an artificial value on the log scale. We would furthermore like to highlight that for statistics we used the true value 0 and not 0.001.

      Figure 2D - it is great that the data from all feeding replicates has been shared, however it is difficult to conclude any meaningful impact in transmission with the knock-out lines when there is so much variation and so few mosquitoes dissected for some datapoints (10 mosquitoes are very small sample sizes). For example, Exp1 shows a clear decrease in mic19- transmission, but then Exp2 does not really show as great effect. Similarly, why does the double knock out have better transmission than the single knockouts? Sure there would be a greater effect?

      We agree with the reviewer and with the new sentence added, as per major point, we hope we clarified the concept. Note that original Figure 2D has been moved to the supplementary information, as per minor comment of another reviewer.

      Figure 3 legend - Please add which statistical test was used and the number of replicates.

      Done

      Figure 4 legend - Please add which statistical test was used and the number of replicates.

      Done. Regarding replicates, note that while we measured over 100 cristae from over 30 mitochondria, these all stem from the same parasite culture.

      Figure 5C - the 3D reconstructions are very nice, but what does the red and yellow coloring show?

      Indeed, the information was missing. We added it to the figure legend.

      Line 352 - "Still, it is striking that, despite the pronounced morphological phenotype, and the possibly high mitochondrial stress levels, the parasites appeared mostly unaffected in life cycle propagation, raising questions about the functional relevance of mitochondria at these stages." How do the authors reconcile this statement with the proven fact that mitochondria-targeted antimalarials (such as atovaquone) are very potent inhibitors of parasite mosquito transmission?

      Our original sentence was reductive. What we wanted to state was related to the functional relevance of crista architecture and overall mitochondrial morphology rather than the general functional relevance of the mitochondria. We changed the sentence accordingly.

      Furthermore, even though we do not discuss this in the article, we are aware of mitochondria targeting drugs that are known to block mosquito transmission. We want to point out that it is difficult to discern the disruption of ETC and therefore an impact on energy conversion with the impact on the essential pathway of pyrimidine synthesis, highly relevant in microgamete formation. Still, a recent paper from Sparkes et al. 2024 showed the essentiality of mitochondrial ATP synthesis during gametogenesis so it is very likely that the mitochondrial energy conversion is highly relevant for transmission to the mosquito.

      Reviewer #1 (Significance (Required)):

      This manuscript is a novel approach to studying mitochondrial biology and does open a lot of unanswered questions for further research directions. Currently there are limitations in the use of statistical tests and detail of methodology, but these could be easily be addressed with a bit more analysis/better explanation in the text. This manuscript could be of interest to readers with a general interest in mitochondrial cell biology and those within the specific field of Plasmodium research. My expertise is in Plasmodium cell biology.

      We thank the reviewer for the praise.

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

      Major comments: 1) In my opinion, the authors tend to sensationalize or overinterpret their results. The title of the manuscript is very misleading. While MICOS is certainly important for crista formation, it is not the only factor, as ATP synthase dimer rows make a highly significant contribution to crista morphology. Thus, one can argue with equal validity that ATP synthase should be considered the 'architect', as it's the conformation of the dimers and rows modulate positive curvature. Secondly, while cristae are still formed upon mic60/mic19 gene knockout (KO), they are severely deformed, and likely dysfunctional (see below). Thus, I do not agree with the title that MICOS is dispensable for crista formation, because the authors results show that it clearly is essential. So, the title should be changed.

      We thank the reviewer for taking the time to review our manuscript.

      Based on the reviewers' interpretation we conclude the title does not come across as intended. We have changed the title to: "The role of MICOS in organizing mitochondrial cristae in malaria parasites"

      The Discussion section starting from line 373 also suffers from overinterpretation as well as being repetitive and hard to understand. The authors infer that MICOS stability is compromised less in the single KOs (sKO) in compared to the mic60/mic19 double KO (dKO). MICOS stability was never directly addressed here and the composition of the MICOS complex is unaddressed, so it does not make sense to speculate by such tenuous connections. The data suggest to me that mic60 and mic19 are equally important for crista formation and crista junction (CJ) stabilization, and the dKO has a more severe phenotype than either KO, further demonstrating neither is epistatic.

      We do agree with the reviewer's notion that we did not address complex stability, and our wording did not make this sufficiently clear. We shortened and rephrased the paragraph in question.

      The following paragraphs (line 387 to 422) continues with such unnecessary overinterpretation to the point that it is confusing and contradictory. Line 387 mentions an 'almost complete loss of CJs' and then line 411 mentions an increase in CJ diameter, both upon Mic60 ablation. I do not think this discussion brings any added value to the manuscript and should be shortened. Yes, maybe there are other putative MICOS subunits that may linger in the KOS that are further destabilized in the dKO, or maybe Mic60 remains in the mic19 KO (and vice versa) to somehow salvage more CJs, which is not possible in the dKO. It is impossible to say with confidence how ATP synthase behaves in the KOs with the current data.

      We shortened this paragraph.

      2) While the authors went through impressive lengths to detect any effect on lifecycle progression, none was found except for a reduction in oocyte count. However, the authors did not address any direct effect on mitochondria, such as OXPHOS complex assembly, respiration, membrane potential. This seems like a missed opportunity, given the team's previous and very nice work mapping these complexes by complexome profiling. However, I think there are some experiments the authors can still do to address any mitochondrial defects using what they have and not resorting to complexome profiling (although this would be definitive if it is feasible):

      i) Quantification of MitoTracker Red staining in WT and KOs. The authors used this dye to visualize mitochondria to assay their gross morphology, but unfortunately not to assay membrane potential in the mutants. The authors can compare relative intensities of the different mitochondria types they categorized in Fig. 3A in 20-30 cells to determine if membrane potential is affected when the cristae are deformed in the mutants. One would predict they are affected.

      Interesting suggestion. As our staining and imaging conditions are suitable for such analysis (as demonstrated by Sarazin et al., 2025, https://www.biorxiv.org/content/10.1101/2025.11.27.690934v1), we performed the measurements on the same dataset which we collected for Figure 3. We did, however, not detect any difference in mitotracker intensity between the different lines. The result of this analysis is included in the new version of Supplementary figure S6.

      ii) Sporozoites are shown in Fig S5. The authors can use the same set up to track their motion, with the hypothesis that they will be slower in the mutants compared to WT due to less ATP. This assumes that sporozoite mitochondria are active as in gametocytes.

      While theoretically plausible and informative, we currently do not know the relevance of mitochondrial energy conversion for general sporozoite biology or specifically features of sporozoite movement. Given the required resources and time to set this experiment up and the uncertainty whether it is a relevant proxy for mitochondrial functioning, we argue it is out of scope for this manuscript.

      iii) Shotgun proteomics to compare protein levels in mutants compared to WT, with the hypothesis that OXPHOS complex subunits will be destabilized in the mutants with deformed cristae. This could be indirect evidence that OXPHOS assembly is affected, resulting in destabilized subunits that fail to incorporate into their respective complexes.

      While this experiment could potentially further our understanding of the interaction between MICOS and levels of OXPHOS complex subunits we argue that the indirect nature of the evidence does not justify the required investments.

      To expedite resubmission, the authors can restrict the cell lines to WT and the dKO, as the latter has a stronger phenotype that the individual KOs and conclusions from this cell line are valid for overall conclusions about Plasmodium MICOS.

      I will also conclude that complexome/shotgun proteomics may be a useful tool also for identifying other putative MICOS subunits by determining if proteins sharing the same complexome profile as PfMic60 and Mic19 are affected. This would address the overinterpretation problem of point 1.

      3) I am aware of the authors previous work in which they were not able to detect cristae in ABS, and thus have concluded that these are truly acristate. This can very well be true, or there can be immature cristae forms that evaded detection at the resolution they used in their volumetric EM acquisitions. The mitochondria and gametocyte cristae are pretty small anyway, so it not unreasonable to assume that putative rudimentary cristae in ABS may be even smaller still. Minute levels of sampled complex III and IV plus complex V dimers in ABS that were detected previously by the authors by complexome profiling would argue for the presence of miniscule and/or very few cristae.

      I think that authors should hedge their claim that ABS is acrisate by briefly stating that there still is a possibility that miniscule cristae may have been overlooked previously.

      We acknowledge that we cannot demonstrate the absolute absence of any membrane irregularities along the inner mitochondrial membrane. At the same time, if such structures were present, they would be extremely small and unlikely to contain the full set of proteins characteristic of mature cristae. For this reason, we consider it appropriate to classify ABS mitochondria as acristate. To reflect the reviewer's point while maintaining clarity for readers, we have slightly adjusted our wording in the manuscript, changing 'fully acristate' to 'acristate'.

      This brings me to the claim that Mic19 and Mic60 proteins are not expressed in ABS. This is based on the lack of signal from the epitope tag; a weak signal is detected in gametocytes. Thus, one can counter that Mic19 and Mic60 are also expressed, but below the expression limits of the assay, as the protein exhibits low expression levels when mitochondrial activity is upregulated.

      We agree with the reviewer that the absence of a detectable epitope‑tag signal does not definitively exclude low‑level expression, and we have therefore replaced the term 'absent' with 'undetectable' throughout the manuscript. In context with previous findings of low-level transcripts of the proteins in a study by Lopez-Berragan et al. and Otto et al., we also added the sentence "The apparent absence could indicate that transcripts are not translated in ABS or that the proteins' expression was below detection limits of western blot analysis." to the discussion. _At the same time, we would like to clarify that transcript levels for both genes fall within the

      To address this point, the authors should determine of mature mic60 and mic19 mRNAs are detected in ABS in comparison to the dKO, which will lack either transcript. RT-qPCR using polyT primers can be employed to detect these transcripts. If the level of these mRNAs are equivalent to dKO in WT ABS, the authors can make a pretty strong case for the absence of cristae in ABS.

      We appreciate the reviewer's suggestion. As noted in the Discussion, existing transcriptomic datasets already show detectable MIC19 and MIC60 mRNAs in ABS. For this reason, we expect RT-qPCR to reveal low (but not absent) levels of both transcripts, unlike the true loss expected to be observed in the dKO. Because such residual signals have been reported previously and their biological relevance remains uncertain, we do not believe transcript levels alone can serve as a definitive indicator of cristae absence in ABS.

      They should highlight the twin CX9C motifs that are a hallmark of Mic19 and other proteins that undergo oxidative folding via the MIA pathway. Interestingly, the Mia40 oxidoreductase that is central to MIA in yeast and animals, is absent in apicomplexans (DOI: 10.1080/19420889.2015.1094593).

      Searching for the CX9C motifs is a valuable suggestion. In response to the reviewer´s suggestion we analysed the conservation of the motif in PfMIC19 and included this in a new figure panel (Figure 1 F).

      Did the authors try to align Plasmodium Mic19 orthologs with conventional Mic19s? This may reveal some conserved residues within and outside of the CHCH domain.

      In response to this comment we made Figure 1 F, where we show conserved residues within the CHCH domains of a broad range of MIC19 annotated sequences across the opisthokonts, and show that the Cx9C motifs are conserved also in PfMIC19. Outside the CHCH domain, we did not find any meaningful conservation, as PfMIC19 heavily diverges from opisthokont MIC19.

      5) Statistcal significance. Sometimes my eyes see population differences that are considered insignificant by the statistical methods employed by the authors, eg Fig. 4E, mutants compared to WT, especially the dKO. Have the authors considered using other methods such as student t-test for pairwise comparisons?

      The graphs in figures 3, 4 and 5 got a makeover, such that they now are in linear scale and violin plots (also following a suggestion from further down in the reviewer's comments). We believe that this improves interpretability. ANOVA was kept as statistical testing to assure the correction for multiple comparisons that cannot be performed with standard t-test. A full overview of statistics and exact p-values can also be found in the newly added supplementary information 2.

      Minor comments: Line 33. Anaerobes (eg Giardia) have mitochondria that do produce ATP, unlike aerobic mitochondria

      We acknowledge that producing ATP via OXPHOS is not a characteristic of all mitochondria-like organelles (e.g. mitosomes), which is why these are typically classified separately from canonical mitochondria. When not considering mitochondria-like organelles, energy conversion is the function that the mitochondrion is most well-known for and the one associated with cristae.

      Line 56: Unclear what authors mean by "canonical model of mitochondria"

      To clarify we changed this to "yeast or human" model of mitochondria.

      Lines 75-76: This applies to Mic10 only

      We removed the "high degree of conservation in other cristate eukaryotes" statement.

      Line 80: Cite DOI: 10.1016/j.cub.2020.02.053

      Done

      Fig 2D: I find this table difficult to read. If authors keep table format, at least get rid of 'mean' column' as this data is better depicted in 2C. I suggest depicted this data either like in 3B depicting portion of infected vs unaffected flies in all experiments, then move modified Table to supplement. Important to point out experiment 5 appears to be an outlier with reduced infectivity across all cell lines, including WT.

      To clarify: the mean reported in the table indicates the mean per replicate while the mean reported in figure 2C is the overall mean for a given genotype that corrects for variability within experiments. We agree that moving the table to the supplementary data is a good idea. We decided to not include a graph for infected and non-infected mosquitoes as this information would be partially misleading, highlighting a phenotype we argue to be influenced by the strong variability.

      Fig. 3C-G: I feel like these data repeatedly lead to same conclusions. These are all different ways of showing what is depicted in Fig 2B: mitochondria gross morphology is affected upon ablation of MICOS. I suggest that these graphs be moved to supplement and replaced by the beautiful images.

      Thank you for the nice comment on our images. We have now moved part of the graphs to supplementary figure 6 and only kept the Relative Frequency, Sphericity and total mitochondria volume per cell in the main figure.

      Line 180: Be more specific with which tubulin isoform is used as a male marker and state why this marker was used in supplemental Fig S6.

      We have now specified the exact tubulin isoform used as the male gametocyte marker, both in the main text and in Supplementary Fig. S6. This is a commercial antibody previously known to work as an effective male marker, which is why we selected it for this experiment. This is now clearly stated in the manuscript.

      Line 196 and Fig 3C: the word 'intensities' in this context is very ambiguous. Please choose a different term (puncta, elements, parts?). This is related to major point 2i above.

      To clarify the biological effect that we can conclude form the measurement, we added an explanation about it in the respective section of the results, and we decided to replace the raw results of the plug-in readout with the deduced relative dispersion.

      Line 222: Report male/female crista measurements

      We added Supplementary information 2, which contains exact statistical test and outcomes on all presented quantifications as well as a per-sex statistical analysis of the data from figure 4. Correspondingly, we extended supplementary information 2 by a per-sex colour code for the thin section TEM data.

      Fig. 4B-E: depict data as violin plots or scatter plots like Fig. 2C to get a better grasp of how the crista coverage is distributed. It seems like the data spread is wider in the double KO. This would also solve the problem with the standard deviation extending beyond 0%.

      We changed this accordingly.

      Lines 331-333: Please clarify that this applies for some, but not all MICOS subunits. Please also see major point 1 above. Also, the authors should point out that despite their structural divergence, trypanosomal cryptic mitofilins Mic34 and Mic40 are essential for parasite growth, in contrast to their findings with PfMic60 (DOI: https://doi.org/10.1101/2025.01.31.635831).

      This has been changed accordingly.

      Line 320: incorrect citation. Related to point 1above.

      Correct citation is now included in the text.

      Lines 333-335. This is related to the above. Again, some subunits appear to affect cell growth under lab conditions, and some do not. This and the previous sentence should be rewritten to reflect this.

      This has been changed accordingly.

      Line 343-345: The sentence and citation 45 are strange. Regarding the former, it is about CHCHD10, whose status as a bona fide MICOS subunit is very tenuous, so I would omit this. About the phenomenon observed, I think it makes more sense to write that Mic60 ablation results in partially fragmented mitochondria in yeast (Rabl et al., 2009 J Cell Biol. 185: 1047-63). A fragmented mitochondria is often a physiological response to stress. I would just rewrite as not to imply that mitochondrial fission (or fusion) is impaired in these KOs, or at least this could be one of several possibilities.

      The sentence has been substituted following the indication of the reviewer. Though we still include the data of the human cells as this has also been shown in Stephens et al. 2020.

      Line 373: 'This indicates' is too strong. I would say 'may suggest' as you have no proof that any of the KOs disrupts MICOS. This hypothesis can be tested by other means, but not by penetrance of a phenotype.

      Done

      Line 376-377; 'deplete functionality' does not make sense, especially in the context of talking about MICOS subunit stability. In my opinion, this paragraph overinterprets the KO effects on MICOS stability. None of the experiments address this phenomenon, and thus the authors should not try to interpret their results in this context. See major point 1. Other suggestions for added value

      We removed the sentence. Also, the entire paragraph has been shortened, restructured and wording was changed to address major point 1.

      1) Does Plasmodium Sam50 co-fractionate with Mic60 and Mic19 in BN PAGE (Fig. 1E)

      While we did identify SAMM50 in our BN PAGE, the protein does not co-migrate with the MICOS components but instead comigrates with other components of a putative sorting and assembly machinery (SAM) complex. As SAMM50, the SAM complex and the overarching putative mitochondrial membrane space bridging (MIB) complex are not mentioned in the manuscript, we decided to not include the information in the figure.

      Reviewer #2 (Significance (Required)):

      The manuscript by Tassan-Lugrezin is predicated on the idea that Plasmodium represents the only system in which de novo crista formation can be studied. They leverage this system to ask the question whether MICOS is essential for this process. They conclude based on their data that the answer is no, which the authors consider unprecedented. But even if their claim is true that ABS is acristate, this supposed advantage does not really bring any meaningful insight into how MICOS works in Plasmodium.

      First the positives of this manuscript. As has been the case with this research team, the manuscript is very sophisticated in the experimental approaches that are made. The highlights are the beautiful and often conclusive microscopy performed by the authors. Only the localization of Mic60 and Mic19 was inconclusive due to their very low expression unfortunately.

      The examination of the MICOS mutants during in vitro life cycle of Plasmodium falciparum is extremely impressive and yields convincing results. Mitochondrial deformation is tolerated by life cycle stage differentiation, with a modest but significant reduction of oocyte production, being observed.

      However, despite the herculean efforts of the authors, the manuscript as it currently stands represents only a minor advance in our understanding of the evolution of MICOS, which from the title and focus of the manuscript, is the main goal of the authors. In its current form, the manuscript reports some potentially important findings:

      1) Mic60 is verified to play a role in crista formation, as is predicted by its orthology to other characterized Mic60 orthologs.

      2) The discovery of a novel Mic19 analog (since the authors maintain there is no significant sequence homology), which exhibits a similar (or the same?) complexome profile with Mic60. This protein was upregulated in gametocytes like Mic60 and phenocopies Mic60 KO.

      3) Both of these MICOS subunits are essential (not dispensable) for proper crista formation

      4) Surprisingly, neither MICOS subunit is essential for in vitro growth or differentiation from ABS to sexual stages, and from the latter to sporozoites. This says more about the biology of plasmodium itself than anything about the essentiality of Mic60, ie plasmodium life cycle progression tolerates defects to mitochondrial morphology. But yes, I agree with the authors that Mic60's apparent insignificance for cell growth in examined conditions does differ with its essentiality in other eukaryotes. But fitness costs were not assayed (eg by competition between mutants and WT in infection of mosquitoes)

      5) Decreased fitness of the mutants is implied by a reduction of oocyte formation.

      While interesting in their own way, collectively they do not represent a major advance in our understanding of MICOS evolution. Furthermore, the findings bifurcate into categories informing MICOS or Plasmodium biology. Both aspects are somewhat underdeveloped in their current form.

      This is unfortunate because there seem to be many missed opportunities in the manuscript that could, with additional experiments, lead to a manuscript with much wider impact. For me, what is remarkable about Plasmodium MICOS that sets it apart from other iterations is the apparent absence of the Mic10 subunit. Purification of plasmodium MICOS via the epitope tagged Mic60 and Mic19 could have verified that MICOS is assembled without this core subunit. Perhaps Mic60 and Mic19 are the vestiges of the complex, and thus operate alone in shaping cristae. Such a reduction may also suggest the declining importance of mitochondria in plasmodium.

      Another missed opportunity was to assay the impact of MICOS-depletion of OXPHOS in plasmodium. This is a salient issue as maybe crista morphology is decoupled from OXPHOS capacity in Plasmodium, which links to the apparent tolerance of mitochondrial morphology in cell growth and differentiation. I suggested in section A experiments to address this deficit.

      Finally, the authors could assay fitness costs of MICOS-ablation and associated phenotypes by assaying whether mosquito infectivity is reduced in the mutants when they are directly competing with WT plasmodium. Like the authors, I am also surprised that MICOS mutants can pass population bottlenecks represented by differentiation events. Perhaps the apparent robustness of differentiation may contribute plasmodium's remarkable ability to adapt.

      I realize that the authors put a lot of efforts into their study and again, I am very impressed by the sophistication of the methods employed. Nevertheless, I think there is still better ways to increase the impact of the study aside from overinterpreting the conclusions from the data. But this would require more experiments along the lines I suggest in Section A and here.

      We thank the reviewer for their extensive analysis of the significance of our findings, including the compliments on our microscopy images and the sophisticated experimental approaches. We hope we have convincingly argued why we could or could not include some of the additional analyses suggested by the reviewer in section 1 above.

      With regard to the significance statement, we want to point out that our finding that PfMICOS is not needed for initial formation of cristae (as opposed to organization thereof), is a confirmation of something that has been assumed by the field, without being the actual focus of studies. We argue that the distinction between formation and organization of cristae is important and deserves some attention within the manuscript. The result of MICOS not being involved in the initial formation of cristae, we argue to be relevant in Plasmodium biology and beyond. As for the insights into how MICOS works in Plasmodium we have confirmed that the previously annotated PfMIC60 is indeed involved in the organization of cristae. Furthermore, we have identified and characterized PfMIC19. These findings, we argue, are indeed meaningful insights into PfMICOS.

      Reviewer #3 (Evidence, reproducibility and clarity (Required)):

      Summary:

      MICOS is a conserved mitochondrial protein complex responsible for organising the mitochondrial inner membrane and the maintenance of cristae junctions. This study sheds first light on the role of two MICOS subunits (Mic60 and the newly annotated Mic19) in the malaria parasite Plasmodium falciparum, which forms cristae de novo during sexual development, as demonstrated by EM of thin section and electron tomography. By generating knockout lines (including a double knockout), the authors demonstrate that knockout of both MICOS subunits leads to defects in cristae morphology and a partial loss of cristae junctions. With a formidable set of parasitological assays, the authors show that despite the metabolically important role of mitochondria for gametocytes, the knockout lines can progress through the life stages and form sporozoites, albeit with diminished infection efficiency.

      We thank the reviewer for their time and compliment.

      Major comments:

      1) The authors should improve to present their findings in the right context, in particular by:

      (i) giving a clearer description in the introduction of what is already known about the role of MICOS. This starts in the introduction, where one main finding is missing: loss of MICOS leads to loss of cristae junctions and the detachment of cristae membranes, which are nevertheless formed, but become membrane vesicles. This needs to be clearly stated in the introduction to allow the reader to understand the consistency of the authors' findings in P. falciparum with previous reports in the literature.

      We extended the introduction to include this information.

      (ii) at the end to the introduction, the motivating hypothesis is formulated ad hoc "conclusive evidence about its involvement in the initial formation of cristae is still lacking" (line 83). If there is evidence in the literature that MICOS is strictly required for cristae formation in any organism, then this should be explained, because the bona fide role of MICOS is maintenance of cristae junctions (the hypothesis is still plausible and its testing important).

      To clarify we rephrased the sentence to: "Although MICOS has been described as an organizer of crista junctions, its role during the initial formation of nascent cristae has not been investigated."

      2) Line 96-97: "Interestingly, PfMIC60 is much larger than the human MICOS counterpart, with a large, poorly predicted N-terminal extension." This statement is lacking a reference and presumably refers to annotated ORFs. The authors should clarify if the true N-terminus is definitely known - a 120kDa size is shown for the P. falciparum but this is not compared to the expected length or the size in S. cerevisiae.

      To solve the reference issue, we added the uniprot IDs we compared to see that the annotated ORF is bigger in Plasmodium. We also changed the comparison to yeast instead of human, because we realized it is confusing to compare to yeast all throughout the figure, but then talk about human in this specific sentence.

      Regarding whether the true N-terminus is known. Short answer: No, not exactly.

      However, we do know that the Pf version is about double the size of the yeast protein.

      As the reviewer correctly states, we show the size of 120kDa for the tagged protein in Figure 1G. Considering that we tagged the protein C-terminally, and observed a 120kDa product on western blot, it is safe to conclude that the true N-terminus does not deviate massively from the annotated ORF, and hence, that there is a considerable extension of the protein beyond a 60kDa protein. We do not directly compare to yeast MIC60 on our western blots, however, that comparison can be drawn from literature: Tarasenko et al., 2017 showed that purified MIC60 running at ~60kDa on SDS-PAGE actively bends membranes, suggesting that in its active form, the monomer of yeast MIC60 is indeed 60kDa in size.

      To clarify, we now emphasize that we ran the Alphafold prediction on the annotated open reading frame (annotated and sequenced by Bohme et al. and Chapell et al. now cited in the manuscript), and revised the wording to make clear what we are comparing in which sentence.

      3) lines 244-245: "Furthermore, our data indicates the effect size increases with simultaneous ablation of both proteins?". The authors should explain which data they are referring to, as some of the data in Fig 3 and 4 look similar and all significance tests relate to the wild type, not between the different mutants, so it is not clear if any overserved differences are significant. The authors repeat this claim in the discussion in lines 368-369 without referring to a specific significance test. This needs to be clarified.

      As a reply to this and other comments from the reviewers we added the multiple testing within all samples. In addition, to clarify statistics used we included a supplementary dataset with all p-values and statistical tests used.

      4) lines 304-306: "Though well established as the cristae organizing system, the role of MICOS in initial formation of cristae remains hidden in model organisms that constitutively display cristae.". This sentence is misleading since even in organisms that display numerous cristae throughout their life cycle, new cristae are being formed as the cells proliferate. Thus, failure to produce cristae in MICOS knockout lines would have been observable but has apparently not been reported in the literature. Thus, the concerted process in P. falciparum makes it a great model organism, but not fundamentally different to what has been studied before in other organisms.

      We deleted this statement.

      5) lines 373-378. "where ablation of just MIC60 is sufficient to deplete functionality of the entire MICOS (11, 15),". The authors' claim appears to be contrary to what is actually stated in ref 15, which they cite:

      "MICOS subunits have non-redundant functions as the absence of both MICOS subcomplexes results in more severe morphological and respiratory growth defects than deletion of single MICOS subunits or subcomplexes."

      This seems in line with what the authors show, rather than "different".

      This sentence has been removed.

      6) lines 380-385: "... thus suggesting that membrane invaginations still arise, but are not properly arranged in these knockout lines. This suggests that MICOS either isn't fully depleted,...". These conclusions are incompatible with findings from ref. 15, which the authors cite. In that study, the authors generated a ∆MICOS line which still forms membrane invaginations, showing that MICOS is not required at all for this process in yeast. Hence the authors' implication that MICOS needs to be fully depleted before membrane invaginations cease to occur is not supported by the literature.

      This sentence has been deleted in the revised version of the manuscript.

      Minor comments:

      7) The authors should consider if the first part of their title could be seen as misleading: It suggests that MICOS is "the architect" in cristae formation, but this is not consistent with the literature nor their own findings.

      Title is changed accordingly

      Minor comments:

      • Line 43, of the three seminal papers describing the discovery of MICOS in 2011, the authors only cite two (refs 6 and 7), but miss the third paper, Hoppins et al, PMID: 21987634, which should probably be corrected.

      Done, the paper is now cited

      • Page 2, line 58: for a more complete picture the authors should also cite the work of others here which shows that although at very low levels, e.g. complex III (a drug target) and ATP synthase do assemble (Nina et al, 2011, JBC).

      Done

      • Page 3, line 80: "Irrespective of the shape of an organism's cristae, the crista junctions have been described as tubular channels that connect the cristae membrane to the inner boundary membrane (22, 24)." This omits the slit-shaped cristae junctions found in yeast (Davies et al, 2011, PNAS), which the authors should include.

      The paper and concept have been added to the manuscript, though the sentence has been moved up in the introduction, when crista junctions are first introduced.

      • Line 97: "poorly predicted N-terminal extension", as there is no experimental structure, we don't know if the prediction is poor. Presumably the authors mean either poorly ordered or the absence of secondary structure elements, or the poor confidence score for that region in the prediction? This should be clarified or corrected.

      We were referring to the poor confidence score. To address this comment as well as major point 2, we rewrote the respective paragraph. It now clearly states that confidence of the prediction is low, and we mention the tool that was used to identify conserved domains (Topology-based Evolutionary Domains).

      • Line 98: "an antiparallel array of ten β-sheets". They are actually two parallel beta-sheets stacked together. The authors could find out the name of this fold, but the confidence of the prediction is marked a low/very low. So, its existence is unknown, not just its "function".

      We adapted the domain description to "a stack of two parallel beta-sheets" and replaced the statement on unknown function by the statement "Because this domain is predicted solely from computational analysis, both its actual existence in the native protein and its biological function remain unknown."

      Fig 1B: The authors show two alphafold predictions of S. cerevisiae and P. falciparum Mic60 structures. There is however an experimental Mic60/19 (fragment) structure from the former organism (PMID: 36044574), which should be included if possible

      We appreciate the reviewer's suggestion and note that the available structural data indeed provides valuable insight into how MIC60 and MIC19 interact. However, these structures represent fusion constructs of limited protein fragments and therefore capture only a small portion of each protein, specifically the interaction interface. Because our aim in Fig. 1B is to compare the overall domain architecture of the full‑length proteins, we believe that including fragment‑based structures would be less informative in this context.

      Line: 318-321: "The same trend was observed for PfMIC19 and PfMIC60. Although transcriptomic data suggested that low-level transcripts of PfMIC19 and PfMIC60 are present in ABS (38), we did not detect either of the proteins in ABS by western blot analysis. While this statement is true, the authors should comment on the sensitivity of the respective methods - how well was the antibody working in their hands and how do they interpret the absence of a WB band compared to transcriptomics data?

      The HA antibody used in our experiments is a standard commercial reagent that performs reliably in both WB and IFA, although it shows a low background signal in gametocytes. We agree that the sensitivity of the method and the interpretation of weak or absent bands should be addressed explicitly. Transcript levels for both PfMIC19 and PfMIC60 in asexual blood stages fall within the

      • Lines 322-323: would the authors not typically have expected an IFA signal given the strength of the band in Western blot? If possible, the authors should comment if the negative fluorescence outcome can indeed be explained with the low abundance or if technical challenges are an equally good explanation.

      Considering the nature of the investigated proteins (embedded in the IMM and spread throughout the mitochondria) difficulties in achieving a clear signal in IFA or U-ExM are not very surprizing. While epitopes may remain buried in IFA, U-ExM usually increases accessibility for the antibodies. However, U-ExM comes at the cost of being prone to dotty background signals, therefore potentially hiding low abundance, naturally dotty signals such as the signal of MICOS proteins that localize to distinct foci (at the CJ) along the mitochondrion. Current literature suggests that, in both human and yeast, STED is the preferred method for accurate spatial resolution of MICOS proteins (https://www.ncbi.nlm.nih.gov/pubmed/32567732,https://www.ncbi.nlm.nih.gov/pubmed/32067344). Unfortunately, we do not have experience with, nor access to, this particular technique/method.

      Lines 357-365: the authors describe limitations of the applied methods adequately. Perhaps it would be helpful to make a similar statement about the analysis of 3D objects like mitochondria and cristae from 2D sections. E.g. the apparent cristae length depends on whether cristae are straight (e.g. coiled structures do not display long cross sections despite their true length in 3D).

      The limitations of other methods are described in the respective results section.

      We added a clarifying sentence in the results section of Figure 4:

      "Note that such measurements do not indicate the true total length or width of cristae, as the data is two-dimensional. The recorded values are to be considered indicative of possible trends, rather than absolute dimensions of cristae."

      This statement refers to the length/width measurements of cristae.

      In the context of Figure 4 D we mention the following (see preprint lines 229 - 230): "We expect this effect to translate into the third dimension and thus conclude that the mean crista volume increases with the loss of either PfMIC19,PfMIC60, or both."

      For Figure 5, we included a clarifying statement in the results section of the preprint (lines 269 - 273): "Note that these mitochondrial volumes are not full mitochondria, but large segments thereof. As a result of the incompleteness of the mitochondria within the section, and the tomography specific artefact of the missing wedge, we were unable to confirm whether cristae were in fact fully detached from the boundary membrane, or just too long to fit within the observable z-range. "

      Line 404: perhaps undetected or similar would be a better description than "hidden"?

      The sentence does not exist in the revised manuscript

      Reviewer #3 (Significance (Required)):

      The main strength of the study is that it provides the first characterisation of the MICOS complex in P. falciparum, a human parasite in which the mitochondrion has been shown to be a drug target. Mic60 and the newly annotated Mic19 are confirmed to be essential for proper cristae formation and morphology, as well as overall mitochondrial morphology. Furthermore, the mutant lines are characterised for their ability to complete the parasite life cycle and defects in infection effectivity are observed. This work is an important first step for deciphering the role of MICOS in the malaria parasite and the composition and function of this complex in this organism. The limitation of the study stems from what is already known about MICOS and its subunits in

      great detail in yeast and humans with similar findings regarding loss of cristae and cristae defects. The findings of this study do not provide dramatic new insight on MICOS function or go substantially beyond the vast existing literature in terms of the extent of the study, which focuses on parasitological assays and morphological analysis. Exploring the role of MICOS in an early-divergent organism and human parasite is however important given the divergence found in mitochondrial biology and P. falciparum is a uniquely suited model system. One aspect that would increase the impact of the paper would be if the authors could mechanistically link the observed morphological defects to the decreased infection efficiency, e.g. by probing effects on mitochondrial function. This will likely be challenging as the morphological defects are diverse and the fitness defects appear moderate/mild.

      As suggested by Reviewer 2, we examined mitochondrial membrane potential in gametocytes using MitoTracker staining and did not observe any obvious differences associated with the morphological defects. At present, additional assays to probe mitochondrial function in P. falciparum gametocytes are not sufficiently established, and developing and validating such methods would require substantial work before they could be applied to our mutant lines. For these reasons, a more detailed mechanistic link between the observed morphological changes and the reduced infection efficiency is currently beyond reach.

      The advance presented in this study is to pioneer the study of MICOS in P. falciparum, thus widening our understanding of the role of this complex to different model organism. This study will likely be mainly of interest for specialised audiences such as basic research parasitologists and mitochondrial biologists. My own field of expertise is mitochondrial biology and structural biology.

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

      Learn more at Review Commons


      Referee #2

      Evidence, reproducibility and clarity

      Major comments:

      1) In my opinion, the authors tend to sensationalize or overinterpret their results. The title of the manuscript is very misleading. While MICOS is certainly important for crista formation, it is not the only factor, as ATP synthase dimer rows make a highly significant contribution to crista morphology. Thus, one can argue with equal validity that ATP synthase should be considered the 'architect', as it's the conformation of the dimers and rows modulate positive curvature. Secondly, while cristae are still formed upon mic60/mic19 gene knockout (KO), they are severely deformed, and likely dysfunctional (see below). Thus, I do not agree with the title that MICOS is dispensable for crista formation, because the authors results show that it clearly is essential. So, the title should be changed.

      The Discussion section starting from line 373 also suffers from overinterpretation as well as being repetitive and hard to understand. The authors infer that MICOS stability is compromised less in the single KOs (sKO) in compared to the mic60/mic19 double KO (dKO). MICOS stability was never directly addressed here and the composition of the MICOS complex is unaddressed, so it does not make sense to speculate by such tenuous connections. The data suggest to me that mic60 and mic19 are equally important for crista formation and crista junction (CJ) stabilization, and the dKO has a more severe phenotype than either KO, further demonstrating neither is epistatic.

      The following paragraphs (line 387 to 422) continues with such unnecessary overinterpretation to the point that it is confusing and contradictory. Line 387 mentions an 'almost complete loss of CJs' and then line 411 mentions an increase in CJ diameter, both upon Mic60 ablation. I do not think this discussion brings any added value to the manuscript and should be shortened. Yes, maybe there are other putative MICOS subunits that may linger in the KOS that are further destabilized in the dKO, or maybe Mic60 remains in the mic19 KO (and vice versa) to somehow salvage more CJs, which is not possible in the dKO. It is impossible to say with confidence how ATP synthase behaves in the KOs with the current data.

      2) While the authors went through impressive lengths to detect any effect on lifecycle progression, none was found except for a reduction in oocyte count. However, the authors did not address any direct effect on mitochondria, such as OXPHOS complex assembly, respiration, membrane potential. This seems like a missed opportunity, given the team's previous and very nice work mapping these complexes by complexome profiling. However, I think there are some experiments the authors can still do to address any mitochondrial defects using what they have and not resorting to complexome profiling (although this would be definitive if it is feasible):

      i) Quantification of MitoTracker Red staining in WT and KOs. The authors used this dye to visualize mitochondria to assay their gross morphology, but unfortunately not to assay membrane potential in the mutants. The authors can compare relative intensities of the different mitochondria types they categorized in Fig. 3A in 20-30 cells to determine if membrane potential is affected when the cristae are deformed in the mutants. One would predict they are affected.

      ii) Sporozoites are shown in Fig S5. The authors can use the same set up to track their motion, with the hypothesis that they will be slower in the mutants compared to WT due to less ATP. This assumes that sporozoite mitochondria are active as in gametocytes.

      iii) Shotgun proteomics to compare protein levels in mutants compared to WT, with the hypothesis that OXPHOS complex subunits will be destabilized in the mutants with deformed cristae. This could be indirect evidence that OXPHOS assembly is affected, resulting in destabilized subunits that fail to incorporate into their respective complexes.

      To expedite resubmission, the authors can restrict the cell lines to WT and the dKO, as the latter has a stronger phenotype that the individual KOs and conclusions from this cell line are valid for overall conclusions about Plasmodium MICOS.

      I will also conclude that complexome/shotgun proteomics may be a useful tool also for identifying other putative MICOS subunits by determining if proteins sharing the same complexome profile as PfMic60 and Mic19 are affected. This would address the overinterpretation problem of point 1.

      3) I am aware of the authors previous work in which they were not able to detect cristae in ABS, and thus have concluded that these are truly acristate. This can very well be true, or there can be immature cristae forms that evaded detection at the resolution they used in their volumetric EM acquisitions. The mitochondria and gametocyte cristae are pretty small anyway, so it not unreasonable to assume that putative rudimentary cristae in ABS may be even smaller still. Minute levels of sampled complex III and IV plus complex V dimers in ABS that were detected previously by the authors by complexome profiling would argue for the presence of miniscule and/or very few cristae.

      I think that authors should hedge their claim that ABS is acrisate by briefly stating that there still is a possibility that miniscule cristae may have been overlooked previously.

      This brings me to the claim that Mic19 and Mic60 proteins are not expressed in ABS. This is based on the lack of signal from the epitope tag; a weak signal is detected in gametocytes. Thus, one can counter that Mic19 and Mic60 are also expressed, but below the expression limits of the assay, as the protein exhibits low expression levels when mitochondrial activity is upregulated.

      To address this point, the authors should determine of mature mic60 and mic19 mRNAs are detected in ABS in comparison to the dKO, which will lack either transcript. RT-qPCR using polyT primers can be employed to detect these transcripts. If the level of these mRNAs are equivalent to dKO in WT ABS, the authors can make a pretty strong case for the absence of cristae in ABS.

      4) The major finding of the manuscript is of a Mic19 analog in plasmodium should be highlighted. As far as I know, this manuscript could represent the first instance of Mic19 outside of opisthokonts that was not found by sensitive profile HMM searches and certainly the first time such a Mic19 was functionally analyzed.

      They should highlight the twin CX9C motifs that are a hallmark of Mic19 and other proteins that undergo oxidative folding via the MIA pathway. Interestingly, the Mia40 oxidoreductase that is central to MIA in yeast and animals, is absent in apicomplexans (DOI: 10.1080/19420889.2015.1094593).

      Did the authors try to align Plasmodium Mic19 orthologs with conventional Mic19s? This may reveal some conserved residues within and outside of the CHCH domain.

      5) Statistcal significance. Sometimes my eyes see population differences that are considered insignificant by the statistical methods employed by the authors, eg Fig. 4E, mutants compared to WT, especially the dKO. Have the authors considered using other methods such as student t-test for pairwise comparisons?

      Minor comments:

      Line 33. Anaerobes (eg Giardia) have mitochondria that do produce ATP, unlike aerobic mitochondria

      Line 56: Unclear what authors mean by "canonical model of mitochondria"

      Lines 75-76: This applies to Mic10 only

      Line 80: Cite DOI: 10.1016/j.cub.2020.02.053

      Fig 2D: I find this table difficult to read. If authors keep table format, at least get rid of 'mean' column' as this data is better depicted in 2C. I suggest depicted this data either like in 3B depicting portion of infected vs unaffected flies in all experiments, then move modified Table to supplement. Important to point out experiment 5 appears to be an outlier with reduced infectivity across all cell lines, including WT.

      Fig. 3C-G: I feel like these data repeatedly lead to same conclusions. These are all different ways of showing what is depicted in Fig 2B: mitochondria gross morphology is affected upon ablation of MICOS. I suggest that these graphs be moved to supplement and replaced by the beautiful images

      Line 180: Be more specific with which tubulin isoform is used as a male marker and state why this marker was used in supplemental Fig S6.

      Line 196 and Fig 3C: the word 'intensities' in this context is very ambiguous. Please choose a different term (puncta, elements, parts?). This is related to major point 2i above.

      Line 222: Report male/female crista measurements

      Fig. 4B-E: depict data as violin plots or scatter plots like Fig. 2C to get a better grasp of how the crista coverage is distributed. It seems like the data spread is wider in the double KO. This would also solve the problem with the standard deviation extending beyond 0%.

      Lines 331-333: Please clarify that this applies for some, but not all MICOS subunits. Please also see major point 1 above. Also, the authors should point out that despite their structural divergence, trypanosomal cryptic mitofilins Mic34 and Mic40 are essential for parasite growth, in contrast to their findings with PfMic60 (DOI: https://doi.org/10.1101/2025.01.31.635831).

      Line 320: incorrect citation. Related to point 1above.

      Lines 333-335. This is related to the above. Again, some subunits appear to affect cell growth under lab conditions, and some do not. This and the previous sentence should be rewritten to reflect this.

      Line 343-345: The sentence and citation 45 are strange. Regarding the former, it is about CHCHD10, whose status as a bona fide MICOS subunit is very tenuous, so I would omit this. About the phenomenon observed, I think it makes more sense to write that Mic60 ablation results in partially fragmented mitochondria in yeast (Rabl et al., 2009 J Cell Biol. 185: 1047-63). A fragmented mitochondria is often a physiological response to stress. I would just rewrite as not to imply that mitochondrial fission (or fusion) is impaired in these KOs, or at least this could be one of several possibilities.

      Line 373: 'This indicates' is too strong. I would say 'may suggest' as you have no proof that any of the KOs disrupts MICOS. This hypothesis can be tested by other means, but not by penetrance of a phenotype.

      Line 376-377; 'deplete functionality' does not make sense, especially in the context of talking about MICOS subunit stability. In my opinion, this paragraph overinterprets the KO effects on MICOS stability. None of the experiments address this phenomenon, and thus the authors should not try to interpret their results in this context. See major point 1.

      Other suggestions for added value

      1) Does Plasmodium Sam50 co-fractionate with Mic60 and Mic19 in BN PAGE (Fig. 1E)

      2) Can Alphafold3 predict a heterotetramer of PfMic60? What about the four Mic19 and Mic60 subunits together. Is this tetramer consistent with the Bock-Bierbaum model. Is this model consistent with the CJ diameter measured in plasmodium, which is perhaps better evidence than that in lines 419-422.

      Significance

      The manuscript by Tassan-Lugrezin is predicated on the idea that Plasmodium represents the only system in which de novo crista formation can be studied. They leverage this system to ask the question whether MICOS is essential for this process. They conclude based on their data that the answer is no, which the authors consider unprecedented. But even if their claim is true that ABS is acristate, this supposed advantage does not really bring any meaningful insight into how MICOS works in Plasmodium.

      First the positives of this manuscript. As has been the case with this research team, the manuscript is very sophisticated in the experimental approaches that are made. The highlights are the beautiful and often conclusive microscopy performed by the authors. Only the localization of Mic60 and Mic19 was inconclusive due to their very low expression unfortunately.

      The examination of the MICOS mutants during in vitro life cycle of Plasmodium falciparum is extremely impressive and yields convincing results. Mitochondrial deformation is tolerated by life cycle stage differentiation, with a modest but significant reduction of oocyte production, being observed.

      The manuscript by Tassan-Lugrezin is predicated on the idea that Plasmodium represents the only system in which de novo crista formation can be studied. They leverage this system to ask the question whether MICOS is essential for this process. They conclude based on their data that the answer is no, which the authors consider unprecedented. But even if their claim is true that ABS is acristate, this supposed advantage does not really bring any meaningful insight into how MICOS works in Plasmodium.

      First the positives of this manuscript. As has been the case with this research team, the manuscript is very sophisticated in the experimental approaches that are made. The highlights are the beautiful and often conclusive microscopy performed by the authors. Only the localization of Mic60 and Mic19 was inconclusive due to their very low expression unfortunately.

      The examination of the MICOS mutants during in vitro life cycle of Plasmodium falciparum is extremely impressive and yields convincing results. Mitochondrial deformation is tolerated by life cycle stage differentiation, with a modest but significant reduction of oocyte production, being observed.

      However, despite the herculean efforts of the authors, the manuscript as it currently stands represents only a minor advance in our understanding of the evolution of MICOS, which from the title and focus of the manuscript, is the main goal of the authors.

      In its current form, the manuscript reports some potentially important findings:

      1) Mic60 is verified to play a role in crista formation, as is predicted by its orthology to other characterized Mic60 orthologs.

      2) The discovery of a novel Mic19 analog (since the authors maintain there is no significant sequence homology), which exhibits a similar (or the same?) complexome profile with Mic60. This protein was upregulated in gametocytes like Mic60 and phenocopies Mic60 KO.

      3) Both of these MICOS subunits are essential (not dispensable) for proper crista formation

      4) Surprisingly, neither MICOS subunit is essential for in vitro growth or differentiation from ABS to sexual stages, and from the latter to sporozoites. This says more about the biology of plasmodium itself than anything about the essentiality of Mic60, ie plasmodium life cycle progression tolerates defects to mitochondrial morphology. But yes, I agree with the authors that Mic60's apparent insignificance for cell growth in examined conditions does differ with its essentiality in other eukaryotes. But fitness costs were not assayed (eg by competition between mutants and WT in infection of mosquitoes)

      5) Decreased fitness of the mutants is implied by a reduction of oocyte formation.

      While interesting in their own way, collectively they do not represent a major advance in our understanding of MICOS evolution. Furthermore, the findings bifurcate into categories informing MICOS or Plasmodium biology. Both aspects are somewhat underdeveloped in their current form.

      This is unfortunate because there seem to be many missed opportunities in the manuscript that could, with additional experiments, lead to a manuscript with much wider impact.

      For me, what is remarkable about Plasmodium MICOS that sets it apart from other iterations is the apparent absence of the Mic10 subunit. Purification of plasmodium MICOS via the epitope tagged Mic60 and Mic19 could have verified that MICOS is assembled without this core subunit. Perhaps Mic60 and Mic19 are the vestiges of the complex, and thus operate alone in shaping cristae. Such a reduction may also suggest the declining importance of mitochondria in plasmodium.

      Another missed opportunity was to assay the impact of MICOS-depletion of OXPHOS in plasmodium. This is a salient issue as maybe crista morphology is decoupled from OXPHOS capacity in Plasmodium, which links to the apparent tolerance of mitochondrial morphology in cell growth and differentiation. I suggested in section A experiments to address this deficit.

      Finally, the authors could assay fitness costs of MICOS-ablation and associated phenotypes by assaying whether mosquito infectivity is reduced in the mutants when they are directly competing with WT plasmodium. Like the authors, I am also surprised that MICOS mutants can pass population bottlenecks represented by differentiation events. Perhaps the apparent robustness of differentiation may contribute plasmodium's remarkable ability to adapt.

      I realize that the authors put a lot of efforts into their study and again, I am very impressed by the sophistication of the methods employed. Nevertheless, I think there is still better ways to increase the impact of the study aside from overinterpreting the conclusions from the data. But this would require more experiments along the lines I suggest in Section A and here.

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

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      Referee #1

      Evidence, reproducibility and clarity

      Summary: This manuscript reports the identification of putative orthologues of mitochondrial contact site and cristae organizing system (MICOS) proteins in Plasmodium falciparum - an organism that unusually shows an acristate mitochondrion during the asexual part of its life cycle and then this develops cristae as it enters the sexual stage of its life cycle and beyond into the mosquito. The authors identify PfMIC60 and PfMIC19 as putative members and study these in detail. The authors at HA tags to both proteins and look for timing of expression during the parasite life cycle and attempt (unsuccessfully) to localise them within the parasite. They also genetically deleted both gene singly and in parallel and phenotyped the effect on parasite development. They show that both proteins are expressed in gametocytes and not asexuals, suggesting they are present at the same time as cristae development. They also show that the proteins are dispensible for the entire parasite life cycle investigated (asexuals through to sporozoites), however there is some reduction in mosquito transmission. Using EM techniques they show that the morphology of gametocyte mitochondria is abnormal in the knock out lines, although there is great variation.

      Major comments: The manuscript is interesting and is an intriguing use of a well studied organism of medical importance to answer fundamental biological questions. My main comments are that there should be greater detail in areas around methodology and statistical tests used. Also, the mosquito transmission assays (which are notoriously difficult to perform) show substantial variation between replicates and the statistical tests and data presentation are not clear enough to conclude the reduction in transmission that is claimed. Perhaps this could be improved with clearer text?

      More specific comments to address:

      Line 101/Fig1E (and figure legend) - What is this heatmap showing. It would be helpful to have a sentence or two linking it to a specific methodology. I could not find details in the M+M section and "specialized, high molecular mass gels" does not adequately explain what experiments were performed. The reference to Supplementary Information 1 also did not provide information. Line 115 and Supplementary Figure 2C + D - The main text says that the transgenic parasites contained a mitochondrially localized mScarlet for visualization and localization, but in the supplementary figure 2 it shows mitotracker labelling rather than mScarlet. This is very confusing. The figure legend also mentions both mScarlet and MitoTracker. I assume that mScarlet was used to view in regular IFAs (Fig S2C) and the MitoTracker was used for the expansion microscopy (Fig S2D)? Please clarify. Figure 2C - what is the statistical test being used (the methods say "Mean oocysts per midgut and statistical significance were calculated using a generalized linear mixed effect model with a random experiment effect under a negative binomial distribution." but what test is this?)? Also the choice of a log10 scale for oocyst intensity is an unusual choice - how are the mosquitoes with 0 oocysts being represented on this graph? It looks like they are being plotted at 10^-1 (which would be 0.1 oocysts in a mosquito which would be impossible). Figure 2D - it is great that the data from all feeding replicates has been shared, however it is difficult to conclude any meaningful impact in transmission with the knock-out lines when there is so much variation and so few mosquitoes dissected for some datapoints (10 mosquitoes are very small sample sizes). For example, Exp1 shows a clear decrease in mic19- transmission, but then Exp2 does not really show as great effect. Similarly, why does the double knock out have better transmission than the single knockouts? Sure there would be a greater effect? Figure 3 legend - Please add which statistical test was used and the number of replicates. Figure 4 legend - Please add which statistical test was used and the number of replicates. Figure 5C - the 3D reconstructions are very nice, but what does the red and yellow coloring show? Line 352 - "Still, it is striking that, despite the pronounced morphological phenotype, and the possibly high mitochondrial stress levels, the parasites appeared mostly unaffected in life cycle propagation, raising questions about the functional relevance of mitochondria at these stages." How do the authors reconcile this statement with the proven fact that mitochondria-targeted antimalarials (such as atovaquone) are very potent inhibitors of parasite mosquito transmission?

      Significance

      This manuscript is a novel approach to studying mitochondrial biology and does open a lot of unanswered questions for further research directions. Currently there are limitations in the use of statistical tests and detail of methodology, but these could be easily be addressed with a bit more analysis/better explanation in the text. This manuscript could be of interest to readers with a general interest in mitochondrial cell biology and those within the specific field of Plasmodium research.

      My expertise is in Plasmodium cell biology.

    1. Author response:

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

      Reviewer #1 (Public review):

      (1) I have to admit that it took a few hours of intense work to understand this paper and to even figure out where the authors were coming from. The problem setting, nomenclature, and simulation methods presented in this paper do not conform to the notation common in the field, are often contradictory, and are usually hard to understand. Most importantly, the problem that the paper is trying to solve seems to me to be quite specific to the particular memory study in question, and is very different from the normal setting of model-comparative RSA that I (and I think other readers) may be more familiar with.

      We have revised the paper for clarity at all levels: motivation, application, and parameterization. We clarify that there is a large unmet need for using RSA in a trial-wise manner, and that this approach indeed offers benefits to any team interested in decoding trial-wise representational information linked to a behavioral responses, and as such is not a problem specific to a single memory study.

      (2) The definition of "classical RSA" that the authors are using is very narrow. The group around Niko Kriegeskorte has developed RSA over the last 10 years, addressing many of the perceived limitations of the technique. For example, cross-validated distance measures (Walther et al. 2016; Nili et al. 2014; Diedrichsen et al. 2021) effectively deal with an uneven number of trials per condition and unequal amounts of measurement noise across trials. Different RDM comparators (Diedrichsen et al. 2021) and statistical methods for generalization across stimuli (Schütt et al. 2023) have been developed, addressing shortcomings in sensitivity. Finally, both a Bayesian variant of RSA (Pattern component modelling, (Diedrichsen, Yokoi, and Arbuckle 2018) and an encoding model (Naselaris et al. 2011) can effectively deal with continuous variables or features across time points or trials in a framework that is very related to RSA (Diedrichsen and Kriegeskorte 2017). The author may not consider these newer developments to be classical, but they are in common use and certainly provide the solution to the problems raised in this paper in the setting of model-comparative RSA in which there is more than one repetition per stimulus.

      We appreciate the summary of relevant literature and have included a revised Introduction to address this bounty of relevant work. While much is owed to these authors, new developments from a diverse array of researchers outside of a single group can aid in new research questions, and should always have a place in our research landscape. We owe much to the work of Kriegeskorte’s group, and in fact, Schutt et al., 2023 served as a very relevant touchpoint in the Discussion and helped to highlight specific needs not addressed by the assessment of the “representational geometry” of an entire presented stimulus set. Principal amongst these needs is the application of trial-wise representational information that can be related to trial-wise behavioral responses and thus used to address specific questions on brain-behavior relationships. We invite the Reviewer to consider the utility of this shift with the following revisions to the Introduction.

      Page 3. “Recently, methodological advancements have addressed many known limitations in cRSA. For example, cross-validated distance measures (e.g., Euclidean distance) have improved the reliability of representational dissimilarities in the presence of noise and trial imbalance (Walther et al., 2016; Nili et al., 2014; Diedrichsen et al., 2021). Bayesian approaches such as pattern component modeling (Diedrichsen, Yokoi, & Arbuckle, 2018) have extended representational approaches to accommodate continuous stimulus features or temporal variation. Further, model comparison RSA strategies (Diedrichsen et al., 2021) and generalization techniques across stimuli (Schütt et al., 2023) have improved sensitivity and inference. Nevertheless, a common feature shared across most of improvements is that they require stimuli repetition to examine the representational structure. This requirement limits their ability to probe brain-behavior questions at the level of individual events”.

      Page 8. “While several extensions of RSA have addressed key limitations in noise sensitivity, stimulus variance, and modeling (e.g., Diedrichsen et al., 2021; Schütt et al., 2023), our tRSA approach introduces a new methodological step by estimating representational strength at the trial level. This accounts for the multi-level variance structure in the data, affords generalizability beyond the fixed stimulus set, and allows one to test stimulus- or trial-level modulations of neural representations in a straightforward way”.

      Page 44. “Despite such prevalent appreciation for the neurocognitive relevance of stimulus properties, cRSA often does not account for the fact that the same stimulus (e.g., “basketball”) is seen by multiple subjects and produces statistically dependent data, an issue addressed by Schütt et al., 2023, who developed cross validation and bootstrap methods that explicitly model dependence across both subjects and stimulus conditions”.

      (3) The stated problem of the paper is to estimate "representational strength" in different regions or conditions. With this, the authors define the correlation of the brain RDM with a model RDM. This metric conflates a number of factors, namely the variances of the stimulus-specific patterns, the variance of the noise, the true differences between different dissimilarities, and the match between the assumed model and the data-generating model. It took me a long time to figure out that the authors are trying to solve a quite different problem in a quite different setting from the model-comparative approach to RSA that I would consider "classical" (Diedrichsen et al. 2021; Diedrichsen and Kriegeskorte 2017). In this approach, one is trying to test whether local activity patterns are better explained by representation model A or model B, and to estimate the degree to which the representation can be fully explained. In this framework, it is common practice to measure each stimulus at least 2 times, to be able to estimate the variance of noise patterns and the variance of signal patterns directly. Using this setting, I would define 'representational strength" very differently from the authors. Assume (using LaTeX notation) that the activity patterns $y_j,n$ for stimulus j, measurement n, are composed of a true stimulus-related pattern ($u_j$) and a trial-specific noise pattern ($e_j,n$). As a measure of the strength of representation (or pattern), I would use an unbiased estimate of the variance of the true stimulus-specific patterns across voxels and stimuli ($\sigma^2_{u}$). This estimator can be obtained by correlating patterns of the same stimuli across repeated measures, or equivalently, by averaging the cross-validated Euclidean distances (or with spatial prewhitening, Mahalanobis distances) across all stimulus pairs. In contrast, the current paper addresses a specific problem in a quite specific experimental design in which there is only one repetition per stimulus. This means that the authors have no direct way of distinguishing true stimulus patterns from noise processes. The trick that the authors apply here is to assume that the brain data comes from the assumed model RDM (a somewhat sketchy assumption IMO) and that everything that reduces this correlation must be measurement noise. I can now see why tRSA does make some sense for this particular question in this memory study. However, in the more common model-comparative RSA setting, having only one repetition per stimulus in the experiment would be quite a fatal design flaw. Thus, the paper would do better if the authors could spell the specific problem addressed by their method right in the beginning, rather than trying to set up tRSA as a general alternative to "classical RSA".

      At a general level, our approach rests on the premise that there is meaningful information present in a single presentation of a given stimulus. This assumption may have less utility when the research goals are more focused on estimating the fidelity of signal patterns for RSA, as in designs with multiple repetitions. But it is an exaggeration to state that such a trial-wise approach cannot address the difference between “true” stimulus patterns and noise. This trial-wise approach has explicit utility in relating trial-wise brain information to trial-wise behavior, across multiple cognitions (not only memory studies, as applied here). We have added substantial text to the Introduction distinguishing cRSA, which is widely employed, often in cases with a single repetition per stimulus, and model comparative methods that employ multiple repetitions. We clarify that we do not consider tRSA an alternative to the model comparative approach, and discuss that operational definitions of representational strength are constrained by the study design.

      Page 3. “In this paper, we present an advancement termed trial-level RSA, or tRSA, which addresses these limitations in cRSA (not model comparison approaches) and may be utilized in paradigms with or without repeated stimuli”.

      Page 4. “Representational geometry usually refers to the structure of similarities among repeated presentations of the same stimulus in the neural data (as captured in the brain RSM) and is often estimated utilizing a model comparison approach, whereas representational strength is a derived measure that quantifies how strongly this geometry aligns with a hypothesized model RSM. In other words, geometry characterizes the pattern space itself, while representational strength reflects the degree of correspondence between that space and the theoretical model under test”.

      Finally, we clarified that in our simulation methods we assume a true underlying activity pattern and a random error pattern. The model RSM is computed based on the true pattern, whereas the brain RSM comes from the noisy pattern, not the model RSM itself.

      Page 9. “Then, we generated two sets of noise patterns, which were controlled by parameters σ<sub>A</sub> and σ<sub>B</sub> , respectively, one for each condition”.

      (4) The notation in the paper is often conflicting and should be clarified. The actual true and measured activity patterns should receive a unique notation that is distinct from the variances of these patterns across voxels. I assume that $\sigma_ijk$ is the noise variances (not standard deviation)? Normally, variances are denoted with $\sigma^2$. Also, if these are variances, they cannot come from a normal distribution as indicated on page 10. Finally, multi-level models are usually defined at the level of means (i.e., patterns) rather than at the level of variances (as they seem to be done here).

      We have added notations for true and measured activity patterns to differentiate it from our notation for variance. We agree that multilevel models are usually defined at the level of means rather than at the level of variances and we include a Figure (Fig 1D) that describes the model in terms of the means. We clarify that the σ ($\sigma$) used in the manuscript were not variances/standard deviations themselves; rather, they were meant to denote components of the actual (multilevel) variance parameter. Each component was sampled from normal distributions, and they collectively summed up to comprise the final variance parameter for each trial. We have modified our notation for each component to the lowercase letter s to minimize confusion. We have also made our R code publicly available on our lab github, which should provide more clarity on the exact simulation process.

      (5) In the first set of simulations, the authors sampled both model and brain RSM by drawing each cell (similarity) of the matrix from an independent bivariate normal distribution. As the authors note themselves, this way of producing RSMs violates the constraint that correlation matrices need to be positive semi-definite. Likely more seriously, it also ignores the fact that the different elements of the upper triangular part of a correlation matrix are not independent from each other (Diedrichsen et al. 2021). Therefore, it is not clear that this simulation is close enough to reality to provide any valuable insight and should be removed from the paper, along with the extensive discussion about why this simulation setting is plainly wrong (page 21). This would shorten and clarify the paper.

      We have added justification of the mixed-effects model given the potential assumption violations. We caution readers to investigate the robustness of their models, and to employ permutation testing that does not make independence assumptions. We have also added checks of the model residuals and an example of permutation testing in the Appendix. Finally, we agree that the first simulation setting does not possess several properties of realistic RDMs/RSMs; however, we believe that there is utility in understanding the mathematical properties of correlations – an essential component of RSA – in a straightforward simulation where the ground truth is known, thus moving the simulation to Appendix 1.

      (6) If I understand the second simulation setting correctly, the true pattern for each stimulus was generated as an NxP matrix of i.i.d. standard normal variables. Thus, there is no condition-specific pattern at all, only condition-specific noise/signal variances. It is not clear how the tRSA would be biased if there were a condition-specific pattern (which, in reality, there usually is). Because of the i.i.d. assumption of the true signal, the correlations between all stimulus pairs within conditions are close to zero (and only differ from it by the fact that you are using a finite number of voxels). If you added a condition-specific pattern, the across-condition RSA would lead to much higher "representational strength" estimates than a within-condition RSA, with obvious problems and biases.

      The Reviewer is correct that the voxel values in the true pattern are drawn from i.i.d. standard normal distributions. We take the Reviewer’s suggestion of “condition-specific pattern” to mean that there could be a condition-voxel interaction in two non-mutually exclusive ways. The first is additive, essentially some common underlying multi-voxel pattern like [6, 34, -52, …, 8] for all condition A trials, and different one such pattern for condition B trials, etc. The second is multiplicative, essentially a vector of scaling factors [x1.5, x0.5, x0.8, …, x2.7] for all condition A trials, and a different one such vector for condition B trials, etc. Both possibilities could indeed affect tRSA as much as it would cRSA.

      Importantly, If such a strong condition-specific pattern is expected, one can build a condition-specific model RDM using one-shot coding of conditions (see example figure; src: https://www.newbi4fmri.com/tutorial-9-mvpa-rsa), to either capture this interesting phenomenon or to remove this out as a confounding factor. This practice has been applied in multiple regression cRSA approaches (e.g., Cichy et al., 2013) and can also be applied to tRSA.

      (7) The trial-level brain RDM to model Spearman correlations was analyzed using a mixed effects model. However, given the symmetry of the RDM, the correlations coming from different rows of the matrix are not independent, which is an assumption of the mixed effect model. This does not seem to induce an increase in Type I errors in the conditions studied, but there is no clear justification for this procedure, which needs to be justified.

      We appreciate this important warning, and now caution readers to investigate the robustness of their models, and consider employing permutation testing that does not make independence assumptions. We have also added checks of the model residuals and an example of permutation testing in the supplement.

      Page 46. “While linear mixed-effects modeling offers a powerful framework for analyzing representational similarity data, it is critical that researchers carefully construct and validate their models. The multilevel structure of RSA data introduces potential dependencies across subjects, stimuli, and trials, which can violate assumptions of independence if not properly modeled. In the present study, we used a model that included random intercepts for both subjects and stimuli, which accounts for variance at these levels and improves the generalizability of fixed-effect estimates. Still, there is a potential for systematic dependence across trials within a subject. To ensure that the model assumptions were satisfied, we conducted a series of diagnostic checks on an exemplar ROI (right LOC; middle occipital gyrus) in the Object Perception dataset, including visual inspection of residual distributions and autocorrelation (Appendix 3, Figure 13). These diagnostics supported the assumptions of normality, homoscedasticity, and conditional independence of residuals. In addition, we conducted permutation-based inference, similar to prior improvements to cRSA (Niliet al. 2014), using a nested model comparison to test whether the mean similarity in this ROI was significantly greater than zero. The observed likelihood ratio test statistic fell in the extreme tail of the null distribution (Appendix 3, Figure 14), providing strong nonparametric evidence for the reliability of the observed effect. We emphasize that this type of model checking and permutation testing is not merely confirmatory but can help validate key assumptions in RSA modeling, especially when applying mixed-effects models to neural similarity data. Researchers are encouraged to adopt similar procedures to ensure the robustness and interpretability of their findings”.

      Exemplar Permutation Testing

      To test whether the mean representational strength in the ROI right LOC (middle occipital gyrus) was significantly greater than zero, we used a permutation-based likelihood ratio test implemented via the permlmer function. This test compares two nested linear mixed-effects models fit using the lmer function from the lme4 package, both including random intercepts for Participant and Stimulus ID to account for between-subject and between-item variability.

      The null model excluded a fixed intercept term, effectively constraining the mean similarity to zero after accounting for random effects:

      ROI ~ 0 + (1 | Participant) + (1 | Stimulus)

      The full model included the same random effects structure but allowed the intercept to be freely estimated:

      ROI ~ 1 + (1 | Participant) + (1 | Stimulus)

      By comparing the fit of these two models, we directly tested whether the average similarity in this ROI was significantly different from zero. Permutation testing (1,000 permutations) was used to generate a nonparametric p-value, providing inference without relying on normality assumptions. The full model, which estimated a nonzero mean similarity in the right LOC (middle occipital gyrus), showed a significantly better fit to the data than the null model that fixed the mean at zero (χ²(1) = 17.60, p = 2.72 × 10⁻⁵). The permutation-based p-value obtained from permlmer confirmed this effect as statistically significant (p = 0.0099), indicating that the mean similarity in this ROI was reliably greater than zero. These results support the conclusion that the right LOC contains representational structure consistent with the HMAXc2 RSM. A density plot of the permuted likelihood ratio tests is plotted along with the observed likelihood ratio test in Appendix 3 Figure 14.

      (8) For the empirical data, it is not clear to me to what degree the "representational strength" of cRSA and tRSA is actually comparable. In cRSA, the Spearman correlation assesses whether the distances in the data RSM are ranked in the same order as in the model. For tRSA, the comparison is made for every row of the RSM, which introduces a larger degree of flexibility (possibly explaining the higher correlations in the first simulation). Thus, could the gains presented in Figure 7D not simply arise from the fact that you are testing different questions? A clearer theoretical analysis of the difference between the average row-wise Spearman correlation and the matrix-wise Spearman correlation is urgently needed. The behavior will likely vary with the structure of the true model RDM/RSM.

      We agree that the comparability between mean row-wise Spearman correlations and the matrix-wise Spearman correlation is needed. We believe that the simulations are the best approach for this comparison, since they are much more robust than the empirical dataset and have the advantage of knowing the true pattern/noise levels. We expand on our comparison of mean tRSA values and matrix-wise Spearman correlations on page 42.

      Page 42. “Although tRSA and cRSA both aim to quantify representational strength, they differ in how they operationalize this concept. cRSA summarizes the correspondence between RSMs as a single measure, such as the matrix-wise Spearman correlation. In contrast, tRSA computes such correspondence for each trial, enabling estimates at the level of individual observations. This flexibility allows trial-level variability to be modeled directly, but also introduces subtle differences in what is being measured. Nonetheless, our simulations showed that, although numerical differences occasionally emerged—particularly when comparing between-condition tRSA estimates to within-condition cRSA estimates—the magnitude of divergence was small and did not affect the outcome of downstream statistical tests”.

      (9) For the real data, there are a number of additional sources of bias that need to be considered for the analysis. What if there are not only condition-specific differences in noise variance, but also a condition-specific pattern? Given that the stimuli were measured in 3 different imaging runs, you cannot assume that all measurement noise is i.i.d. - stimuli from the same run will likely have a higher correlation with each other.

      We recognize the potential of condition-specific patterns and chose to constrain the analyses to those most comparable with cRSA. However, depending on their hypotheses, researchers may consider testing condition RSMs and utilizing a model comparison approach or employ the z-scored approach, as employed in the simulations above. Regarding the potential run confounds, this is always the case in RSA and why we exclude within-run comparisons. We have also added to the Discussion the suggestion to include run as a covariate in their mixed-effects models. However, we do not employ this covariate here as we preferred the most parsimonious model to compare with cRSA.

      Page 46 - 47. “Further, while analyses here were largely employed to be comparable with cRSA, researchers should consider taking advantage of the flexibility of the mixed-effects models and include co variates of non-interest (run, trial order etc.)”.

      (10) The discussion should be rewritten in light of the fact that the setting considered here is very different from the model-comparative RSA in which one usually has multiple measurements per stimulus per subject. In this setting, existing approaches such as RSA or PCM do indeed allow for the full modelling of differences in the "representational strength" - i.e., pattern variance across subjects, conditions, and stimuli.

      We agree that studies advancing designs with multiple repetitions of a given stimulus image are useful in estimating the reliability of concept representations. We would argue however that model comparison in RSA is not restricted to such data. Many extant studies do not in fact have multiple repetitions per stimulus per subject (Wang et al., 2018 https://doi.org/10.1088/1741-2552/abecc3, Gao et al, 2022 https://doi.org/10.1093/cercor/bhac058, Li et al, 2022 https://doi.org/10.1002/hbm.26195, Staples & Graves, 2020 https://doi.org/10.1162/nol_a_00018) that allow for that type of model-comparative approach. While beneficial in terms of noise estimation, having multiple presentations was not a requirement for implementing cRSA (Kriegeskorte, 2008 https://doi.org/10.3389/neuro.06.004.2008). The aim of this manuscript is to introduce the tRSA approach to the broad community of researchers whose research questions and datasets could vary vastly, including but not limited to the number of repeated presentations and the balance of trial counts across conditions.

      (11) Cross-validated distances provide a powerful tool to control for differences in measurement noise variances and possible covariances in measurement noise across trials, which has many distinct advantages and is conceptually very different from the approach taken here.

      We have added language on the value of cross-validation approaches to RSA in the Discussion:

      Page 47. “Additionally, we note that while our proposed tRSA framework provides a flexible and statistically principled approach for modeling trial-level representational strength, we acknowledge that there are alternative methods for addressing trial-level variability in RSA. In particular, the use of cross-validated distance metrics (e.g., crossnobis distance) has become increasingly popular for controlling differences in measurement noise variance and accounting for possible covariance structures across trials (Walther et al., 2016). These metrics offer several advantages, including unbiased estimation of representational dissimilarities under Gaussian noise assumptions and improved generalization to unseen data. However, cross-validated distances are conceptually distinct from the approach taken here: whereas cross-validation aims to correct for noise-related biases in representational dissimilarity matrices, our trial-level RSA method focuses on estimating and modeling the variability in representation strength across individual trials using mixed-effects modeling. Rather than proposing a replacement for cross-validated RSA, tRSA adds a complementary tool to the methodological toolkit—one that supports hypothesis-driven inference about condition effects and trial-level covariates, while leveraging the full structure of the data”.

      (12) One of the main limitations of tRSA is the assumption that the model RDM is actually the true brain RDM, which may not be the case. Thus, in theory, there could be a different model RDM, in which representational strength measures would be very different. These differences should be explained more fully, hopefully leading to a more accessible paper.

      Indeed, the chosen model RSM may not be the true RSM, but as the noise level increases the correlation between RSMs practically becomes zero. In our simulations we assume this to be true as a straightforward way to manipulate the correspondence between the brain data and the model. However, just like cRSA, tRSA is constrained by the model selections the researchers employ. We encourage researchers to have carefully considered theoretically-motivated models and, if their research questions require, consider multiple and potentially competing models. Furthermore, the trial-wise estimates produced by tRSA encourage testing competing models within the multiple regression framework. We have added this language to the Discussion.

      Page 46. ..”choose their model RSMs carefully. In our simulations, we designed our model RSM to be the “true” RSM for demonstration purposes. However, researchers should consider if their models and model alternatives”.

      Pages 45-46. “While a number of studies have addressed the validity of measuring representational geometry using designs with multiple repetitions, a conceptual benefit of the tRSA approach is the reliance on a regression framework that engenders the testing of competing conceptual models of stimulus representation (e.g., taxonomic vs. encyclopedic semantic features, as in Davis et al., 2021)”.

      Reviewer #2 (Public review):

      (1)  While I generally welcome the contribution, I take some issue with the accusatory tone of the manuscript in the Introduction. The text there (using words such as 'ignored variances', 'errouneous inferences', 'one must', 'not well-suited', 'misleading') appears aimed at turning cRSA in a 'straw man' with many limitations that other researchers have not recognized but that the new proposed method supposedly resolves. This can be written in a more nuanced, constructive manner without accusing the numerous users of this popular method of ignorance.

      We apologize for the unintended accusatory tone. We have clarified the many robust approaches to RSA and have made our Introduction and Discussion more nuanced throughout (see also 3, 11 and16).

      (2) The described limitations are also not entirely correct, in my view: for example, statistical inference in cRSA is not always done using classic parametric statistics such as t-tests (cf Figure 1): the rsatoolbox paper by Nili et al. (2014) outlines non-parametric alternatives based on permutation tests, bootstrapping and sign tests, which are commonly used in the field. Nor has RSA ever been conducted at the row/column level (here referred to by the authors as 'trial level'; cf King et al., 2018).

      We agree there are numerous methods that go beyond cRSA addressing these limitations and have added discussion of them into our manuscript as well as an example analysis implementing permutation tests on tRSA data (see response to 7). We thank the reviewer for bringing King et al., 2014 and their temporal generalization method to our attention, we added reference to acknowledge their decoding-based temporal generalization approach.

      Page 8. “It is also important to note that some prior work has examined similarly fine-grained representations in time-resolved neuroimaging data, such as the temporal generalization method introduced by King et al. (see King & Dehaene, 2014). Their approach trains classifiers at each time point and tests them across all others, resulting in a temporal generalization matrix that reflects decoding accuracy over time. While such matrices share some structural similarity with RSMs, they do not involve correlating trial-level pattern vectors with model RSMs nor do their second-level models include trial-wise, subject-wise, and item-wise variability simultaneously”.

      (3) One of the advantages of cRSA is its simplicity. Adding linear mixed effects modeling to RSA introduces a host of additional 'analysis parameters' pertaining to the choice of the model setup (random effects, fixed effects, interactions, what error terms to use) - how should future users of tRSA navigate this?

      We appreciate the opportunity to offer more specific proscriptions for those employing a tRSA technique, and have added them to the Discussion:

      Page 46. “While linear mixed-effects modeling offers a powerful framework for analyzing representational similarity data, it is critical that researchers carefully construct and validate their models and choose their model RSMs carefully. In our simulations, we designed our model RSM to be the “true” RSM for demonstration purposes. However, researchers should consider if their models and model alternatives. However, researchers should always consider if their models match the goals of their analysis, including 1) constructing the random effects structure that will converge in their dataset and 2) testing their model fits against alternative structures (Meteyard & Davies, 2020; Park et al., 2020) and 3) considering which effects should be considered random or fixed depending on their research question”.

      (4) Here, only a single real fMRI dataset is used with a quite complicated experimental design for the memory part; it's not clear if there is any benefit of using tRSA on a simpler real dataset. What's the benefit of tRSA in classic RSA datasets (e.g., Kriegeskorte et al., 2008), with fixed stimulus conditions and no behavior?

      To clarify, our empirical approach uses two different tasks: an Object Perception task more akin to the classic RSA datasets employing passive viewing, and a Conceptual Retrieval task that more directly addresses the benefits of the trialwise approach. We felt that our Object Perception dataset is a simpler empirical fMRI dataset without explicit task conditions or a dichotomous behavioral outcome, whereas the Retrieval dataset is more involved (though old/new recognition is the most common form of memory retrieval testing) and  dependent on behavioral outcomes. However, we recognize the utility of replication from other research groups and do invite researchers to utilize tRSA on their datasets.

      (5) The cells of an RDM/RSM reflect pairwise comparisons between response patterns (typically a brain but can be any system; cf Sucholutsky et al., 2023). Because the response patterns are repeatedly compared, the cells of this matrix are not independent of one another. Does this raise issues with the validity of the linear mixed effects model? Does it assume the observations are linearly independent?

      We recognize the potential danger for not meeting model assumptions. Though our simulation results and model checks suggest this is not a fatal flaw in the model design, we caution readers to investigate the robustness of their models, and consider employing permutation testing that does not make independence assumptions. We have also added checks of the model residuals and an example of permutation testing in the Appendix. See response to R1.

      (6) The manuscript assumes the reader is familiar with technical statistical terms such as Type I/II error, sensitivity, specificity, homoscedasticity assumptions, as well as linear mixed models (fixed effects, random effects, etc). I am concerned that this jargon makes the paper difficult to understand for a broad readership or even researchers currently using cRSA that might be interested in trying tRSA.

      We agree this jargon may cause the paper to be difficult to understand. We have expanded/added definitions to these terms throughout the methods and results sections.

      Page 12. “Given data generated with 𝑠<sub>𝑐𝑜𝑛𝑑,𝐴</sub> = 𝑠<sub>𝑐𝑜𝑛𝑑,B</sub>, the correct inference should be a failure to reject the null hypothesis of ; any significant () result in either direction was considered a false positive (spurious effect, or Type I error). Given data generated with , the inference was considered correct if it rejected the null hypothesis of  and yielded the expected sign of the estimated contrast (b<sub>B-𝐴</sub><0). A significant result with the reverse sign of the estimated contrast (b<sub>B-𝐴</sub><0) was considered a Type I error, and a nonsignificant (𝑝 ≥ 0.05) result was considered a false negative (failure to detect a true effect, or Type II error)”.

      Page 2. “Compared to cRSA, the multi-level framework of tRSA was both more theoretically appropriate and significantly sensitive (better able to detect) to true effects”.

      Page 25.”The performance of cRSA and tRSA were quantified with their specificity (better avoids false positives, 1 - Type I error rate) and sensitivity (better avoids false negatives 1 - Type II error rate)”.

      Page 6. “One of the fundamental assumptions of general linear models (step 4 of cRSA; see Figure 1D) is homoscedasticity or homogeneity of variance — that is, all residuals should have equal variance” .

      Page11. “Specifically, a linear mixed-effects model with a fixed effect  of condition (which estimates the average effect across the entire sample, capturing the overall effect of interest) and random effects of both subjects and stimuli (which model variation in responses due to differences between individual subjects and items, allowing generalization beyond the sample) were fitted to tRSA estimates via the `lme4 1.1-35.3` package in R (Bates et al., 2015), and p-values were estimated using Satterthwaites’s method via the `lmerTest 3.1-3` package (Kuznetsova et al., 2017)”.

      (7) I could not find any statement on data availability or code availability. Given that the manuscript reuses prior data and proposes a new method, making data and code/tutorials openly available would greatly enhance the potential impact and utility for the community.

      We thank the reviewer for raising our oversight here. We have added our code and data availability statements.

      Page 9. “Data is available upon request to the corresponding author and our simulations and example tRSA code is available at https://github.com/electricdinolab”.

      Reviewer #1 (Recommendations for the authors):

      (13) Page 4: The limitations of cRSA seem to be based on the assumption that within each different experimental condition, there are different stimuli, which get combined into the condition. The framework of RSA, however, does not dictate whether you calculate a condition x condition RDM or a larger and more complete stimulus x stimulus RDM. Indeed, in practice we often do the latter? Or are you assuming that each stimulus is only shown once overall? It would be useful at this point to spell out these implicit assumptions.

      We agree that stimulus x stimulus RDMs can be constructed and are often used. However, as we mentioned in the Introduction, researchers are often interested in the difference between two (or more) conditions, such as “remembered” vs. “forgotten” (Davis et al., https://doi.org/10.1093/cercor/bhaa269) or “high cognitive load” vs. “low cognitive load” (Beynel et al., https://doi.org/10.1523/JNEUROSCI.0531-20.2020). In those cases, the most common practice with cRSA is to construct condition-specific RDMs, compute cRSA scores separately for each condition, and then compare the scores at the group level. The number of times each stimulus gets presented does not prevent one from creating a model RDM that has the same rows and columns as the brain RDM, either in the same condition (“high load”) or across different conditions.

      (14) Page 5: The difference between condition-level and stimulus-level is not clear. Indeed, this definition seems to be a function of the exact experimental design and is certainly up for interpretation. For example, if I conduct a study looking at the activity patterns for 4 different hand actions, each repeated multiple times, are these actions considered stimuli or conditions?

      We have added clarifying language about what is considered stimuli vs conditions. Indeed, this will depend on the specific research questions being employed and will affect how researchers construct their models. In this specific example, one would most likely consider each different hand action a condition, treating them as fixed effects rather than random effects, given their very limited number and the lack of need to generalize findings to the broader “hand actions” category.

      Page 5. “Critically, the distinction between condition-level and stimulus level is not always clear as researchers may manipulate stimulus-level features themselves. In these cases, what researchers ultimately consider condition-level and stimulus-level will depend on their specific research questions. For example, researchers intending to study generalized object representation may consider object category a stimulus-level feature, while researchers interested in if/how object representation varies by category may consider the same category variable condition-level”.

      (15) Page 5: The fact that different numbers of trials / different levels of measurement noise / noise-covariance of different conditions biases non-cross-validated distances is well known and repeatedly expressed in the literature. We have shown that cross-validation of distances effectively removes such biases - of course, it does not remove the increased estimation variability of these distances (for a formal analysis of estimation noise on condition patterns and variance of the cross-nobis estimator, see (Diedrichsen et al. 2021)).

      We thank the reviewer for drawing our attention to this literature and have added discussions of these methods.

      (16). Page 5: "Most studies present subjects with a fixed set of stimuli, which are supposedly samples representative of some broader category". This may be the case for a certain type of RSA experiments in the visual domain, but it would be unfair to say that this is a feature of RSA studies in general. In most studies I have been involved in, we use a "stimulus" x "stimulus" RDM.

      We have edited this sentence to avoid the “most” characterization. We also added substantial text to the introduction and discussion distinguishing cRSA, which is nonetheless widely employed, especially in cases with a single repetition per stimulus (Macklin et al., 2023, Liu et al, 2024) and the model comparative method and explicitly stating that we do not consider tRSA an alternative to the model comparative approach.

      (17). Page 5: I agree that "stimuli" should ideally be considered a random effect if "stimuli" can be thought of as sampled from a larger population and one wants to make inferences about that larger population. Sometimes stimuli/conditions are more appropriately considered a fixed effect (for example, when studying the response to stimulation of the 5 fingers of the right hand). Techniques to consider stimuli/conditions as a random effect have been published by the group of Niko Kriegeskorte (Schütt et al. 2023).

      Indeed, in some cases what may be thought of as “stimuli” would be more appropriately entered into the model as a fixed effect; such questions are increasingly relevant given the focus on item-wise stimulus properties (Bainbridge et al., Westfall & Yarkoni). We have added text on this issue to the Discussion and caution researchers to employ models that most directly answer their research questions.

      Page 46. “However, researchers should always consider if their models match the goals of their analysis, including 1) constructing the random effects structure that will converge in their dataset and 2) testing their model fits against alternative structures (Meteyard & Davies, 2020; Park et al., 2020) and 3) considering which effects should be considered random or fixed depending on their research question. An effect is fixed when the levels represent the specific conditions of theoretical interest (e.g., task condition) and the goal is to estimate and interpret those differences directly. In contrast, an effect is random when the levels are sampled from a broader population (e.g., subjects) and the goal is to account for their variability while generalizing beyond the sample tested. Note that the same variable (e.g., stimuli) may be considered fixed or random depending on the research questions”.

      (18) Page 6: It is correct that the "classical" RSA depends on a categorical assignment of different trials to different stimuli/conditions, such that a stimulus x stimulus RDM can be computed. However, both Pattern Component Modelling (PCM) and Encoding models are ideally set up to deal with variables that vary continuously on a trial-by-trial or moment-by-moment basis. tRSA should be compared to these approaches, or - as it should be clarified - that the problem setting is actually quite a different one.

      We agree that PCM and encoding models offer a flexible approach and handle continuous trial-by-trial variables. We have clarified the problem setting in cRSA is distinct on page 6, and we have added the robustness of encoding models and their limitations to the Discussion.

      Page 6. “While other approaches such as Pattern Component Modeling (PCM) (Diedrichsen et al., 2018) and encoding models (Naselaris et al., 2011) are well-suited to analyzing variables that vary continuously on a trial-by-trial or moment-by-moment basis, these frameworks address different inferential goals. Specifically, PCM and encoding models focus on estimating variance components or predicting activation from features, while cRSA is designed to evaluate representational geometry. Thus, cRSA as well as our proposed approach address a problem setting distinct from PCM and encoding models”.

      (19) Page 8: "Then, we generated two noise patterns, which were controlled by parameters 𝜎 𝐴 and 𝜎𝐵, respectively, one for each condition." This makes little sense to me. The noise patterns should be unique to each trial - you should generate n_a + n_b noise patterns, no?

      We clarify that the “noise patterns” here are n_voxel x n_trial in size; in other words, all trial-level noise patterns are generated together and each trial has their own unique noise pattern. We have revised our description as “two sets of noise patterns” for clarity starting on page 9.

      (20) Page 9: First, I assume if this is supposed to be a hierarchical level model, the "noise parameters" here correspond to variances? Or do these \sigma values mean to signify standard deviations? The latter would make little sense. Or is it the noise pattern itself?

      As clarified in 4., the σ values are meant to denote hierarchical components of the composite standard deviation; we have updated our notation to use lower case letter s instead for clarity.

      (21) Page 10: your formula states "𝜎<sub>𝑠𝑢𝑏𝑗</sub>~ 𝙽(0, 0.5^2)". This conflicts with your previous mention that \sigmas are noise "levels" are they the noise patterns themselves now? Variances cannot be normally distributed, as they cannot be negative.

      As clarified in 4., the σ values are meant to denote hierarchical components of the composite standard deviation; we have updated our notation to use lower case letter s instead for clarity.

      (22) Page 13: What was the task of the subject in the Memory retrieval task? Old/new judgements relative to encoding of object perception?

      We apologize for the lack of clarity about the Memory Retrieval task and have added that information and clarified that the old/new judgements were relative to a separate encoding phase, the brain data for which has been reported elsewhere.

      Page 14. “Memory Retrieval took place one day after Memory Encoding and involved testing participants’ memory of the objects seen in the Encoding phase. Neural data during the Encoding phase has been reported elsewhere. In the main Memory Retrieval task, participants were presented with 144 labels of real-world objects, of which 114 were labels for previously seen objects and 30 were unrelated novel distractors. Participants performed old/new judgements, as well as their confidence in those judgements on a four-point scale (1 = Definitely New, 2 = Probably New, 3 = Probably Old, 4 = Definitely Old)”.

      (23) Page 13: If "Memory Retrieval consisted of three scanning runs", then some of the stimulus x stimulus correlations for the RSM must have been calculated within a run and some between runs, correct? Given that all within-run estimates share a common baseline, they share some dependence. Was there a systematic difference between the within-run and the between-run correlations?

      We have clarified in this portion of the methods that within run comparisons were excluded from our analyses. We also double-checked that the within-run exclusion was included in the description of the Neural RSMs.

      Page 14. “Retrieval consisted of three scanning runs, each with 38 trials, lasting approximately 9 minutes and 12 seconds (within-run comparisons were later excluded from RSA analyses)”.

      Page 18. “This was done by vectorizing the voxel-level activation values within each region and calculating their correlations using Pearson’s r, excluding all within-run comparisons.”

      (24) Page 20: It is not clear why the mean estimate of "representational strength" (i.e., model-brain RSM correlations) is important at all. This comes back to Major point #2, namely that you are trying to solve a very different problem from model-comparative RSA.

      We have clarified that our approach is not an alternative to model-comparative RSA, and that depending on the task constraints researchers may choose to compare models with tRSA or other approaches requiring stimulus repetition (see 3).

      (25) Page 21: I believe the problems of simulating correlation matrices directly in the way that the authors in their first simulation did should be well known and should be moved to an appendix at best. Better yet, the authors could start with the correct simulation right away.

      We agree the paper is more concise with these simulations being moved to the appendix and more briefly discussed. We have implemented these changes (Appendix 1). However, we are not certain that this problem is unknown, and have several anecdotes of researchers inquiring about this “alternative” approach in talks with colleagues, thus we do still discuss the issues with this method.

      (26) Page 26: Is the "underlying continuous noise variable 𝜎𝑡𝑟𝑖𝑎𝑙 that was measured by 𝑣𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 " the variance of the noise pattern or the noise pattern itself? What does it mean it was "measured" - how?

      𝜎𝑡𝑟𝑖𝑎𝑙 is a vector of standard deviations for different trials, and 𝜎𝑡𝑟𝑖𝑎𝑙 i would be used to generate the noise patterns for trial i. v_measured is a hypothetical measurement of trial-level variability, such as “memorability” or “heartbeat variability”. We have revised our description to clarify our methods.

      Reviewer #2 (Recommendations for the authors):

      (8) It would be helpful to provide more clarity earlier on in the manuscript on what is a 'trial': in my experience, a row or column of the RDM is usually referred to as 'stimulus condition', which is typically estimated on multiple trials (instances or repeats) of that stimulus condition (or exemplars from that stimulus class) being presented to the subject. Here, a 'trial' is both one measurement (i.e., single, individual presentation of a stimulus) and also an entry in the RDM, but is this the most typical scenario for cRSA? There is a section in the Discussion that discusses repetitions, but I would welcome more clarity on this from the get-go.

      We have added discussion of stimulus repetition methods and datasets to the Introduction and clarified our use of the terms.

      Page 8. “Critically, in single-presentation designs, a “trial” refers to one stimulus presentation, and corresponds to a row or column in the RSM. In studies with repeated stimuli, these rows are often called “conditions” and may reflect aggregated patterns across trials. tRSA is compatible with both cases: whether rows represent individual trials or averaged trials that create “conditions”, tRSA estimates are computed at the row level”.

      (9) The quality of the results figures can be improved. For example, axes labels are hard to read in Figure 3A/B, panels 3C/D are hard to read in general. In Figure 7E, it's not possible to identify the 'dark red' brain regions in addition to the light red ones.

      We thank the reviewer for raising these and have edited the figures to be more readable in the manner suggested.

      (10) I would be interested to see a comparison between tRSA and cRSA in other fMRI (or other modality) datasets that have been extensively reported in the literature. These could be the original Kriegeskorte 96 stimulus monkey/fMRI datasets, commonly used open datasets in visual perception (e.g., THINGS, NSD), or the above-mentioned King et al. dataset, which has been analyzed in various papers.

      We recognize the great utility of replication from other research groups and do invite researchers to utilize tRSA on their datasets.

      (11) On P39, the authors suggest 'researchers can confidently replace their existing cRSA analysis with tRSA': Please discuss/comment on how researchers should navigate the choice of modeling parameters in tRSA's linear mixed effects setting.

      We have added discussion of the mixed-effects parameters and the various and encourage researchers to follow best practices for their model selection.

      Page 46. “However, researchers should always consider if their models match the goals of their analysis, including 1) constructing the random effects structure that will converge in their dataset and 2) testing their model fits against alternative structures (Meteyard & Davies, 2020; Park et al., 2020) and 3) considering which effects should be considered random or fixed depending on their research question”.

      (12) The final part of the Results section, demonstrating the tRSA results for the continuous memorability factor in the real fMRI data, could benefit from some substantiation/elaboration. It wasn't clear to me, for example, to what extent the observed significant association between representational strength and item memorability in this dataset is to be 'believed'; the Discussion section (p38). Was there any evidence in the original paper for this association? Or do we just assume this is likely true in the brain, based on prior literature by e.g. Bainbridge et al (who probably did not use tRSA but rather classic methods)?

      Indeed, memorability effects have been replicated in the literature, but not using the tRSA method. We have expanded our discussion to clarify the relationship of our findings and the relevant literature and methods it has employed.

      Page 38. “Critically, memorability is a robust stimulus property that is consistent across participants and paradigms (Bainbridge, 2022). Moreover, object memorability effects have been replicated using a variety of methods aside from tRSA, including univariate analyses and representational analyses of neural activity patterns where trial-level neural activity pattern estimates are correlated directly with object memorability (Slayton et al, 2025).”

      (13) The abstract could benefit from more nuance; I'm not sure if RSA can indeed be said to be 'the principal method', and whether it's about assessing 'quality' of representations (more commonly, the term 'geometry' or 'structure' is used).

      We have edited the abstract to reflect the true nuisance in the current approaches.

      Abstract. Neural representation refers to the brain activity that stands in for one’s cognitive experience, and in cognitive neuroscience, a prominent method of studying neural representations is representational similarity analysis (RSA). While there are several recent advances in RSA, the classic RSA (cRSA) approach examines the structure of representations across numerous items by assessing the correspondence between two representational similarity matrices (RSMs): usually one based on a theoretical model of stimulus similarity and the other based on similarity in measured neural data.

      (14) RSA is also not necessarily about models vs. neural data; it can also be between two neural systems (e.g., monkey vs. human as in Kriegeskorte et al., 2008) or model systems (see Sucholutsky et al., 2023). This statement is also repeated in the Introduction paragraph 1 (later on, it is correctly stated that comparing brain vs. model is most likely the 'most common' approach).

      We have added these examples in our introduction to RSA.

      Page 3.”One of the central approaches for evaluating information represented in the brain is representational similarity analysis (RSA), an analytical approach that queries the representational geometry of the brain in terms of its alignment with the representational geometry of some cognitive model (Kriegeskorte et al., 2008; Kriegeskorte & Kievit, 2013), or, in some cases, compares the representational geometry of two neural systems (e.g., Kriegeskorte et al., 2008) or two model systems (Sucholutsky et al., 2023)”.

      (15) 'theoretically appropriate' is an ambiguous statement, appropriate for what theory?

      We apologize for the ambiguous wording, and have corrected the text:

      Page 11. “Critically, tRSA estimates were submitted to a mixed-effects model which is statistically appropriate for modeling the hierarchical structure of the data, where observations are nested within both subjects and stimuli (Baayen et al., 2008; Chen et al., 2021)”.

      (16) I found the statement that cRSA "cannot model representation at the level of individual trials" confusing, as it made me think, what prohibits one from creating an RDM based on single-trial responses? Later on, I understood that what the authors are trying to say here (I think) is that cRSA cannot weigh the contributions of individual rows/columns to the overall representational strength differently.

      We thank the reviewer for their clarifying language and have added it to this section of the manuscript.

      “Abstract. However, because cRSA cannot weigh the contributions of individual trials (RSM rows/columns), it is fundamentally limited in its ability to assess subject-, stimulus-, and trial-level variances that all influence representation”.

      (17) Why use "RSM" instead of "RDM"? If the pairwise comparison metric is distance-based (e..g, 1-correlation as described by the authors), RDM is more appropriate.

      We apologize for the error, and have clarified the Methods text:

      Page3-4. First, brain activity responses to a series of N trials are compared against each other (typically using Pearson’s r) to form an N×N representational similarity matrix.

      (18) Figure 2: please write 'Correlation estimate' in the y-axis label rather than 'Estimate'.

      We have edited the label in Figure 2.

      (19) Page 6 'leaving uncertain the directionality of any findings' - I do not follow this argument. Obviously one can generate an RDM or RSM from vector v or vector -v. How does that invalidate drawing conclusions where one e.g., partials out the (dis)similarity in e.g., pleasantness ratings out of another RDM/RSM of interest?

      We agree such an approach does not invalidate the partial method; we have clarified what we mean by “directionality”.

      Page 8. ”For instance, even though a univariate random variable , such as pleasantness ratings, can be conveniently converted to an RSM using pairwise distance metrics (Weaverdyck et al., 2020), the very same RSM would also be derived from the opposite random variable , leaving uncertain of the directionality (or if representation is strongest for pleasant or unpleasant items) of any findings with the RSM (see also Bainbridge & Rissman, 2018)”.

      (20) P7 'sampled 19900 pairs of values from a bi-variate normal distribution', but the rows/columns in an RDM are not independent samples - shouldn't this be included in the simulation? I.e., shouldn't you simulate first the n=200 vectors, and then draw samples from those, as in the next analysis?

      This section has been moved to Appendix 1 (see responses to Reviewer 1.13).

      (21) Under data acquisition, please state explicitly that the paper is re-using data from prior experiments, rather than collecting data anew for validating tRSA.

      We have clarified this in the data acquisition section.

      Page 13. “A pre-existing dataset was analyzed to evaluate tRSA. Main study findings have been reported elsewhere (S. Huang, Bogdan, et al., 2024)”.

      (22) Figure 4 could benefit from some more explanation in-text. It wasn't clear to me, for example, how to interpret the asterisks depicted in the right part of the figure.

      We clarified the meaning of the asterisks in the main text in addition to the existent text in the figure caption.

      Page 26. “see Figure 4, off-diagonal cells in blue; asterisks indicate where tRSA was statistically more sensitive then cRSA)”.

      (23) Page 38 "the outcome of tRSA's improved characterization can be seen in multiple empirical outcomes:" it seems there is one mention of 'outcomes' too many here.

      We have revised this sentence.

      Page 41. “tRSA's improved characterization can be seen in multiple empirical outcomes”.

      (24) Page 38 "model fits became the strongest" it's not clear what aspect of the reported results in the paragraph before this is referring to - the Appendix?

      Yes, the model fits are in the Appendix, we have added this in text citation.

      Moreover, model-fits became the strongest when the models also incorporated trial-level variables such as fMRI run and reaction time (Appendix 3, Table 6).

      References

      Diedrichsen, J., Berlot, E., Mur, M., Schütt, H. H., Shahbazi, M., & Kriegeskorte, N. (2021). Comparing representational geometries using whitened unbiased-distance-matrix similarity. Neurons, Behavior, Data and Theory, 5(3). https://arxiv.org/abs/2007.02789

      Diedrichsen, J., & Kriegeskorte, N. (2017). Representational models: A common framework for understanding encoding, pattern-component, and representational-similarity analysis. PLoS Computational Biology, 13(4), e1005508.

      Diedrichsen, J., Yokoi, A., & Arbuckle, S. A. (2018). Pattern component modeling: A flexible approach for understanding the representational structure of brain activity patterns. NeuroImage, 180, 119-133.

      Naselaris, T., Kay, K. N., Nishimoto, S., & Gallant, J. L. (2011). Encoding and decoding in fMRI. NeuroImage, 56(2), 400-410.

      Nili, H., Wingfield, C., Walther, A., Su, L., Marslen-Wilson, W., & Kriegeskorte, N. (2014). A toolbox for representational similarity analysis. PLoS Computational Biology, 10(4), e1003553.

      Schütt, H. H., Kipnis, A. D., Diedrichsen, J., & Kriegeskorte, N. (2023). Statistical inference on representational geometries. ELife, 12. https://doi.org/10.7554/eLife.82566

      Walther, A., Nili, H., Ejaz, N., Alink, A., Kriegeskorte, N., & Diedrichsen, J. (2016). Reliability of dissimilarity measures for multi-voxel pattern analysis. NeuroImage, 137, 188-200.

      King, M. L., Groen, I. I., Steel, A., Kravitz, D. J., & Baker, C. I. (2019). Similarity judgments and cortical visual responses reflect different properties of object and scene categories in naturalistic images. NeuroImage, 197, 368-382.

      Kriegeskorte, N., Mur, M., Ruff, D. A., Kiani, R., Bodurka, J., Esteky, H., ... & Bandettini, P. A. (2008). Matching categorical object representations in inferior temporal cortex of man and monkey. Neuron, 60(6), 1126-1141.

      Nili, H., Wingfield, C., Walther, A., Su, L., Marslen-Wilson, W., & Kriegeskorte, N. (2014). A toolbox for representational similarity analysis. PLoS computational biology, 10(4), e1003553.

      Sucholutsky, I., Muttenthaler, L., Weller, A., Peng, A., Bobu, A., Kim, B., ... & Griffiths, T. L. (2023). Getting aligned on representational alignment. arXiv preprint arXiv:2310.13018.

    1. Dans quelle mesure ces produits qui, pour beaucoup d’enfants d’adolescents relèvent de la consommation courante, leur servent-ils à établir et entretenir des relations d’amitié et quel impact ont-ils sur elles ?

      Problématique

    1. Author response:

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

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      In this fMRI study, the authors wished to assess neural mechanisms supporting flexible "temporal construals". For this, human participants learned a story consisting of fifteen events. During fMRI, events were shown to them, and they were instructed to consider the event from "an internal" or from "an external" perspective. The authors found opposite patterns of brain activity in the posterior parietal cortex and the anterior hippocampus for the internal and the external viewpoint. They conclude that allocentric sequences are stored in the hippocampus, whereas egocentric sequences are used in the parietal cortex. The claims align with previous fMRI work addressing this question.

      We appreciate the reviewer's concise summary of our research. We would like to offer two clarifications to prevent any potential misunderstandings.

      First, the activity patterns in the parietal cortex and hippocampus are not entirely opposite across internal and external perspectives. Specifically, the activation level in the posterior parietal cortex shows a positive correlation with sequential distance during external-perspective tasks, but a negative correlation during internal-perspective tasks. In contrast, the activation level in the anterior hippocampus positively correlates with sequential distance, irrespective of the observer's perspective. Therefore, our results suggest that the parietal cortex, with its perspective-dependent activity, supports egocentric representation; the hippocampus, with its consistent activity across perspectives, supports allocentric representation.

      Second, while some of our findings align with previous fMRI studies, to our knowledge, no prior research has explicitly investigated how the neural representation of time may vary depending on the observer's viewpoint. This gap in the literature is the primary motivation for our current study.

      Strengths:

      The research topic is fascinating, and very few labs in the world are asking the question of how time is represented in the human brain. Working hypotheses have been recently formulated, and this work seems to want to tackle some of them.

      We appreciate the reviewer's acknowledgment of the theoretical significance of our study.

      Weaknesses:

      The current writing is fuzzy both conceptually and experimentally. I cannot provide a sufficiently well-informed assessment of the quality of the experimental work because there is a paucity of details provided in the report. Any future revisions will likely improve transparency.

      (1) Improving writing and presentation:

      The abstract and the introduction make use of loaded terms such as "construals", "mental timeline", "panoramic views" in very metaphoric and unexplained ways. The authors do not provide a comprehensive and scholarly overview of these terms, which results in verbiage and keywords/name-dropping without a clear general framework being presented. Some of these terms are not metaphors. They do refer to computational concepts that the authors should didactically explain to their readership. This is all the more important that some statements in the Introduction are misattributed or factually incorrect; some statements lack attributions (uncited published work). Once the theory, the question, and the working hypothesis are clarified, the authors should carefully explain the task.

      We appreciate the reviewer's critics.

      The formulation of the scientific question in the introduction is grounded in the spatial construals of time hypothesis and conceptual metaphor theory (e.g., Traugott, 1978; Lakoff & Johnson, 1980; see recent reviews by Núñez & Cooperrider, 2013; Bender & Beller, 2014). These frameworks were originally developed through analyses of how spatial metaphors are used to describe temporal concepts in natural language. Consequently, it is theoretically motivated and largely unavoidable to introduce the two primary temporal construals—mental time travel and mental time watching— using metaphorical expressions.

      However, we do agree with the reviewer that the introduction in the original manuscript was overly long and that the working hypothesis was not clearly stated. In the revised manuscript, we have streamlined the introduction and substantially revised the following two paragraphs to clarify the formulation of our working hypothesis (Pages 5-6):

      “Recent studies have already begun to investigate the neural representation of the memorized event sequence (e.g., Deuker et al., 2016; Thavabalasingam et al., 2018; Bellmund et al., 2019, 2022; see reviews by Cohn-Sheehy & Ranganath, 2017; Bellmund et al., 2020). Yet, the neural mechanisms that enable the brain to construct distinct construals of an event sequence remain largely unknown. Valuable insights may be drawn from research in the spatial domain, which diPerentiates the neural representation in allocentric and egocentric reference frames. According to an influential neurocomputational model (Byrne et al., 2007; Bicanski & Burgess, 2018; Bicanski & Burgess, 2020), allocentric and egocentric spatial representations are dissociable in the brain—they are respectively implemented in the medial temporal lobe (MTL)—including the hippocampus—and the parietal cortex. Various egocentric representations in the parietal cortex derived from diPerent viewpoints can be transformed and integrated into a unified allocentric representation and stored in the MTL (i.e., bottom-up process). Conversely, the allocentric representation in the MTL can serve as a template for reconstructing diverse egocentric representations across diPerent viewpoints in the parietal cortex (i.e., top-down process).”

      “In line with the spatial construals of time hypothesis, several authors have recently suggested that such mutually engaged egocentric and allocentric reference frames (in the parietal cortex and the medial temporal lobe, respectively) proposed in the spatial domain might also apply to the temporal one (e.g., Gauthier & van Wassenhove, 2016ab; Gauthier et al., 2019, 2020; Bottini & Doeller, 2020). If this hypothesis holds, it could explain how the brain flexibly generates diverse construals of the same event sequence. Specifically, the hippocampus may encode a consistent representation of an event sequence that is independent of whether an individual adopts an internal or external perspective, reflecting an allocentric representation of time. In contrast, parietal cortical representations are expected to vary flexibly with the adopted perspective that is shaped by task demands, reflecting an egocentric representation of time.”

      In the revised manuscript, we also corrected statements in the Introduction that may have been misattributed (see Reviewer 2, comment 4(ii)) and added several relevant and important publications.

      (2) The experimental approach lacks sufficient details to be comprehensible to a general audience. In my opinion, the results are thus currently uninterpretable. I highlight only a couple of specific points (out of many). I recommend revision and clarification.

      (a) No explanation of the narrative is being provided. The authors report a distribution of durations with no clear description of the actual sequence of events. The authors should provide the text that was used, how they controlled for low-level and high-level linguistic confounds.

      We thank the reviewer for the suggestions. The event sequence for the odd-numbered participants is shown in the original Figure 1. In the revised manuscript, we added to Figure 1 the figure supplement 1 to illustrate the actual sequence of events for the participants with both odd and even numbers. We also added the narratives used in the reading phase of the learning procedures for the participants with both odd and even numbers (Figure 1—source data 1).

      To control for low-level linguistic confounds, we included the number of syllables as a covariate in the first-level general linear model in the fMRI analysis. To address high-level linguistic confounds, such as semantic information (which is difficult to quantify), we randomly assigned event labels to the 15 events twice, creating two counterbalanced versions for participants with even and odd numbers (see Comment 2b below).

      (b) The authors state, "we randomly assigned 15 phrases to the events twice". It is impossible to comprehend what this means. Were these considered stimuli? Controls? IT is also not clear which event or stimulus is part of the "learning set" and whether these were indicated to be such to participants.

      We apologize for any confusion in the Results section and the legend of Figure 1. Our motivation was explained in the "Stimuli" section of the Methods. In the revised manuscript, we have clarified this by adding an explanation to the legend of Figure 1 and including the supplementary Figure 1: " To minimize potential confounds between the semantic content of the event phrases and the temporal structure of the events, we randomly assigned the phrases to the events, creating two versions for participants with even and odd ID numbers. Both versions can be seen in Figure1—figure supplement 1 and Figure 1—source data 1."

      (c) The left/right counterbalancing is not being clearly explained. The authors state that there is counterbalancing, but do not sufficiently explain what it means concretely in the experiment. If a weak correlation exists between sequential position and distance, it also means that the position and the distance have not been equated within. How do the authors control for these?

      We thank the reviewer for highlighting this point and apologize for the lack of clarity in the original manuscript. In the current version (Page 40), we have provided further clarification: “We carefully selected two sets of 20 event pairs from the 210 possible combinations, assigning them to the odd and even runs of the fMRI experiment. Using a brute-force search, we identified 20 pairs in which sequential distance showed only weak correlations with positional information for both reference and target events (ranging from 1 to 15), as well as with behavioral responses (Same vs. Different or Future vs. Past, coded as 0 and 1), with all correlation coefficients below 0.2. At the same time, we balanced the proportion of correct responses across conditions: for the external-perspective task, Same/Different = 11/9 and 12/8; for the internal-perspective task, Future/Past = 12/8 and 8/12. Under these constraints, the sequential distances in both sets ranged from 1 to 5. To further mitigate spatial response biases, we pseudorandomized the left/right on-screen positions of the two response options within each task block, while ensuring an equal number of correct responses mapped to the left and right buttons (i.e., 10 per block).”

      The event pairs we selected already represent the best possible choice given all the criteria we aimed to satisfy. It is impossible to completely eliminate all potential correlations. For instance, if the target event occurs near the beginning of the day, it will tend to fall in the past, whereas if it occurs near the end of the day, it is more likely to fall in the future. To further ensure that the significant results were not driven by these weak confounding factors, we constructed another GLM that included three additional parametric modulators: the sequence position of the target event (ranging from 1 to 15) and the behavioral responses (Future vs. Past in the internal-perspective task; Same vs. Different in the external-perspective task, coded as 0 and 1). The significant findings were unaffected.

      (d) The authors used two tasks. In the "external perspective" one, the authors asked participants to report whether events were part of the same or a different part of the day. In the "internal perspective one", the authors asked participants to project themselves to the reference event and to determine whether the target event occurred before or after the projected viewpoint. The first task is a same/different recognition task. The second task is a temporal order task (e.g., Arzy et al. 2009). These two asks are radically different and do not require the same operationalization. The authors should minimally provide a comprehensive comparison of task requirements, their operationalization, and, more importantly, assess the behavioral biases inherent to each of these tasks that may confound brain activity observed with fMRI.

      We understand the reviewer’s concern. We agree that there is a substantial difference between the two tasks. However, the primary goal of this study was not to directly compare these tasks to isolate a specific cognitive component. Rather, the neural correlates of temporal distance were first identified as brain regions showing a significant correlation between neural activity and temporal distance using the parametric modulation analysis. We then compared these neural correlates between the two tasks. Therefore, any general differences between the tasks should not be a confound for our main results. Our aim was to examine whether the hippocampal representation of temporal distance remains consistent across different perspectives, and whether the parietal representation of temporal distance varies as a function of the perspective adopted.

      Therefore, the main aim of our task manipulation was to ensure that participants adopted either an external or an internal perspective on the event sequence, depending on the task condition. In the Introduction (Pages 6–7), we clarify this manipulation as follows: “In the externalperspective task, participants localized events with respect to external temporal boundaries, judging whether the target event occurred in the same or a different part of the day as the reference event. In the internal-perspective task, participants were instructed to mentally project themselves into the reference event and localize the target event relative to their own temporal point, judging whether the target event happened in the future or the past of the reference event (see Methods for details of the scanning procedure).”

      We believe this task manipulation was successful. Behaviorally, the two tasks showed opposite correlations between reaction time and temporal distance, resembling the symbolic distance versus mental scanning effect. Neurally, contrasting the internal- and external-perspective tasks revealed activation of the default mode network, which is known to play a central role in self-projection (Buckner et al., 2017).

      (e) The authors systematically report interpreted results, not factual data. For instance, while not showing the results on behavioral outcomes, the authors directly interpret them as symbolic distance effects.

      Thank you for this comment. In the original paper, we reported the relevant statistics before our interpretation: “Sequential Distance was correlated positively with RT in the external-perspective task (z = 3.80, p < 0.001) but negatively in the internal-perspective task (z = -3.71, p < 0.001).” However, they may have been difficult to notice, and we are including a figure for the RT analysis in the revised manuscript.

      Crucially, the authors do not comment on the obvious differences in task difficulty in these two tasks, which demonstrates a substantial lack of control in the experimental design. The same/different task (task 1 called "external perspective") comes with known biases in psychophysics that are not present in the temporal order task (task 2 called " internal perspective"). The authors also did not discuss or try to match the performance level in these two tasks. Accordingly, the authors claim that participants had greater accuracy in the external (same/different) task than in the internal task, although no data are shown and provided to support this report. Further, the behavioral effect is trivialized by the report of a performance accuracy trade off that further illustrates that there is a difference in the task requirements, preventing accurate comparison of the two tasks.

      As noted in Question 2d, we acknowledge the substantial difference between the two tasks. However, the primary goal of this study was not to directly compare these tasks to isolate a specific cognitive component. Instead, we first identified the neural correlates of temporal distance as brain regions showing a significant correlation between neural activity and temporal distance, independent of task demands. We then compared these neural correlates across the two task conditions, which were designed to engage different temporal perspectives. Therefore, any general differences between the tasks should not be a confound for our main findings and interpretation.

      Our aim was to investigate whether the hippocampal representation of temporal distance remains consistent across different perspectives and whether the parietal representation of temporal distance varies as a function of the perspective adopted. We do not see how this doubledissociation pattern could be explained by differences in task difficulty.

      While we do not consider the overall difference in task difficulty between the two tasks to be a confounding factor, we acknowledge the potential confound posed by variations in task difficulty across temporal distances (1 to 5). This concern arises from the similarity between the activity patterns in the posterior parietal cortex and reaction time across temporal distances. To address this, we conducted control analyses to test this hypothesis (see the second and third points from Reviewer 2 for details).

      On page 8, we present the behavioral accuracy data: “Participants showed significantly higher accuracy in the external-perspective task than in the internal-perspective task (external-perspective task: M = 93.5%, SD = 4.7%; internal-perspective task: M = 89.5%, SD = 8.1%; paired t(31) = 3.33, p = 0.002).”

      All fMRI contrasts are also confounded by this experimental shortcoming, seeing as they are all reported at the interaction level across a task. For instance, in Figure 4, the authors report a significant beta difference between internal and external tasks. It is impossible to disentangle whether this effect is simply due to task difference or to an actual processing of the duration that differs across tasks, or to the nature of the representation (the most difficult to tackle, and the one chosen by the authors).

      We thank the reviewer for pointing out this important issue. Like temporal distance, the neural correlates of duration were not derived from a direct contrast between the two tasks. Instead, they were identified by detecting brain regions showing a significant correlation between neural activity and the implied duration of each event using the parametric modulation analysis. Therefore, what is shown in Figure 4 reflects the significant differences in these neural correlations with duration between the two tasks.

      The observed difference in the neural representation of duration between the two tasks was unexpected. In the original manuscript, we provided a post hoc explanation: “Since the externalperspective task in the current study encouraged the participants to compare the event sequence with the external parallel temporal landmarks, duration representation in the hippocampus may be dampened.”

      However, we agree that this difference might also arise from other factors distinguishing the two tasks. In the revised manuscript, we have clarified this possibility as follows: “The difference in duration representation between the two tasks remains open to interpretation. One possible explanation is that the hippocampus is preferentially involved in memory for durations embedded within event sequences (see review by Lee et al., 2020). In the internal-perspective task, participants indeed localized events within the event sequence itself. In contrast, the externalperspective task encouraged participants to compare the event sequence with external temporal landmarks, which may have attenuated the hippocampal representation of duration.”

      Conclusion:

      In conclusion, the current experimental work is confounded and lacks controls. Any behavioral or fMRI contrasts between the two proposed tasks can be parsimoniously accounted for by difficulty or attentional differences, not the claim of representational differences being argued for here.

      We hope that our explanations and clarifications above adequately address the reviewer’s concerns. We would like to reiterate that we did not directly compare the two tasks. Rather, we first identified the neural representations of sequential distance and duration, and then examined how these representations differed across tasks. It is unclear to us how the overall difference in task difficulty or attentional demands could lead to the observed pattern of results.

      By determining where the neural representations were consistent and where they diverged, we were able to differentiate brain regions that encode temporal information allocentrically from those that represent temporal information in a perspective-dependent manner, modulated by task demands.

      Reviewer #2 (Public review):

      Summary:

      Xu et al. used fMRI to examine the neural correlates associated with retrieving temporal information from an external compared to internal perspective ('mental time watching' vs. 'mental time travel'). Participants first learned a fictional religious ritual composed of 15 sequential events of varying durations. They were then scanned while they either (1) judged whether a target event happened in the same part of the day as a reference event (external condition); or (2) imagined themselves carrying out the reference event and judged whether the target event occurred in the past or will occur in the future (internal condition). Behavioural data suggested that the perspective manipulation was successful: RT was positively correlated with sequential distance in the external perspective task, while a negative correlation was observed between RT and sequential distance for the internal perspective task. Neurally, the two tasks activated different regions, with the external task associated with greater activity in the supplementary motor area and supramarginal gyrus, and the internal condition with greater activity in default mode network regions. Of particular interest, only a cluster in the posterior parietal cortex demonstrated a significant interaction between perspective and sequential distance, with increased activity in this region for longer sequential distances in the external task, but increased activity for shorter sequential distances in the internal task. Only a main effect of sequential distance was observed in the hippocampus head, with activity being positively correlated with sequential distance in both tasks. No regions exhibited a significant interaction between perspective and duration, although there was a main effect of duration in the hippocampus body with greater activity for longer durations, which appeared to be driven by the internal perspective condition. On the basis of these findings, the authors suggest that the hippocampus may represent event sequences allocentrically, whereas the posterior parietal cortex may process event sequences egocentrically.

      We sincerely appreciate the reviewers for providing an accurate, comprehensive, and objective summary of our study.

      Strengths:

      The topic of egocentric vs. allocentric processing has been relatively under-investigated with respect to time, having traditionally been studied in the domain of space. As such, the current study is timely and has the potential to be important for our understanding of how time is represented in the brain in the service of memory. The study is well thought out, and the behavioural paradigm is, in my opinion, a creative approach to tackling the authors' research question. A particular strength is the implementation of an imagination phase for the participants while learning the fictional religious ritual. This moves the paradigm beyond semantic/schema learning and is probably the best approach besides asking the participants to arduously enact and learn the different events with their exact timings in person. Importantly, the behavioural data point towards successful manipulation of internal vs. external perspective in participants, which is critical for the interpretation of the fMRI data. The use of syllable length as a sanity check for RT analyses, as well as neuroimaging analyses, is also much appreciated.

      We thank the reviewer for the positive and encouraging comments.

      Weaknesses/Suggestions:

      Although the design and analysis choices are generally solid, there are a few finer details/nuances that merit further clarification or consideration in order to strengthen the readers' confidence in the authors' interpretation of their data.

      (1) Given the known behavioural and neural effects of boundaries in sequence memory, I was wondering whether the number of traversed context boundaries (i.e., between morning-afternoon, and afternoon-evening) was controlled for across sequential length in the internal perspective condition? Or, was it the case that reference-target event pairs with higher sequential numbers were more likely to span across two parts of the day compared to lower sequential numbers? Similarly, did the authors examine any potential differences, whether behaviourally or neurally, for day part same vs. day part different external task trials?

      We thank the reviewer for the thoughtful comments. When we designed the experiment, we minimized the correlation between the sequential distance between the target and reference events and whether the reference and target events occurred within the same or different parts of the day (coded as Same = 0, Different = 1). The point-biserial correlation coefficient between these two variables across all the trials within the same run were controlled below 0.2.

      To investigate the effect of day-part boundaries on behavior, as well as the contribution of other factors, we conducted a new linear mixed-effects model analysis incorporating four additional variables. They are whether the target and the reference events are within the same or different parts of the day (i.e., Same vs. Different), whether the target event is in the future or the past of the reference event (i.e., Future vs. Past), and the interactions of the two factors with Task Type (i.e., internal- vs. external-perspective task).

      The results are largely the same as the original one in the table: There was a significant main effect of Syllable Length, and the interaction effects between Task Type and Sequence Distance and between Task Type and Duration remain significant. What's new is we also found a significant interaction effect between Task Type and Same vs. Different.

      As shown in the Figure 2—figure supplement 1, this Same vs. Different effect was in line with the effect of Sequential Distance, with two events in the same and different parts of the day corresponding to the short and long sequential distances. Given that Sequential Distance had already been considered in the model, the effect of parts of the day should result from the boundary effect across day parts or the chunking effect within day parts, i.e., the sequential distance across different parts of the day was perceived longer while the sequential distance within the same parts of the day was perceived shorter. We have incorporated these findings into the manuscript.

      Neurally, to further verify that the significant effects of sequential distance were not driven by its weak correlation with the Same/Different judgment or other potential confounding factors, we constructed another GLM that incorporated three additional parametric modulators: the sequence position of the target event (ranging from 1 to 15) and the behavioral responses (Future vs. Past in the internal-perspective task; Same vs. Different in the external-perspective task, coded as 0 and 1). The significant findings were unaffected.

      (2) I would appreciate further insight into the authors' decision to model their task trials as stick functions with duration 0 in their GLMs, as opposed to boxcar functions with varying durations, given the potential benefits of the latter (e.g., Grinband et al., 2008). I concur that in certain paradigms, RT is considered a potential confound and is taken into account as a nuisance covariate (as the authors have done here). However, given that RTs appear to be critical to the authors' interpretation of participant behavioural performance, it would imply that variations in RT actually reflect variations in cognitive processes of interest, and hence, it may be worth modelling trials as boxcar functions with varying durations.

      We appreciate the reviewer’s insightful comment on this important issue. Whether to control for RT’s influence on fMRI activation is indeed a long-standing paradox. On the one hand, RT reflects underlying cognitive processes and therefore should not be fully controlled for. On the other hand, RT can independently influence neural activity, as several brain networks vary with RT irrespective of the specific cognitive process involved—a domain-general effect. For example, regions within the multiple-demand network are often positively correlated with RT across different cognitive domains.

      Our strategy in the manuscript is to first present the results without including RT as a control variable and then examine whether the effects are preserved after controlling for RT. In the revised manuscript, we have clarified this approach (Page 13): “Here, changes in activity levels within the PPC were found to align with RT. Whether to control for RT’s influence on fMRI activation represents a well-known paradox. On the one hand, RT reflects underlying cognitive processes and therefore should not be fully controlled for. On the other hand, RT can independently influence neural activity, as several brain networks vary with RT irrespective of the specific cognitive process involved—a domain-general effect. For instance, regions within the multiple-demand network are often positively correlated with RT and task difficulty across diverse cognitive domains (e.g., Fedorenko et al., 2013; Mumford et al., 2024). To evaluate the second possibility, we conducted an additional control analysis by including trial-by-trial RT as a parametric modulator in the first-level model (see Methods). Notably, the same PPC region remained the only area in the entire brain showing a significant interaction between Task Type and Sequential Distance (voxel-level p < 0.001, clusterlevel FWE-corrected p < 0.05). This finding indicates that PPC activity cannot be fully attributed to RT. Furthermore, we do not interpret the effect as reflecting a domain-general RT influence, as regions within the multiple-demand system—typically sensitive to RT and task difficulty—did not exhibit significant activation in our data.”

      The reason we did not use boxcar functions with varying durations in our original manuscript is that we also applied parametric modulation in the same model. In the parametric modulation, all parametric modulators inherit the onsets and durations of the events being modulated. Consequently, the modulators would also take the form of boxcar functions rather than stick functions—the height of each boxcar reflecting the parameter value and its length reflecting the RT. We were uncertain whether this approach would be appropriate, as we have not encountered other studies implementing parametric modulation in this manner.

      For exploratory purposes, we also conducted a first-level analysis using boxcar functions with variable durations. The same PPC region remained the strongest area in the entire brain that shows an interaction effect between Task Type and Sequential Distance. However, the cluster size was slightly reduced (voxel-level p < 0.001, cluster-level FWE-corrected p = 0.0610; see the Author response image 1 below). The cross indicates the MNI coordinates at [38, –69, 35], identical to those shown in the main results (Figure 4A).

      Author response image 1.

      (3) The activity pattern across tasks and sequential distance in the posterior parietal cortex appears to parallel the RT data. Have the authors examined potential relationships between the two (e.g., individual participant slopes for RT across sequential distance vs. activity betas in the posterior parietal cortex)?

      We thank the reviewer for this helpful suggestion. As shown in the Author response image 2, the interaction between Task Type and Sequential Distance was a stronger predictor of PPC activation than of RT. Because PPC activation and RT are measured on different scales, we compared their standardized slopes (standardized β) measuring the change in a dependent variable in terms of standard deviations for a one-standard-deviation increase in an independent variable. The standardized β for the Task Type × Sequential Distance interaction was −0.30 (95% CI [−0.42, −0.19]) for PPC activation and −0.21 (95% CI [−0.30, −0.13]) for RT. The larger standardized effect for PPC activation indicates that the Task Type × Sequential Distance interaction was a stronger predictor of neural activation than of behavioral RT.

      Author response image 2.

      A more relevant question is whether PPC activation can be explained by temporal information (i.e., the sequential distance) independently of RT. To test this, we included both Sequential Distance and RT in the same linear mixed-effects model predicting PPC Activation Level. As shown in the Author response table 1, although RT independently influenced PPC activation (F(1, 288) = 4.687, p = 0.031), the interaction between Task Type and Sequential Distance was a much stronger independent predictor (F(1, 290) = 19.319, p < 0.001).

      Author response table 1.

      PPC Activation Level Predicted by Sequential Distance and RT

      (3) Linear Mixed Model Formula: PPC Activation Level ~ 1 + Task Type * (Sequential Distance + RT) + (1 | Participant)

      (4) There were a few places in the manuscript where the writing/discussion of the wider literature could perhaps be tightened or expanded. For instance:

      (i) On page 16, the authors state 'The negative correlation between the activation level in the right PPC and sequential distance has already been observed in a previous fMRI study (Gauthier & van Wassenhove, 2016b). The authors found a similar region (the reported MNI coordinate of the peak voxel was 42, -70, 40, and the MNI coordinate of the peak voxel in the present study was 39, -70, 35), of which the activation level went up when the target event got closer to the self-positioned event. This finding aligns with the evidence suggesting that the posterior parietal cortex implements egocentric representations.' Without providing a little more detail here about the Gauthier & van Wassenhove study and what participants were required to do (i.e., mentally position themselves at a temporal location and make 'occurred before' vs. 'occurred after' judgements of a target event), it could be a little tricky for readers to follow why this convergence in finding supports a role for the posterior parietal cortex in egocentric representations.

      We appreciate the reviewer’s comments. In the revised manuscript, we have provided a more detailed explanation of Gauthier and van Wassenhove’s study (Page 17): “The negative correlation between the activation level in the right PPC and sequential distance has already been observed in a previous fMRI study by Gauthier & van Wassenhove (2016b). In their study, the participants were instructed to mentally position themselves at a specific time point and judge whether a target event occurred before or after that time point. The authors identified a similar brain region (reported MNI coordinates of the peak voxel: 42, −70, 40), closely matching the activation observed in the present study (MNI coordinates of the peak voxel: 39, −70, 35). In both studies, activation in this region increased as the target event approached the self-positioned time point, which aligns with the evidence suggesting that the posterior parietal cortex implements egocentric representations.”

      (ii) Although the authors discuss the Lee et al. (2020) review and related studies with respect to retrospective memory, it is critical to note that this work has also often used prospective paradigms, pointing towards sequential processing being the critical determinant of hippocampal involvement, rather than the distinction between retrospective vs. prospective processing.

      We sincerely thank the reviewer for highlighting these important points. In response, we have revised the section of the Introduction discussing the neural underpinnings of duration (Pages 3-4). “Neurocognitive evidence suggests that the neural representation of duration engages distinct brain systems. The motor system—particularly the supplementary motor area—has been associated with prospective timing (e.g., Protopapa et al., 2019; Nani et al., 2019; De Kock et al., 2021; Robbe, 2023), whereas the hippocampus is considered to support the representation of duration embedded within an event sequence (e.g., Barnett et al., 2014; Thavabalasingam et al., 2018; see also the comprehensive review by Lee et al., 2020).”

      (iii) The authors make an interesting suggestion with respect to hippocampal longitudinal differences in the representation of event sequences, and may wish to relate this to Montagrin et al. (2024), who make an argument for the representation of distant goals in the anterior hippocampus and immediate goals in the posterior hippocampus.

      We thank the reviewer for bringing this intriguing and relevant study to our attention. In the Discussion of the manuscript, we have incorporated it into our discussion (Page 21): “Evidence from the spatial domain has suggested that the anterior hippocampus (or the ventral rodent hippocampus) implements global and gist-like representations (e.g., larger receptive fields), whereas the posterior hippocampus (or the dorsal rodent hippocampus) implements local and detailed ones (e.g., finer receptive fields) (e.g., Jung et al., 1994; Kjelstrup et al., 2008; Collin et al., 2015; see reviews by Poppenk et al., 2013; Robin & Moscovitch, 2017; see Strange et al., 2014 for a different opinion). Recent evidence further shows that the organizational principle observed along the hippocampal long axis may also extend to the temporal domain (Montagrin et al., 2024). In that study, the anterior hippocampus showed greater activation for remote goals, whereas the posterior hippocampus was more strongly engaged for current goals, which are presumed to be represented in finer detail.”

      Reviewing Editor Comments:

      While both reviewers acknowledged the significance of the topic, they raised several important concerns. We believe that providing conceptual clarification, adding important methodological details, as well as addressing potential confounds will further strengthen this paper.

      We thank the editor for the suggestions.

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      (1) Please, provide the actual ethical approval #.

      We have added the ethical approval number in the revised manuscript (P 36): “The ethical committee of the University of Trento approved the experimental protocol (Approval Number 2019-018),”

      (2) Thirty-two participants were tested. Please report how you estimated the sample size was sufficient to test your working hypothesis.

      We thank the editor for pointing out this omission. In the revised manuscript, we have added an explanation for our choice of sample size (p. 36): “The sample size was chosen to align with the upper range of participant numbers reported in previous fMRI studies that successfully detected sequence or distance effects in the hippocampus (N = 15–34; e.g., Morgan et al., 2011; Howard et al., 2014; Deuker et al., 2016; Garvert et al., 2017; Theves et al., 2019; Park et al., 2021; Cristoforetti et al., 2022).”

      (3) All MRI figures: please orient the reader; left/right should be stated.

      In the revised manuscript, we have added labels to all MRI figures to indicate the left and right hemispheres.

      (4) In Figure 3A-B, the clear lateralization of the activation is not discussed in the Results or in the Discussion. Was it predicted?

      We thank the editors for highlighting this important point regarding hemispheric lateralization. The right-lateralization observed in our findings is indeed consistent with previous literature. In the revised manuscript, we have expanded our discussion to emphasize this aspect more clearly.

      For the parietal cortex, we now note (Page 17-18): “The negative correlation between activation in the right posterior parietal cortex (PPC) and sequential distance has previously been reported in an fMRI study by Gauthier and van Wassenhove (2016b). In their paradigm, participants were instructed to mentally position themselves at a specific time point and judge whether a target event occurred before or after that point. The authors identified a similar region (peak voxel MNI coordinates: 42, −70, 40), closely corresponding to the activation observed in the present study (peak voxel MNI coordinates: 39, −70, 35). In both studies, activation in this region increased as the target event approached the self-positioned time point, consistent with evidence suggesting that the posterior parietal cortex supports egocentric representations. Neuropsychological studies have further shown that patients with lesions in the bilateral or right PPC exhibit ‘egocentric disorientation’ (Aguirre & D’Esposito, 1999), characterized by an inability to localize objects relative to themselves (e.g., Case 2: Levine et al., 1985; Patient DW: Stark, 1996; Patients MU: Wilson et al., 1997, 2005).”

      For the hippocampus, we have added (Page 19): “Previous research has shown that hippocampal activation correlates with distance (e.g., Morgan et al., 2011; Howard et al., 2014; Garvert et al., 2017; Theves et al., 2019; Viganò et al., 2023), and that distributed hippocampal activity encodes distance information (e.g., Deuker et al., 2016; Park et al., 2021). Most studies have reported hippocampal ePects either bilaterally or predominantly in the right hemisphere, whereas only one study (Morgan et al., 2011) found the ePect localized to the left hippocampus.”

    1. Author response:

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

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      This is a well-structured and interesting manuscript that investigates how herbivorous insects, specifically whiteflies and planthoppers, utilize salivary effectors to overcome plant immunity by targeting the RLP4 receptor.

      Strengths:

      The authors present a strong case for the independent evolution of these effectors and provide compelling evidence for their functional roles.

      Weaknesses:

      Western blot evidence for effector secretion is weak. The possibility of contamination from insect tissues during the sample preparation should be avoided.

      Below are some specific comments and suggestions to strengthen the manuscript.

      Thank you very much for your comments. We have carefully revised the MS following your valuable suggestions and comments.

      (1) Western blot evidence for effector secretion:

      The western blot evidence in Figure 1, which aims to show that the insect protein is secreted into plants, is not fully convincing. The band of the expected size (~30 kDa) in the infested tissues is very weak. Furthermore, the high and low molecular weight bands that appear in the infested tissues do not match the size of the protein in the insects themselves, and a high molecular weight band also appears in the uninfested control tissues. It is difficult to draw a definitive conclusion that this protein is secreted into the plants based on this evidence. The authors should also address the possibility of contamination from insect tissues during the sample preparation and explain how they have excluded this possibility.

      Thank you for pointing out this. One or two bands between 25-35kDa were specifically identified in B. tabaci-infested plants, but not the non-infested plants, and the smaller high intensity band is the same size as that of BtRDP in salivary glands. This experiment has been repeated for six times. In the current version, we reperformed this experiment, and provided salivary gland sample as a positive control, which showed the same molecular weight with a specific band in infested sample. It is noteworthily that in the experiment of current version, only the smaller high intensity band appear, while the low intensity band did not appear. The detection of a protein within infested plant tissue is a key criterion for validating the secretion of salivary effectors, an approach supported by numerous studies in this field. Furthermore, our previous LC-MS/MS analysis of B. tabaci watery saliva identified six unique peptides matching BtRDP, providing independent evidence for its presence in saliva. Therefore, as we now state in the manuscript “the detection of BtRDP in infested plants (Fig. 1a) and in watery saliva (Fig. S1) collectively indicates that BtRDP is a salivary protein”.

      Regarding the higher molecular weight band that present in both infested and non-infested samples, we agree that it most likely represents a non-specific band, which is a common occurrence in Western blot assays. Such bands are sometimes used to indicate comparable sample loading. To address the possibility of contamination by insect tissues, we wish to clarify that all insects and deposited eggs were carefully removed from the infested leaves prior to sample processing. Moreover, BtRDP is undetectable at the egg stage, and no BtRDP-associated band can be detected even in egg contamination. We have revised the Methods section to explicitly state this procedure:

      “After feeding, the eggs deposited on the infested tobacco leaves were removed. The leaves showing no visible insect contamination were immediately frozen in liquid nitrogen and ground to a fine powder.”

      (2) Inconsistent conclusion (Line 156 and Figure 3c):

      The statement in line 156 is inconsistent with the data presented in Figure 3c. The figure clearly shows that the LRR domain of the protein is the one responsible for the interaction with BtRDP, not the region mentioned in the text. This is a critical misrepresentation of the experimental findings and must be corrected. The conclusion in the text should accurately reflect the data from the figure.

      We apologize for any confusion caused by the original phrasing. In our previous manuscript, the description “NtRLP4 without signal peptides and transmembrane domains” referred specifically to the truncated construct NtRLP4<sub>(23-541)</sub> used in the experiment. To prevent any misunderstanding, we have revised the sentence in the updated version to state explicitly: “Point-to-point Y2H assays reveal that NtRLP4<sub>(23-541)</sub> (a truncated version lacking the signal peptide and transmembrane domains) interacts with BtRDP<sup>-sp</sup>”.

      (3) Role of SOBIR1 in the RLP4/SOBIR1 Complex:

      The authors demonstrate that the salivary effectors destabilize the RLP4 receptor, leading to a decrease in its protein levels and a reduction in the RLP4/SOBIR1 complex. A key question remains regarding the fate of SOBIR1 within this complex. The authors should clarify what happens to the SOBIR1 protein after the destabilization of RLP4. Does SOBIR1 become unbound, targeted for degradation itself, or does it simply lose its function without RLP4? This would provide further insight into the mechanism of action of the effectors.

      Thank you for suggestion. In the current version, we assessed the impact of BtRDP on NtSOBIR1 following NtRLP4 destabilization. The results showed that while the NtRLP4-myc accumulation was markedly reduced, NtSOBIR1-flag levels remained unchanged, suggesting that destabilization of NtRLP4 did not affect NtSOBIR1 accumulation.

      (4) Clarification on specificity and evolutionary claims:

      The paper's most significant claim is that the effectors from both whiteflies and planthoppers "independently evolved" to target RLP4. While the functional data is compelling, this evolutionary claim would be more convincing with stronger evidence. Showing that two different effector proteins target the same host protein is a fascinating finding but without a robust phylogenetic analysis, the claim of independent evolution is not fully supported. It would be valuable to provide a more detailed evolutionary analysis, such as a phylogenetic tree of the effector proteins, showing their relationship to other known insect proteins, to definitively rule out a shared, but highly divergent, common ancestor.

      We appreciate the reviewer’s valuable suggestion to investigate a potential evolutionary link between BtRDP and NlSP104. Our initial analysis already indicated no detectable sequence similarity. To address this point more thoroughly, we attempted a phylogenetic analysis. However, we were unable to generate a meaningful alignment due to a complete lack of conserved amino acid sequences. Therefore, we conducted a comparative genomics analysis by blasting both proteins against the genomic or transcriptomic data of 30 diverse insect species. This analysis revealed that RDP is exclusively present in Aleyrodidae species, and SP104 is exclusively present in Delphacidae species (Table S1). Taken together, the absence of sequence similarity, their distinct protein structure, and their lineage-specific distributions, we conclude that BtRDP and NlSP104 are highly unlikely to be homologous and thus did not originate from a common ancestor.

      (5) Role of SOBIR1 in the interaction:

      The results suggest that the effectors disrupt the RLP4/SOBIR1 complex. It is not entirely clear if the effectors are specifically targeting RLP4, SOBIR1, or both. Further experiments, such as a co-immunoprecipitation assay with just RLP4 and the effector, could clarify if the effector can bind to RLP4 in the absence of SOBIR1. This would help to definitively place RLP4 as the primary target.

      We appreciate the reviewer’s insightful comments regarding whether the effector preferentially targets RLP4, SOBIR1, or both. In our study, we conducted reciprocal co-immunoprecipitation assays using RLP4 and BtRDP as controls. These assays showed that BtRDP interacts with RLP4 but does not interact with SOBIR1, supporting the conclusion that SOBIR1 is unlikely to be a direct target of BtRDP. We fully agree that testing the interaction between RLP4 and BtRDP in the absence of SOBIR1 would further strengthen the conclusion. However, we were unable to obtain N. tabacum SOBIR1 knockout mutants, and therefore could not experimentally assess whether the RLP4–BtRDP interaction persists in planta without SOBIR1. Nevertheless, our yeast two-hybrid assays demonstrate that RLP4 and BtRDP can directly interact, indicating that their association does not strictly depend on SOBIR1. Together, these results support the interpretation that RLP4 is the primary target of BtRDP, while SOBIR1 is not directly engaged by the effector.

      (6) Transcriptome analysis (Lines 130-143):

      The transcriptome analysis section feels disconnected from the rest of the manuscript. The findings, or lack thereof, from this analysis do not seem to be directly linked to the other major conclusions of the paper. This section could be removed to improve the manuscript's overall focus and flow. If the authors believe this data is critical, they should more clearly and explicitly connect the conclusions of the transcriptome analysis to the core findings about the effector-RLP4 interaction.

      Thank you for suggestion. As you and Reviewer #2 pointed, the transcriptomic analysis did not closely link to the major conclusions of the paper, and we got little information from the transcriptomic analysis. Therefore, we remove these analyses to improve the manuscript’s overall focus and flow.

      (7) Signal peptide experiments (Lines 145 and beyond):

      The experiments conducted with the signal peptide (SP) are questionable. The SP is typically cleaved before the protein reaches its final destination. As such, conducting experiments with the SP attached to the protein may have produced biased observations and could lead to unjustified conclusions about the protein's function within the plant cell. We suggest the authors remove the experiments that include the signal peptide.

      Thank you for pointing out this. The SP was retained to direct the target proteins to the extracellular space of plant cells. Theoretically, the SP is cleaved in the mature protein. This methodology is widely used in effector biology. For example, the SP directs Meloidogyne graminicola Mg01965 to the apoplast, where it functions in immune suppression, whereas Mg01965 without the SP fails to exert this function (10.1111/mpp.12759). In our study, the SP of BtRDP was expected to guide the target protein to the extracellular space, facilitating its interaction with RLP4. Moreover, the observed protein sizes of BtRDP with and without the SP in transgenic plants were identical, suggesting successful SP cleavage. Therefore, we have retained the experiments involving the SP in the current version.

      (8) Overly strong conclusion and unclear evidence (Line 176):

      The use of the word "must" on line 176 is very strong and presents a definitive conclusion without sufficient evidence. The authors state that the proteins must interact with SOBIR1, but they do not provide a clear justification for this claim. Is SOBIR1 the only interaction partner for NtRLP4? The authors should provide a specific reason for focusing on SOBIR1 instead of demonstrating an interaction with NtRLP4 first. Additionally, do BtRDP or NlSP694 also interact with SOBIR1 directly? The authors should either tone down their language to reflect the evidence or provide a clearer justification for this strong claim.

      Thank you for pointing this out. In the current version, the word “must” has been toned down to “may” due to insufficient supporting evidence. In this study, SOBIR1 was chosen because it has been widely reported to be required for the function of several RLPs involved in innate immunity. However, it remains unclear whether SOBIR1 is the only interaction partner of NtRLP4. In the current version, we have clarified the rationale for focusing on SOBIR1 prior to the experiments “The receptor-like kinase SOBIR1, which contains a kinase domain, has been widely reported to be required for the function of RLPs involved in innate immunity (Gust & Felix, 2014)” and discussed that “Although NtRLP4 interacts with SOBIR1, this alone does not confirm that it operates strictly through this canonical module. Evidence from other RLPs shows that co-receptor usage can be flexible, and some RLPs function partly or conditionally independent of SOBIR1. Therefore, a more definitive assessment of NtRLP4 signaling will therefore require genetic dissection of its co-receptor dependencies, including but not limited to SOBIR1.”. In addition, the direct interaction between BtRDP and SOBIR1 was experimentally tested, and the results showed that BtRDP failed to interact with SOBIR1.

      Minor Comments

      (9) The statement in the abstract, "However, it remains unclear how these invaders are able to overcome receptor perception and disable the plant signaling pathways," is not entirely accurate. The fields of effector biology and host-pathogen interactions have provided significant insight into how pathogens and pests manipulate both Pattern-Triggered Immunity (PTI) and Effector-Triggered Immunity (ETI). While the specific mechanism described in this paper is novel, the broader claim that the field is unclear on these processes weakens the initial hook of the paper. A more precise framing of the problem would be beneficial, perhaps by stating that the specific mechanisms used by these particular herbivores to target RLP4 were previously unknown.

      Thank you for this insightful comment. We agree that the original statement in the abstract overstated the lack of understanding in the field. In the current version, we have refined the sentence to more accurately reflect the current state of knowledge, emphasizing that while microbial suppression of plant immunity has been extensively studied, the strategies used by herbivorous insects to overcome receptor-mediated defenses remain less understood. The revised sentence now reads as follows: “Although the mechanisms used by microbial pathogens to suppress plant immunity are well studied, how herbivorous insects overcome receptor-mediated defenses remains unclear”.

      (10) The introduction is heavily focused on Pattern Recognition Receptors (PRRs), which, while central to the paper's findings, gives a somewhat narrow view of the plant's defense against herbivores. It would be beneficial to briefly acknowledge the broader context of plant defenses, such as physical barriers, direct chemical toxicity, and indirect defenses, before narrowing the focus to the specific molecular interactions of PRRs that are the core of this study. This would provide a more complete picture of the "arms race" between plants and herbivores.

      Thank you for this valuable suggestion. We agree that the original introduction focused too narrowly on pattern-recognition receptors (PRRs). In the current version, we have expanded the introductory section to provide a broader overview of plant defense mechanisms. Specifically, we now acknowledge the multiple layers of plant defenses, including physical barriers (e.g., cuticle and cell wall), chemical defenses (e.g., toxic secondary metabolites and anti-nutritive compounds), and indirect defenses mediated by herbivore-induced volatiles. This addition provides a more complete context for understanding the molecular interactions discussed in this study. The revised paragraph now reads as follows: “Plants have evolved sophisticated defense systems to survive constant attacks from pathogens and herbivorous insects. These defenses operate at multiple levels, including physical barriers such as the cuticle and cell wall, chemical defenses involving toxic secondary metabolites and anti-nutritive compounds, and indirect defenses that attract natural enemies of herbivores through the emission of herbivore-induced volatiles. Beyond these general strategies, plants also rely on highly specialized molecular immune responses that allow them to detect and respond rapidly to invaders.”

      (11) The figure legends are generally clear, but some could be more detailed. For instance, in Figure 2, it would be helpful to explicitly state what each bar represents in the graph and to include the statistical test used. Please ensure all panels in all figures have clear labels.

      Thank you for this helpful suggestion. We have revised the legend of Fig. 2 and other figures to provide more detailed information for each panel. Specifically, we now explicitly describe what each bar represents in the graphs and specify the statistical test used. In addition, we ensured that all panels are clearly labeled. These changes improve clarity and allow readers to better interpret the data.

      (12) The methods section is comprehensive, but it would be helpful to include more specifics on the statistical analyses used. For example, the type of statistical test (e.g., t-test, ANOVA) and the software used should be mentioned for each experiment.

      Thank you for your suggestion. We have revised the Methods section (Statistical analysis) to provide more detailed information on the statistical analysis used for each experiment.

      (13) The manuscript's overall impact is weakened by the inclusion of unnecessary words and a few grammatical issues. A focused revision to tighten the language would make the major findings stand out more clearly. For example, on page 2, line 18, "in whitefly Bemisia tabaci, BtRDP is an Aleyrod..." seems to have an incomplete sentence. A thorough proofreading for typos and grammatical errors is highly recommended to improve the overall readability.

      Thank you for your suggestion. We have carefully revised the abstract and the manuscript to improve clarity, readability, and grammatical correctness. In addition, we sought the assistance of a professional English editor to thoroughly proofread and polish the manuscript, ensuring that the language meets high academic standards.

      (14) The discussion section is strong, but it could benefit from a more explicit connection between the findings and the broader ecological implications. For instance, how might the independent evolution of these effectors in different insect species impact plant-insect co-evolutionary dynamics?

      We thank the reviewer for the valuable suggestion. In the current version, we have added a paragraph in the Discussion section highlighting the broader ecological and evolutionary implications of our findings. Specifically, we discuss how the independent evolution of RLP4-targeting effectors in different insect lineages may drive plant-insect co-evolution, influence selection pressures on both plants and herbivores, and potentially shape defense diversification across plant communities. This addition helps to link our molecular findings to ecological outcomes and co-evolutionary dynamics.

      (15) The sentence on line 98, which reads " A few salivary proteins have been reported to attach to salivary sheath after secretion" seems to serve an unclear purpose in the introduction. It would be helpful for the authors to clarify its relevance to the surrounding context or to the paper's overall argument. Its inclusion currently disrupts the flow of the introduction and makes it difficult for the reader to understand its intended purpose.

      We thank the reviewer for the comment. We have revised the paragraph to clarify the relevance of salivary sheath localization to the study. Specifically, we now introduce the role of the salivary sheath as a potential scaffold for effector delivery and explicitly link previous reports of sheath-associated salivary proteins to our observation that BtRDP localizes to the salivary sheath after secretion.

      (16) The writing in lines 104-106 is both grammatically inconsistent and overly wordy. The authors switch between present and past tense ("is" and "was"), and the sentences could be made more concise to improve the clarity and flow of the text. Also check entire paper.

      We thank the reviewer for pointing this out. We have revised the sentence to improve grammatical consistency and clarity, and also checked the manuscript for similar issues. The sentence is now split into two concise statements. In addition, we have thoroughly checked the entire manuscript for similar tense inconsistencies and overly wordy sentences, and have made revisions throughout to ensure consistent past tense usage and improved readability.

      (16) The sentences on lines 111-113 are quite wordy. The core conclusion, which is that the protein affects the insect's feeding probe, could be expressed more simply and directly to improve clarity and flow. I suggest rephrasing this section to be more concise and to highlight the primary finding without the added language.

      We thank the reviewer for the helpful suggestion. We have revised the sentences to make them more concise and to emphasize the main finding that BtRDP influences the whitefly’s feeding behavior as follow: “Compared with the dsGFP control, dsBtRDP-treated B. tabaci showed a marked reduction in phloem ingestion and a longer pathway duration, indicating that BtRDP is required for efficient feeding (Fig. 2c).”

      (17) On line 118, the authors mention "subcellular location." It is not clear where the protein is localized. The authors should explicitly state the specific subcellular compartment of the protein, as this is crucial for understanding its function and interaction with other proteins.

      We thank the reviewer for this valuable comment. To clarify the subcellular localization of BtRDP, we have revised the manuscript accordingly. The transgenic line overexpressing the full-length BtRDP including the signal peptide (oeBtRDP) is expected to localize in the apoplast (extracellular space), whereas the line expressing BtRDP without the signal peptide (oeBtRDP<sup>-sp</sup>) is likely retained in the cytoplasm.

      (18) Lines 121-128, the description of the fecundity and choice assays in this section is overly wordy. The authors should present the main conclusion of these experiments more directly and concisely. The key finding is that the protein affects feeding behavior; this central point is somewhat lost in the detailed, and sometimes repetitive, phrasing.

      We thank the reviewer for this suggestion. In the revised manuscript, we have simplified the description of the fecundity and two-choice assays to highlight the main conclusion as follow: “Fecundity and two-choice assays showed that BtRDP, whether localized in the apoplast (oeBtRDP) or cytoplasm (oeBtRDP<sup>-sp</sup>), enhanced whitefly settling and oviposition compared with EV controls (Fig. 2d-i; Fig. S10), indicating that BtRDP promotes whitefly feeding behavior regardless of its subcellular location.”

      (19) Line 148, the manuscript mentions experiments involving transformation, but the transformation efficiency is not provided. Please include the transformation efficiency for all transformation experiments, as this is crucial for the reproducibility of the results.

      We thank the reviewer for raising this point. We would like to clarify that no transformation experiments were performed in this section. The experiments described involved Y2H screening using BtRDP<sup>-sp</sup> as a bait to identify interacting proteins from a N. benthamiana cDNA library. Therefore, there is no transformation efficiency to report.

      (20) Line 159, the manuscript refers to a sequence similarity around line 159 but does not provide the specific data. It is important to show the actual sequence similarity, perhaps in a supplementary figure or table, to support the claims being made.

      We thank the reviewer for this suggestion. To support our statement regarding sequence similarity, we have added the corresponding alignment figure in the Fig. S11.

      (21) Line 159, the manuscript refers to "three randomly selected salivary proteins." It is unclear from where these proteins were selected. The authors should clarify the source of this selection (e.g., a specific database or a previous study) to ensure the methodology is transparent and the results are reproducible.

      We thank the reviewer for raising this point. These proteins were selected based on previously reports (10.1093/molbev/msad221; 10.1111/1744-7917.12856). In the current version, we provide the accession of these proteins in the MS.

      (22) Line 160, the description "NtcCf9 without signal peptide and transmembrane domains" is difficult to understand. It would be clearer and more consistent to use a term like "truncated NtcCf9" and then specify which domains were removed, as this is a standard practice in molecular biology for describing protein constructs.

      We thank the reviewer for this suggestion. We have revised the manuscript to describe the construct as “truncated NtCf9” and specified that the signal peptide and transmembrane domains were removed

      (23) The phrase "incubated with anti-flag beads" on line 172 is a detail of a routine method. Such details are more appropriate for the Methods section rather than the main text, which should focus on the results and their implications. Please remove such descriptions from the main text to improve readability and flow.

      We thank the reviewer for this suggestion. We have removed the methodological detail from the main text to improve readability. We also check this throughout the MS.

      I am excited about the potential of this work and look forward to seeing the current version.

      We sincerely thank the reviewer for the positive feedback and encouragement. We appreciate your time and thoughtful comments.

      Reviewer #2 (Public review):

      Summary:

      The authors tested an interesting hypothesis that white flies and planthoppers independently evolved salivary proteins to dampen plant immunity by targeting a receptor-like protein.

      Strengths:

      The authors used a wide range of methods to dissect the function of the white fly protein BtRDP and identify its host target NtRLP4.

      Thank you very much for your comments. We have carefully revised the MS following your valuable suggestions and comments.

      Weaknesses:

      (1) Serious concerns about protein work.

      I did not find the indicated protein bands for anti-BtRDP in Figures 1a and 1b in the original blot pictures shown in Figure S30. In Figure 1a, I can't get the point of showing an unspecific protein band with a size of ~190 kD as a loading control for a protein of ~ 30 kD.

      The data discrepancy led me to check other Western blot pictures. Similarly, Figures 2d, 3b, 3d, and S15b (anti-Myc) do not correspond to the original blots shown. In addition, the anti-Myc blot in Figure 4i, all blot pictures in Figures 5b, 5h, and S19a appeared to be compressed vertically. These data raised concerns about the quality of the manuscript.

      Blots shown in Figure 3d, 4f, 4g, and 4h appeared to be done at a different exposure rate compared to the complete blot shown in Figure S30. The undesirable connection between Western blot pictures shown in the figures and the original data might be due to the reduced quality of compressed figures during submission. Nevertheless, clarification will be necessary to support the strength of the data provided.

      We sincerely thank the reviewer for carefully examining our Western blot data and for pointing out these inconsistencies. The discrepancy between the figures in the main text and the original blots (Figure S30) resulted from an oversight during manuscript revision. This manuscript had undergone multiple rounds of revision after submission to another journal. During this process, the main figures and supplementary figures were updated separately, and we mistakenly failed to replace the original blot files with the corresponding current versions.

      For the different exposure rate, the blots shown in the main text were adjusted for overall contrast and brightness to enhance band visibility and presentation clarity, whereas the original images in Figure S30 were raw, unprocessed scans directly from the imaging system. For example, in the Author response image 1 below, to visualize the loading of the input sample, the output figure was adjusted for overall contrast and brightness. This was acceptable for image processing (https://www.nature.com/nature-portfolio/editorial-policies/image-integrity)

      Author response image 1.

      The same figure with brightness and contrast changes across the entire image.

      For the vertical compression, in the previous version, some images were vertically compressed for layout purposes to make the composite figures appear more visually balanced. However, after consulting relevant publication guidelines, we realized that such one-dimensional compression is not encouraged by certain journals as it may alter the original aspect ratio of the image. Therefore, in the manuscript, we have avoided any non-proportional scaling and retained the original aspect ratio of all images.

      We have now carefully rechecked all Western blot data, replaced the outdated raw blot images with the correct corresponding ones, avoid vertical compression, and ensured that the processed figures in the main text match their original data. The revised supplementary figures now accurately reflect the raw experimental results.

      (2) Misinterpretation of data.

      I am afraid the authors misunderstood pattern-triggered immunity through receptor-like proteins. It is true that several LRR-type RLPs constitutively associate with SOBIR1, and further recruit BAK1 or other SERKs upon ligand binding. One should not take it for granted that every RLP works this way. To test the hypothesis that NtRLP4 confers resistance to B.tabaci infestation, the author compared transcriptional profiles between an EV plant line and an RLP4 overexpression line. If I understood the methods and figure legends correctly, this was done without B. tabaci treatment. This experimental design is seriously flawed. To provide convincing genetic evidence, independent mutant lines (optionally independent overexpression lines) in combination with different treatments will be necessary. Otherwise, one can only conclude that overexpressing the RLP4 protein generated a nervous plant. In addition, ROS burst, but not H2O2 accumulation, is a common immune response in pattern-triggered immunity.

      We agree with the reviewer that not every RLP functions through the same mechanism as the canonical SOBIR1–BAK1 pathway. In the current version, we further examined the interaction between the whitefly salivary protein and SOBIR1, and found that they do not interact. However, our interaction assays clearly demonstrated that NtRLP4 does interact with SOBIR1. Whether NtRLP4 functions through, or exclusively through, SOBIR1 remains uncertain, and we have emphasized this limitation in the Discussion section as follow: “Although NtRLP4 interacts with SOBIR1, this alone does not confirm that it operates strictly through this canonical module. Evidence from other RLPs shows that co-receptor usage can be flexible, and some RLPs function partly or conditionally independent of SOBIR1 [39]. Therefore, a more definitive assessment of NtRLP4 signaling will therefore require genetic dissection of its co-receptor dependencies, including but not limited to SOBIR1.”

      Regarding the transcriptome analysis, our original aim was to explore why B. tabacishowed such a pronounced preference among tobacco plants. As this preference was assessed using uninfested plants, we also performed transcriptome sequencing using plants without B. tabaci treatment. The enrichment analysis demonstrated that the majority of up-regulated DEGs were associated with plant–pathogen interaction, environmental adaptation, MAPK signaling, and signal transduction pathways, while down-regulated DEGs were enriched in glutathione, carbohydrate, and amino acid metabolism. Notably, many DEGs were annotated as RLK/RLPs or WRKY transcription factors, most of which were upregulated, suggesting an enhanced defense state in the NtRLP4-overexpressing plants. The altered expression of JA- and SA-related genes (e.g., upregulation of FAD7 and downregulation of PAL and NPR1) further supported this enhanced defense and hormonal crosstalk. We agree that combining overexpression or knockout lines with insect infestation treatments would provide more direct genetic evidence for NtRLP4-mediated resistance, and we have acknowledged this as an important future direction. Nevertheless, our current data are consistent with the conclusion that NtRLP4 overexpression confers increased resistance to B. tabaci infestation.

      Finally, DAB staining for H<sub>2</sub>O<sub>2</sub> accumulation is also a well-established indicator of PTI responses, and many studies have shown that overexpression of salivary elicitors can trigger such accumulation.

      (3) Lack of logic coherence.

      The written language needs substantial improvement. This impeded the readability of the work. More importantly, the logic throughout the manuscript appeared scattered. The choice of testing protein domains for protein-protein interactions, using plants overexpressing an insect protein to study its subcellular localization, switching back and forth between using proteins with signal peptides and without signal peptides, among others, lacks a clear explanation.

      We appreciate the reviewer’s careful reading and valuable comments regarding the logical coherence of our manuscript.

      (1) To improve the English quality, the entire manuscript has been professionally edited by a certified language-editing service.

      (2) Regarding the rationale for testing protein domains in the protein–protein interaction assays: NtRLP4 is a membrane-anchored receptor-like protein composed of extracellular, transmembrane, and short intracellular domains. We aimed to determine which region of NtRLP4 is responsible for interacting with the salivary protein, as this would help infer the likely site of interaction in planta. In addition, not all RLPs contain a malectin-like domain, and we sought to verify whether the BtRDP–NtRLP4 interaction depends on this domain. To enhance the logical flow, we introduced a brief statement explaining the experimental purpose before presenting the interaction assays in the current version as follow: “These findings raised the question of which domain of NtRLP4 is responsible for binding BtRDP, as identifying the interacting domain could help infer where the salivary protein contacts the receptor in planta. We therefore dissected the NtRLP4 domains accordingly.”

      (3) With respect to using plants overexpressing an insect protein to examine subcellular localization: since both the brown planthopper and the whitefly are non-model species for which stable genetic transformation is technically unfeasible, many previous studies have used Agrobacterium-mediated transient expression or transgenic plant systems to investigate the subcellular localization of insect salivary proteins within host cells. Following these precedents, our study also employed plant systems to determine the localization of the insect protein and to assess how different localizations affect plant defense responses.

      (4) As for switching between constructs with or without signal peptides: the subcellular localization of effectors can influence their biological activity and interactions. Previous studies have used the presence or absence of signal peptides, or replacement with a PR1 signal peptide, to direct protein targeting (for example, Frontiers in Plant Science, 2022, 13:813181). Because salivary sheaths are generally considered to localize in the apoplastic space, we generated two transgenic N. tabacum lines overexpressing BtRDP: one carrying the full-length coding sequence including the signal peptide (oeBtRDP), expected to be secreted into the apoplast, and another lacking the signal peptide (oeBtRDP-sp), likely retained in the cytoplasm. In the current version, we clarified this rationale and added references to similar studies to improve the manuscript’s logic and readability. Details are as follow: “To investigate the role of BtRDP in different subcellular location of host plants, we constructed two transgenic N. tabacum lines overexpressing BtRDP: one carrying the full-length coding sequence including the signal peptide (oeBtRDP), which is expected to be secreted into the apoplast (extracellular space), and the other lacking the signal peptide (oeBtRDP<sup>-sp</sup>), which is likely retained in the cytoplasm.”

      Reviewer #3 (Public review):

      Summary:

      In this study, Wang et al. investigate how herbivorous insects overcome plant receptor-mediated immunity by targeting plant receptor-like proteins. The authors identify two independently evolved salivary effectors, BtRDP in whiteflies and NlSP694 in brown planthoppers, that promote the degradation of plant RLP4 through the ubiquitin-dependent proteasome pathway. NtRLP4 from tobacco and OsRLP4 from rice are shown to confer resistance against herbivores by activating defense signaling, while BtRDP and NlSP694 suppress these defenses by destabilizing RLP4 proteins.

      Strengths:

      This work highlights a convergent evolutionary strategy in distinct insect lineages and advances our understanding of insect-plant coevolution at the molecular level.

      Thank you very much for your comments. We have carefully revised the MS following your valuable suggestions and comments.

      Weaknesses:

      (1) I found the naming of BtRDP and NlSP694 somewhat confusing. The authors defined BtRDP as "B. tabaci RLP-degrading protein," whereas NlSP694 appears to have been named after the last three digits of its GenBank accession number (MF278694, presumably). Is there a standard convention for naming newly identified proteins, for example, based on functional motifs or sequence characteristics? As it stands, the inconsistency makes it difficult for readers to clearly distinguish these proteins from those reported in other studies.

      Thank you for your comment. These are species-specific salivary proteins that have not been reported or annotated in previous studies. Because no homologous genes could be identified in other species, there are no existing names or annotations for these proteins. For such lineage-specific salivary proteins, it is common in recent studies to name them according to their experimentally identified functions. For example, a recently reported salivary protein was named SR45-interacting salivary protein (SISP) based on its function (10.1111/nph.70668). Following this convention, we adopted a similar functional naming strategy in this study. We acknowledge that there may not yet be a standardized rule for naming such proteins, and we would be glad to follow a more authoritative naming guideline if possible.

      (2) Figure 2 and other figures. Transgenic experiments require at least two independent lines, because results from a single line may be confounded by position effects or unintended genomic alterations, and multiple lines provide stronger evidence for reproducibility and reliability.

      We appreciate the reviewer’s suggestion. In our study, two independent transgenic lines were used to ensure the reproducibility and reliability of the results. One representative line was presented in the main figures, while data from the second independent line were included in the supplementary figures. To make this clearer, we have emphasized in the manuscript that bioassays were conducted using two independent transgenic lines.

      (3) Figure 3e. Quantitative analysis of NtRLP4 was required. Additionally, since only one band was observed in oeRLP, were any tags included in the construct?

      Thank you for your comment. In the current version, quantitative analysis of NtRLP4 expression has been performed and is now presented in Figure 3. For the oeRLP plants, no tag was fused to NtRLP4; thus, anti-RLP serum was used to detect the target bands. In contrast, oeBtRDP and oeBtRDP-sp were fused with C-terminal FLAG tags, and their detection was carried out using anti-FLAG serum. This information has been clarified in the revised Methods section as follows: “The oeBtRDP and oeBtRDP<sup>-sp</sup> were fused with C-terminal FLAG tags, while no tag was fused to oeNtRLP4.”

      (4) Figure 4a. The RNAi effect appears to be well rescued in Line 1 but poorly in Line 2. Could the authors clarify the reason for this difference?

      Thank you for pointing this out. We also noticed that the RNAi effect appeared to be better rescued in Line 2 than in Line 1. Based on our measurements, the silencing efficiency of NtRLP4 in RNAi-RLP4 Line 1 was markedly weaker than in Line 2, which likely explains the difference in rescue efficiency. In the current version, we have clarified this point as follows: “Both RNAi-RLP lines showed reduced NtRLP4 levels compared with EV plants, with RNAi-RLP#2 exhibiting a stronger silencing effect (Fig. S19a).” “The differential rescue effect between the two RNAi lines likely resulted from their different NtRLP4 silencing efficiencies, with the lower NtRLP4 level in RNAi-RLP#2 leading to a more complete rescue phenotype.”

      (5) ROS accumulation is shown for only a single leaf. A quantitative analysis of ROS accumulation across multiple samples would be necessary to support the conclusion. The same applies to Figure 16f.

      Thank you for pointing this out. The H<sub>2</sub>O<sub>2</sub> accumulation experiments have been repeated for 5 times in Figure 4 and Figure S16f. In the current version, we addressed that “the experiment is repeated five times with similar results” in the figure legends.

      (6) Figure 4f: NtRLP4 abundance was significantly reduced in oeBtRDP plants but not in oeBtRDP-SP. Although coexpression analysis suggests that BtRDP promotes NtRLP4 degradation in an ubiquitin-dependent manner, the reduced NtRLP4 levels may not result from a direct interaction between BtRDP and NtRLP4. It is possible that BtRDP influences other factors that indirectly affect NtRLP4 abundance. The authors should discuss this possibility.

      Thank you for your valuable suggestion. We agree that the reduced NtRLP4 abundance may not necessarily result from a direct interaction between BtRDP and NtRLP4. In the manuscript, we have further discussed this possibility as follows: “Notably, BtRDP and NlSP104 shared no sequence or structural similarity and lack resemblance to known eukaryotic ubiquitin-ligase domains. Their interaction with RLP4s occurs in the extracellular space (Fig. 3d; Fig. 5c), whereas the ubiquitin-proteasome system primarily functions in the cytosol and nucleus [46]. Furthermore, NtRLP4 reduction is observed only in oeBtRDP transgenic plants, not in oeBtRDP-sp plants (Fig. 4f), suggesting that BtRDP exerts its influence on NtRLP4 in the extracellular space. These observations collectively argue against the possibility that BtRDP or NlSP694 possesses intrinsic E3 ligase activity capable of directly ubiquitinating RLP4s within plant cells. Importantly, the reduced NtRLP4 levels may not result from a direct physical interaction between BtRDP and NtRLP4. Instead, BtRDP may indirectly affect RLP4 post-translational modification, thereby accelerating its degradation, which warrants further investigation”

      (7) The statement in lines 335-336 that 'Overexpression of NtRLP4 or NtSOBIR1 enhances insect feeding, while silencing of either gene exerts the opposite effect' is not supported by the results shown in Figures S16-S19. The authors should revise this description to accurately reflect the data.

      Thank you for pointing this out. We agree that our original statement was not precise, as we measured the insect settling preference and oviposition on transgenic plants, but did not directly assess the feeding behavior of B. tabaci. Therefore, we have revised the description in the manuscript to more accurately reflect our data as follows: “Overexpression of NtRLP4 or NtSOBIR1 in N. tabacum is attractive to B. tabaci and promotes insect reproduction, whereas silencing of either gene exerts the opposite effect.”

      (8) BtRDP is reported to attach to the salivary sheath. Does the planthopper NlSP694 exhibit a similar secretion localization (e.g., attachment to the salivary sheath)? The authors should supplement this information or discuss the potential implications of any differences in secretion localization between BtRDP and NlSP694 for their respective modes of action.

      Thank you for your insightful suggestion. We agree that determining the secretion localization of NlSP694 would provide valuable information for understanding its potential mode of action. Immunohistochemical (IHC) staining is indeed a critical approach for such analysis. However, in this study, we were unable to express NlSP694 in Escherichia coli, and the antibody generated using a synthesized peptide did not show sufficient specificity or sensitivity for IHC detection. Consequently, we were unable to determine whether NlSP694 is attached to the salivary sheath. Therefore, whether BtRDP and NlSP694 acted in different mode require further investigation.

      Recommendations for the authors:

      Reviewer #3 (Recommendations for the authors):

      (1) Figure 1e. The BtRDP-labeled fluorescent signal is difficult to discern. An enlarged view of the target region would be helpful for clarity.

      Thank you for your suggestion. In the current version, an enlarged view of the target region was provided below the figure.

      (2) The finding that BtRDP accumulates in the salivary sheath secreted by Bemisia tabaci is important for understanding the subcellular localization of this protein during actual insect feeding. I suggest moving Figure S5 to the main text.

      Thank you for your suggestion. Figure S5 has been moved to Fig. 1f in the current version.

      (3) Please carefully cross-check the figure numbering to ensure that all in-text citations correspond to the correct figures and panels. i.e., lines 136,188,192, and 194.

      Thank you for pointing this out. We corrected them in the current version.

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      Referee #2

      Evidence, reproducibility and clarity

      In their study, Haghighi et al. seek to build upon prior literature linking alterations in mitochondrial network distribution with various kinds of psychosis. Correlations between subcellular mitochondrial localization and different psychological states is an interesting and potentially fruitful frontier and should be explored; however, despite their ambitious strategy to screen 168 skin fibroblasts from patients experiencing psychosis, and examine various online image databases, there is a concerning number of issues related to the image-analysis approach. The foremost of these is a lack of direct measures of mitochondrial distribution, which might serve to validate their proposed MITO-SLOPE protocol. There is also a worrisome lack of robust controls, which are critical in light of how admittedly subtle some of the distribution phenotypes may be. Overall, the aim to screen differences in mitochondrial distribution is a laudable goal and, in the context of psychological disorders, could be helpful in identifying new therapeutic targets; but the methodology employed in this study does not seem to be sufficiently rigorous to be able to leverage this approach for screening purposes.

      I have extensive experience investigating mitochondria with advanced imaging technologies, including super-resolution microscopy as well as high-throughput and 4D imaging modalities. I am also familiar with standard as well as machine-learning approaches for quantifying mitochondrial morphology as well as distribution or trafficking. In my opinion, this study requires substantial revision, both in terms of the indirect and often opaque image-analysis pipeline as well as the inclusion of orthogonal experiments, which could serve to lessen concerns regarding purported differences in mitochondrial distribution, which are so difficult to discern as to be imperceptible. It is worth noting, too, that this study appears to be predicated, in many ways, upon a 2010 study (Cataldo et al.) of mitochondria in patients with bipolar disorder, which appears to reflect its own lack of critical controls for cell size.

      Major comments:

      The authors state, in the first paragraph of the results section: "By eye, we observed that samples from patients in the control and MDD categories show a more fine-grained, dispersed mitochondrial network extending to the edges of the cell, whereas patients in the categories experiencing psychosis tend to show an agglomerated, thicker network more concentrated around the nucleus. The pattern is subtle and heterogeneous across a cell population." The pattern is indeed subtle. I am concerned that it is so subtle as to be imperceptible. Firstly, it is important to note that the mitochondrial reticulum in BP, SZ, and SZA is more difficult to differentiate, by eye, because the signal appears to be saturated in places, such that the boundaries of individual mitochondria are indistinguishable due to differences in contrast or possibly from the fluorescence intensity itself. Although the authors indicate in the legend that the intensity of the mitochondrial fluorescence was adjusted "for visual clarity," it appears that the contrast needs to be decreased in the BP, SZ, and SZA conditions. It is also important to note that MitoTrackers load into mitochondria in a membrane-potential-dependent fashion. Did the authors detect differences in membrane potential between these groups? While imaging, was the same laser power and gain utilized from condition to condition? With this being said, it is not clear that mitochondria in control and MDD categories have different morphologies from the other conditions. It is also not clear what "fine-grained" means in this context. Is this a comment on aspect ratio? If so, it would be better to use standard terminology. (Why are there large red circular structures in the nucleus? These are likely not mitochondria, so why are they showing up in the channel with MitoTracker?) It is also not evident that one condition has more dispersed mitochondria than another. Given that the authors appear to be making this a central claim of their manuscript, it would seem appropriate to highlight specifically the regions of the different cells that they believe exhibit meaningful differences. If I attempt to look at the merged image, which is important because it is really the only way that one can gauge the relative distance of the mitochondrial network from the edge of the cell, there would seem to be no obvious differences between the conditions. Another key point that I think important to mention, given that it is frequently referenced in this manuscript, Cataldo et al., 2010 indicate that mitochondria in patient fibroblasts with bipolar disorder (BD) are more perinuclear than those in control. However, a cursory inspection of the images from this study (e.g., Figure 2A-B; Figure 4A-D; and Figure 6A-H) unambiguously demonstrate that the BD cells are smaller than the control cells. Of course, if the cells are smaller, the distance from the nucleus will tend to be shorter. In Cataldo et al., 2010, the authors state, "We also measured cell area, cell length, cell width, and cell perimeter of the fibroblasts used in this analysis to verify that the observed mitochondrial distributional differences were not simply a result of BD cells being smaller, shorter, or fatter. No significant differences in any of these measurements were seen based on diagnosis after two sample t tests." Notably, the data is not shown, so it is difficult to appreciate what the variance of the population of cells from control and BD would look like, but it must be said, nevertheless, that the representative images in this paper all point to the BD cells being smaller. In light of this, it would be helpful if Haghighi et al. could add scale bars to all the images (e.g., in Figure 2), so readers can ascertain whether all the cells are portrayed at the same scale and are of similar areas.

      As the authors indicate, interpretable measures of mitochondrial morphology include values like size and shape. It is concerning, therefore, that Figure 3 purports to identify a number of significantly different mitochondrial "features" in the patient groups experiencing psychosis, but they do not appear to make an effort to clarify how any of these features might reflect ground truths of mitochondrial architecture, which can be understood directly by values such as aspect ratio, circularity, area, number organelles, number of nodes or branching points in a network, etc. Unless the authors can specifically tie their machine-learning classifications to standard mitochondrial shape descriptors, their classifications will remain opaque and therefore of limited credibility or value. One way to improve the validation of their machine-learning classification methods would be to use empirically sound methods for manipulating a mitochondrial morphology and distribution, which could serve as positive or negative controls. For example, treatment of cells with the uncoupler FCCP would induce mitochondrial fragmentation, treatment with cycloheximide results in stress-induced mitochondrial hyperfusion (SIMH), or treatment with Nocodazole would block mitochondrial trafficking. Treating control cells with these chemicals would help to establish baseline measurements for how far the patient cells are deviating from untreated controls, in one direction or another. Such considerations, I think, are especially important when the mitochondrial phenotypes are so subtle. I agree with the authors' argument that, for the purposes of screening, it is best to focus on a single metric. Based on their apparent discernment of the subtle differences in mitochondrial distribution in patients experiencing psychosis, they opted to examine possible differences in network density. To this end, they developed "MITO-SLOPE." Out of multiple categories of features, they highlight the following as the most powerful for establishing differences in mitochondrial network density:

      "(a) A subset of texture measures in the nuclei and cytoplasm area of the mito channel. (b) A subset of features measuring the intensity of the mitochondria area across the cell."

      Within the concentric bins around the cell nuclei, they measure:

      • FracAtD: Fraction of total stain in an object at a given radius.
      • MeanFrac: Mean fractional intensity at a given radius, calculated as the fraction of total intensity normalized by the fraction of pixels at a given radius.
      • RadialCV: Coefficient of variation of intensity within a ring, calculated across 8 slices."

      While the authors have recommended the use of a single metric for purposes of screening, MITO-SLOPE appears to represent a bundle of metrics, which, in the end, do not amount to a clear readout of what is being measured. From my point of view, if one were interested in measuring mitochondrial distribution, then, in an ideal situation, one would measure the average distance of all the mitochondria from the center of the nucleus. And, since the size of the cell is critical for establishing relative distances to the boundaries or periphery of the cell, one would normalize this metric by cellular area. Thus, the readout would be: [average mitochondrial distance from the nuclear center (µm)]/[cellular area (µm2)]. An even simpler metric could be: [average mitochondrial distance from nuclear center (µm)]/[average cytoplasmic radius (µm)]. When talking about mitochondrial distribution, we typically think in terms of where is the mitochondrial network, on average, in relation to the nucleus (perinuclear) or to the edge of the cell (peripheral). By quantifying the actual mean distance of the mitochondrial network in relation to both the nucleus and the bona fide cell extremities, via the metrics I described above, one can obtain direct measurements of the truly meaningful values related to mitochondrial distribution. It seems deviating from these approaches introduces more and more opportunities for confounding variables.

      However, the MITO-SLOPE analysis does not seem to consider this metric. Is this, or a similar variation, not the most direct way to establish differences in the mitochondrial network distribution? I would, of course, at least want to see a discussion of why the authors have not chosen to use the most direct form of quantification for this purely spatial value. Why opt for a multifaceted measurement of a relatively straightforward quantity, when a simpler form of quantification would not only suffice but arguably be more likely to capture the ground truth? With this being said, it is not clear to me why, within MITO-SLOPE there seems to be a reliance on measuring the "intensity" of the mitochondria. (And what intensity is it? Mean intensity per ROI?) Of course, particularly if MitoTrackers were used for staining mitochondria, there will be heterogeneity in fluorescence intensity from organelle to organelle, which introduces potential confounders into the workflow. Furthermore, as indicated above, to know if the subcellular distribution of mitochondria is truly altered, it is essential to know if the cell size has likewise changed. Therefore, any unbiased measure of mitochondrial distribution must take into consideration the size of the cell; however, based on the information provided about MITO-SLOPE, it does not appear that the authors are accounting for possible variations in cell size that might account for alterations in mitochondrial network distribution - i.e., a smaller cell will have a more constrained area in which mitochondria will be able to disperse - thus, not accounting for cell size (area) will yield ambiguous results. For example, how can we know if mitochondrial motility is impaired or if the cell is simply smaller and there is less space in which to move? Another complexity, here, is if the cell boundaries were not accounted for via staining of actin, etc., then establishing a true cell boundary will be very challenging. How many bins are sufficient to capture the whole cell? Just 12? Furthermore, human fibroblasts have a tendency to be quite large (sometimes several hundred microns from end to end); how can the authors account for the whole cell, particularly in cases where part of the cell is beyond the field of view or cells are growing on top of each other, as is often the case?

      In Figure 6, there is no control image that could be used as a frame of reference. I have extensive experience imaging A549 cells. The mitochondria in these images appear to be highly fragmented. The staining patterns, particularly of the cells treated with divalproex-sodium, are quite dim, indicating mitochondrial depolarization. Of course, depolarization affects the fluorescence intensity of mitochondria stained with vital dyes, such as MitoTrackers, which will, in turn, presumably affect the values obtained from MITO-SLOPE, which appear to rely on intensity gradients, rather than more concrete spatial coordinates. Also, as indicated above, it is unclear how the authors are establishing the edges of cells without a marker of the plasma membrane or cytoskeleton.

      The authors note that "Divalproex-sodium is a benzodiazepine receptor agonist and HDAC inhibitor (Rahman et al. 2025) used to manage a variety of seizure disorders (Willmore 2003) and bipolar disorder(Bond et al. 2010; Cipriani et al. 2013); it shows a positive MITO-SLOPE which is the direction expected to normalize the centralized mitochondrial localization associated with psychosis." Insofar as this recommends the drug for use in "normalizing" perinuclear mitochondria within neurons, it would seem only prudent to mention that this drug also appears to induce mitochondrial depolarization and fragmentation, which are both associated with a range of severe human pathologies. I would caution the authors to not highlight one potential benefit while omitting an obvious side effect involving what appears to be significant perturbation of mitochondrial structure and function. What is the point of normalizing mitochondrial distribution if the mitochondria being redistributed are dysfunctional?

      The authors note, in Figure 7, that their MITO-SLOPE analysis was unable to discern a statistically significant difference in cells with specific knockouts of genes associated with mitochondrial trafficking. If the MITO-SLOPE cannot discern a difference in the context of a substantial abrogation of mitochondrial transport capacity, how is it that it could detect meaningful differences where there is only a "subtle" change in distribution? This result would seem to militate strongly against the efficacy of this analysis pipeline and would not recommend its use for unbiased screening and discovery.

      Minor comments:

      For Figure 6 b and c, "µm" should be "µM."

      The introduction and discussion could be more concise.

      Significance

      This study attempts to fill an important gap in knowledge relating to mitochondrial distribution and psychological disorders. It aims to perform an initial screen to try to validate a novel analysis pipeline called MITO-SLOPE, however, the study appears to lack analytical rigor, both in terms of the underlying cell biology together with the approach for quantification, itself. Conceptually, this study has great promise, but the authors will need to improve their pipeline prior to publication, which will likely require fundamental revisions, including an array of orthogonal measures (largely lacking here) as well as detailed demonstrations of how the segmentation actually works and ultimately yields data reflecting demonstrable mitochondrial trafficking/distribution defects.

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      Referee #1

      Evidence, reproducibility and clarity

      Summary:

      The manuscript by Haghighi and McPhie et al. builds upon their previous findings by exploring the mitochondrial localization as a disease-associated phenotype in mental disorders, particularly in psychotic disorders. They recruited a cohort of patients diagnosed with schizophrenia, schizoaffective disorder, bipolar disorder and MDD. By taking advantage of skin biopsies, they screened patient-derived fibroblasts for aberrant mitochondrial localization and morphology using common staining techniques. Then, they use a machine learning approach to classify patients into their respective groups, which was effective for BP, SZA and pooled psychotic patients. Authors then develop a single feature for phenotyping, Mito-SLOPE, a metric of mitochondria density distribution across a cell by radial areas. With this metric, psychotic patients tend to have more nuclear-localized than edge-localized mitochondria; whereas MDD patients show a trend for higher edge-to-nucleus distribution. To find candidate drugs, authors screen publicly available datasets of cells treated with small compounds using mito-SLOPE. Furthermore, authors then apply mitoSLOPE on a CRISPR screen dataset, showcasing the role of mitochondrial dynamics genes and three genes of interest because of their association with psychosis. Finally, they identified the top genes whose KO or overexpression may explain (or reverse) the mitoSLOPE phenotype.

      Overall, the manuscript is well-written, the conclusions are supported within their limitations and this work represents an advancement in the field. I recommend it for publication provided these concerns are addressed:

      Major comments:

      1. The mitoSLOPE measure is very interesting and most likely reflects a subtle changes in mitochondrial transport. How does the microtubule network look like in the patient fibroblasts, are there obvious alterations in e.g. their posttranslational modifications? Is there a difference in mito transport speed or pausing frequency?
      2. I concur with the exclusion of compounds that obviously alter cell shape, as the authors mention for the cancer therapeutics. Some cancer therapeutics actually affect microtubule dynamics (see 1st point), which may underlie their effect on both cell shape and mitoSLOPE. To undertand the mechanism of action, the top hits should also be tested for the integrity of the microtubular network and mitochondrial transport parameters.
      3. While I agree with the authors' reasoning that the observed phenotype could be a result of the disease or the result of a compensatory mechanism, their hypothesis could be experimentally tested by addition of any of the top hits in order to reverse mitoSLOPE in their patient cell lines. It may not have worked for Lithium in their last manuscript, but the mechanism of action of the novel compounds could be cell intrinsic.
      4. Does recreation of the CRISPR cell line in their hands produce the same phenotype?
      5. Additionally, the observed phenotypes could also be a product of the medication taken by the patients. Deeper patient data from the cohort may be relevant to put the findings in context. How were patients diagnosed? Which medications were the patients taking? Was substance abuse present? In Mertens et al, Lithium responders and Lithium non-responders showed a differential mitochondrial response, how does this affect their dataset?
      6. While MDD itself is not a psychotic disorder, it can still present with psychotic features. Was this evaluated during the recruitment? Also important, were they on antipsychotic medication in addition to antidepressant therapy?
      7. The fact that CACNA1C is excluded from the "unbiased" hit discovery (Fig 8) undermines the power of the filtering criteria selected by the authors. Authors should include some discussion around this.

      Minor comments:

      1. Colored images should be made colorblind-accessible. This applies to microscopy images and graphs.
      2. Fig 3: Exact p-values should be reported in the graphs
      3. Fig. 5 and Fig 7a-b: It is not immediately clear what the lines in these graphs represent. Is it the individual drug/gene hits in a pre-ranked manner?
      4. Fig 6 b-c: should the "m" be capitalized for Molarity?
      5. The annotation of divalproex/valproic acid as a "benzodiazepine receptor agonist" is incorrect. While it is known to enhance GABAergic neurotransmission, the mechanism is supported to be through GABA synthesis rather than being a GABA-A receptor agonist (see eg. PMID: 23407051).
      6. Supplementary Fig 3 and 4 could be swapped to match the main text order.
      7. One reference was inaccessible: Anon, Phenomics-Enabled Discovery and Optimization of Small Molecule RBM39 Degraders as Alternative to CDK12 Targeting in High-Grade Serious Ovarian Cancer (HGSOC).

      Significance

      Recently, mitochondria have emerged as mediators of anxious behavior and are increasingly studied in the context of neuropsychiatric disorders. However, the molecular mechanisms that connect altered mitochondrial performance to specific neuropathological conditions are unknown. This study extends our knowledge in this realm. While it is in principle an extension of earlier work from the authors (Cataldo, A.M. et al. Am. J. Pathol. 2010), it has added value due to the application of their automated analysis to publicly available datasets, providing a clear technical advance. This identified known as well as novel compounds that could revert the mitochondrial phenotype and makes this study specifically interesting to an audience interested in translational research. The strength of the manuscript certainly lies in the large number of examples studied and their well-rounded discussion of their findings. It is limited by the fact that the phenotype of neuropsychiatric conditions is studied in peripheral cells, and thus may not be a simple cell-autonomous response but a compensatory, systemic response that is not easy to replicate in a fibroblast in isolation. No mechanistic insight is gained on the underlying cell biology in the current format.

    1. Reviewer #1 (Public review):

      Summary:

      In this manuscript, Seegren and colleagues demonstrate that in a mouse model of neonatal E. coli meningitis, loss of endothelial toll-like receptor 4 (TLR4) leads to a marked decrease in transcriptional dysregulation across multiple leptomeningeal cell types, a decrease in vascular permeability, and a decrease in macrophage abundance. In contrast, loss of macrophage TLR4 had less pronounced effects. Using cultured wild-type and TLR4-knockout endothelial cells, the authors further demonstrate that TLR4-NF-κB signaling leads to reversible internalization of the tight junction protein claudin-5, establishing a potential mechanism of increased vascular permeability. Finally, the authors use RNA-sequencing of wild-type and TLR4-knockout endothelial cells to define the TLR4-dependent cell-autonomous transcriptional response to E. coli.

      Strengths:

      (1) The authors address an important, well-motivated hypothesis related to the cellular and molecular mechanisms of leptomeningeal inflammation.

      (2) The authors use model systems (mouse conditional knockouts and cultured endothelial cells) that are appropriate to address their hypotheses. The data are of high quality.

      Weaknesses:

      (1) The authors perform single-nucleus RNA-seq on dissected leptomeninges from control and E. coli-infected mice across three genotypes (WT, Tlr4MKO, and Tlr4ECKO). A major discovery from this experiment, as summarized by the authors, is: "Tlr4ECKO mice exhibited a global attenuation of infection-induced transcriptional responses across all major leptomeningeal cell types, as judged by the positions of cell clusters in the UMAP." This conclusion could be considerably strengthened by improving the qualitative and quantitative analysis.

      (2) The authors interpret E. coli infection-induced increases in leptomeningeal sulfo-NHS-biotin as evidence of compromised BBB integrity (i.e., extravasation from the vasculature) (Results, page 7), but another possible route in this context is sulfo-NHS-biotin entry from the dura across a compromised arachnoid barrier. The complete rescue in Tlr4ECKOs is strongly suggestive that the vascular route dominates, but it would strengthen the work if the authors could assess arachnoid barrier fidelity (e.g. via immunohistochemistry). At a minimum, authors should mention that the sulfo-NHS-biotin signal in this context may represent both vascular and arachnoid barrier extravasation.

      (3) The authors state that "deletion of TLR4 prevented both NF-κB nuclear translocation and Cldn5 internalization in response to E. coli (Figure 4A-D)" (Results, page 9). In Figures 4C and D, however, there is no indicator of a statistical test directly comparing the two genotypes. A comparison of within-genotype P-values should not be used to support a genotype difference (PMID: 34726155).

      (4) In the first paragraph of the Results, the authors summarize the meningeal layers as (1) pia, (2) subarachnoid space, (3) arachnoid, and (4) dura, and then state "The second and third layers constitute the leptomeninges." This definition of leptomeninges seems to omit the pia, which is widely considered part of the leptomeninges (PMID: 37776854).

      (5) The Cdh5-CreER/+;Tlr4 fl/- mouse lacks TLR4 in all endothelial cells (i.e., in peripheral organs as well as CNS/leptomeninges), and, as the authors note, the periphery is exposed to E. coli. It would be helpful if the authors could comment in the Discussion on the possibility that peripheral effects (e.g., peripheral endothelial cytokine production, changes to blood composition as a result of changes to peripheral endothelial permeability) may contribute to the observed leptomeningeal phenotypes.

    1. PDE4B knockout (KO) mice (46) were generated by Drs. S.-L. Catherine Jin and Marco Conti (Stanford University, CA), and kindly distributed via the Mutant Mouse Resource and Research Centers (MMRRC)

      DOI: 10.1152/ajpendo.00215.2025

      Resource: Mutant Mouse Regional Resource Center (RRID:SCR_002953)

      Curator: @AleksanderDrozdz

      SciCrunch record: RRID:SCR_002953


      What is this?

    1. eLife Assessment

      The importance of uterine natural killer (NK) cells in reproductive success has been demonstrated in mice and humans; however, it is still unclear how uterine NK cells are developed. In this valuable manuscript, the authors provide convincing evidence that TGF-b signaling in NK cells supports normal pregnancy in mice by the conversion of conventional NK cells into uterine tissue-resident NK cells. There are some concerns about the paper, particularly around Figures 1A, 1C, and 2E.

    2. Reviewer #1 (Public review):

      This is an excellent paper from Dr. Yokoyama and colleagues. The experiments are technically demanding, given the very low cell numbers and the challenges of working with implantation sites at gestational days 6.5, 10.5, and 14.5. Overall, the impact of TGF-β receptor II deficiency in the NK lineage on uterine trNK cell numbers and litter size is convincing, and the authors' conclusions are well supported by the data. Less convincing, however, is the claim that the decrease in trNK cells is compensated by an increase in cNK cells; rather, the absence of TGF-β receptor II appears to result in an overall reduction of NK/ILC1 cells.

      Major Points:

      (1) Figure 1A and B

      Although a trend is evident, it does not appear that the absolute number of cNK cells at day 14 is significantly changed from day 6.5?

      (2) Figure 2E

      The authors state, "This reduction of uterine trNK cells was accompanied by a concomitant increase in the absolute number and frequency of CD49b+Eomes+ cNK cells within the pregnant uterus of TGF-βRIINcr1Δ dams (Figure 2 D, E). The number of cNK cells appears relatively low (visually ~1,000-1,300), and although the difference is statistically significant, its physiological relevance is unclear. More importantly, this modest increase does not correlate with the marked decrease in trNK and ILC1 populations, as cNK cells do not appear to accumulate. In my opinion, the conclusion "Collectively, these findings indicate that a TGF-β-driven differentiation pathway directs the conversion of peripheral cNK cells into uterine trNK cells during murine pregnancy" should be slightly toned down.

      (3) Figures 2-4

      It is unclear whether the littermate controls are floxed mice or floxhet-Ncr1iCre mice? This distinction is important, as Ncr1iCre expression itself could potentially lead to a phenotype.

    3. Reviewer #2 (Public review):

      In their manuscript "TGF-β drives the conversion of conventional NK cells into uterine tissue-resident NK cells to support murine pregnancy", Yokoyama and colleagues investigate the role of Tgfbr2 expression by NK cells in the formation of tissue-resident uterine NK cells and subsequent importance in murine pregnancy. By transferring congenic splenic conventional NK cells into pregnant mice, they show conversion of circulating NK cells into uterine ivCD45 negative tissue-resident NK cells. When interfering with the formation of uterine trNK cells, spiral artery remodelling was impaired, fetal resorption rates were increased, and litter sizes were reduced.

      Generally, this is a research topic of high interest, yet the manuscript is lacking detailed mechanistic insights, and some questions remain open. At the current state, the data represent an interesting characterisation of the Tgfbr2-fl/fl Ncr1-Cre mice in pregnancy, but considering (a) the recent publication by the group (Reference 17) on the role of Eomes+ cNK cells during pregnancy, (b) the previously described role of Tgfbr2 and autocrine TGFb expression for uterine NK cell differentiation in virgin mice (also cited by the authors), and (c) the well-known relevance of uterine NK cells during pregnancy, additional experiments addressing the specific role of Tgfb during pregnancy would help to improve novelty and significance of the manuscript. To this end, the following aspects should be discussed and, where applicable, experimentally addressed by the authors:

      (1) The authors suggest cNK extravasation and local differentiation into iv- trNK.

      Can it be estimated how much this process contributes to the trNK pool vs. a potential local proliferation of already existing trNK? How do absolute numbers of CD49a+ Eomes+ trNK change during pregnancies? (In Figure 1A, the cell numbers of CD49a+ Eomes+ trNK seem to go down dramatically between gd 6.5 and 14.5). The plot in 1B could also include absolute numbers of ILC1s and trNKs. Would recruited cNK cells compensate for a potential loss of CD49a+ Eomes+ trNK?

      (2) Figure 1C: 2.5

      Mio cNK cells have been transferred, but only very few cells can be detected within the uterus (concatenated FACS plot shown). What may represent the limit to generate uterine trNK out of cNK? Is the niche supporting cNK-trNK differentiation limited? Is it only a specific subset of (splenic) cNK capable of differentiating into trNK? Is gd 0.5 the optimal timepoint for the transfer? Is there continuous recruitment of cNK into the uterus and differentiation into trNK, or is it enhanced at specific timepoints of pregnancy? Could there be local proliferation of cNK-derived trNK? This could be studied by proliferation dye dilution of WT cNK cells in this transfer-setup.

      (3) The authors should consider inducible Tgfbr2 deletion (e.g. with Tamoxifen-inducible Cre) to enable development of the uterine NK compartment in virgin mice and only ablate trNK differentiation during pregnancy. This could help to estimate the turnover of cNK into trNK, or to understand if constant cNK recruitment is required to form the uterine trNK compartment during pregnancy.

      (4) Did the authors consider transfer of Tgfbr2-floxed Ncr1-Cre cNK in the same setup as in Fig. 1C? This experiment could confirm the requirement of Tgfbr-dependent signalling for cNK to trNK conversion during pregnancy versus effects of Tgfb signals on trNK numbers in the uterus at steady state (before pregnancy).

      (5) Figures 2D/E

      The authors should state that ILC1s are reduced in the virgin uterus of female Tgfbr2-floxed or Tgfb1-floxed Ncr1-Cre mice and cite the relevant work (the Ref #29 discussed in this context did not show that?). It would be helpful to include an analysis of all three uterine ILC subsets in steady state. This could help to answer the question if the cNK cell changes are pregnancy-specific or a general phenomenon in Tgfbr2-floxed Ncr1-Cre mice.

      (6) Figure 2E

      Please phrase more carefully about the "concomitant increase" of cNKs, since this increase is much less pronounced compared to the very strong reduction (absence) of trNKs in Tgfbr2-floxed Ncr1-Cre mice. Do the authors suggest that cNKs are halted at this stage and cannot differentiate into trNK, based on these data?

      (7) Figure 3/4

      Can the reduced litter size and the abnormal spiral artery formation be rescued by transfer of WT cNK into Tgfbr2-floxed Ncr1-Cre mice?

    1. Partenariat Parents-École : Un Pilier pour la Réussite Scolaire

      Résumé Analytique

      Ce document de synthèse analyse les points clés de la conférence organisée par Parents Partenaires en Éducation (PPE) Ontario, portant sur l'importance cruciale du partenariat entre les familles et les institutions scolaires.

      Le message central est que la réussite des élèves ne repose pas uniquement sur l'école, mais sur une collaboration étroite et proactive où les parents agissent en tant que « co-éducateurs ».

      L'engagement parental est structuré autour de trois dimensions : l'investissement personnel, l'investissement cognitif et l'engagement institutionnel.

      Pour les familles, particulièrement celles issues de l'immigration, cette implication est un levier majeur pour déconstruire les biais inconscients, valoriser l'identité culturelle et assurer une intégration réussie.

      L'analyse démontre que l'inclusion est un choix délibéré et que le sentiment d'appartenance ne peut émerger que lorsque les voix des parents participent activement aux processus de décision au sein des conseils d'école et des comités.

      --------------------------------------------------------------------------------

      1. Cadre Conceptuel de l'Engagement Parental

      L'engagement parental ne se limite pas à la supervision des devoirs ; il s'agit d'un investissement multidimensionnel qui influence directement les performances académiques et le bien-être socio-affectif de l'enfant.

      Les Trois Dimensions de l'Engagement

      Selon la littérature scientifique citée, l'engagement se décline comme suit :

      | Dimension | Description | Exemples concrets | | --- | --- | --- | | Investissement personnel | Aspirations et intérêt manifesté pour la vie scolaire de l'enfant. | Discussions sur la journée, intérêt pour les camarades et les activités. | | Investissement cognitif | Accompagnement dans les tâches et respect des structures scolaires. | Supervision des devoirs, fréquentation de la bibliothèque, respect des règles (ex: usage des appareils électroniques). | | Engagement institutionnel | Présence effective et participation aux processus de décision. | Participation aux conseils d'école, comités de parents, réunions et bénévolat actif. |

      --------------------------------------------------------------------------------

      2. L'Identité et les Valeurs : Fondements du Partenariat

      L'identité et les valeurs des parents ne doivent pas rester à la porte de l'école. Elles constituent les filtres à travers lesquels le partenariat s'exprime.

      L'identité comme outil de décodage : Le système scolaire a besoin de connaître l'identité socioculturelle des familles pour adapter son offre de services (enseignants, travailleurs sociaux).

      La décolonisation de l'esprit : Pour les parents immigrants, il est essentiel d'articuler leur identité face au choc culturel et de valoriser leurs origines pour que l'enfant se sente en sécurité dans son environnement scolaire.

      Le filtre des valeurs : Les décisions majeures concernant l'éducation de l'enfant doivent être passées au filtre des valeurs familiales. L'implication dans les conseils d'école permet de challenger l'approche « taille unique » (one size fits all) des politiques scolaires.

      --------------------------------------------------------------------------------

      3. Analyse des Bénéfices de la Collaboration

      La collaboration entre les parents et l'école crée une dynamique « gagnant-gagnant » pour toutes les parties prenantes.

      Pour l'Élève

      Renforcement de la confiance : L'enfant est fier de voir sa famille impliquée et valorisée.

      Motivation accrue : La proximité des parents stimule l'engagement de l'élève dans ses propres apprentissages.

      Réduction des biais : Une collaboration étroite permet de changer le regard du personnel scolaire sur l'enfant, transformant parfois une perception négative (ex: hyperactivité perçue comme un trouble) en une reconnaissance de traits positifs (ex: curiosité et créativité).

      Pour les Parents

      Fluidité de la communication : Les échanges directs avec les enseignants facilitent la résolution rapide des problématiques.

      Acteur du changement : Les parents peuvent influencer les politiques (ex: code vestimentaire, introduction de l'uniforme, littératie financière).

      Lutte contre l'isolement : L'implication favorise l'intégration sociale et culturelle, surtout pour les nouveaux arrivants.

      Pour le Personnel Scolaire

      Meilleure compréhension culturelle : Les parents aident les enseignants à décoder les comportements des élèves sous un angle culturellement adapté.

      Soutien opérationnel : Le bénévolat parental (ex: accompagnement au musée) enrichit l'expérience pédagogique.

      --------------------------------------------------------------------------------

      4. Diversité, Inclusion et Appartenance

      Une distinction cruciale est faite entre ces trois concepts pour guider l'action parentale :

      1. La Diversité : Un fait statistique (nombres, quotas, pluralité linguistique et culturelle).

      2. L'Inclusion : Un choix individuel et collectif. C'est la volonté d'accueillir et de s'intégrer activement.

      3. L'Appartenance : Le stade ultime, atteint uniquement lorsque les voix des minorités sont intégrées aux discussions et aux processus de décision.

      --------------------------------------------------------------------------------

      5. Exemples d'Impact par l'Engagement Proactif

      La source met en lumière plusieurs cas où l'initiative parentale a transformé l'environnement scolaire :

      Adaptation culturelle : La proposition d'un coin calme pour la prière a permis à un élève de vivre sa foi en sécurité, harmonisant les valeurs de la maison et de l'école.

      Valorisation identitaire : Une séance de lecture de contes et de danses africaines a transformé la perception d'une élève sur ses vêtements traditionnels, passant de la honte à la fierté.

      Innovation curriculaire : L'initiative d'un parent a mené à l'adoption de la littératie financière comme priorité au sein d'un conseil d'école.

      Réorientation stratégique : La proximité entre une mère et une enseignante a permis de rediriger un élève vers un programme plus adapté à son profil (Baccalauréat International), modifiant ainsi sa trajectoire académique.

      --------------------------------------------------------------------------------

      6. Conclusion et Appel à l'Action

      Le document conclut que le manque de temps est souvent une barrière perçue plutôt que réelle. Une heure par mois offerte au conseil d'école peut suffire pour exercer une influence positive.

      Messages clés pour l'avenir :

      • Les parents sont les premiers éducateurs ; l'école fournit l'instruction, les parents fournissent l'éducation.

      • L'implication des parents est le seul moyen efficace pour que le système scolaire connaisse et respecte l'identité des familles qu'il sert.

      • Chaque parent possède un pouvoir d'influence et doit choisir d'être un acteur du changement pour garantir une société pluraliste et enrichie par ses différences.

    1. datos

      estos dos últimos párrafos no acaban de reflejar bien cómo estas dos obras ponen en danza lo que expones en el anterior ("A partir del análisis..."). Yo insistiría más en la idea de datos capturados como base de la performance, y por qué esto es relevante en el contexto de la propuesta del CFP, que mirar a captura con una mirada muy ámplia. La especificidad de tu propuesta puede hacerla destacar pero también es necesario marcar bien por qué encaja con el conjunto.

    2. desarrollado en mi tesis doctoral a partir de los planteamientos de Elsa Muñiz (2010). Propongo entenderlas como modos intencionales de intervenir o usar el cuerpo mediante tecnologías de comunicación digital en el contexto de la vida hiperconectada.

      Como decía Paloma, mejor reformular esto. Obviar la tesis, y explicarlo como investigación sin más.

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      Reply to the reviewers

      General statements.

      We thank the reviewers for their positive response and useful suggestions on our manuscript. They recognize the ‘proof of concept’ nature of the work and the importance of extending the number of human mutation-specific DMD mouse models from one to five for preclinical research. We feel that the quality of the manuscript has been improved upon implementation of the reviewer’s suggestions.

      Reviewer 1.

      OPTIONAL - From the point of view of the reviewer, it seems plausible to use CRISP/Cas9 to "clean up" the original hDMDmdx mouse line by selectively removing one of the YACs forming the tail-to-tail tandem in the mouse genome. Once such single copy mouse line is generated (and proven viable?) any subsequent rearrangement of the hDMD transgene would prove much less challenging. Such mouse line would also better represent human model where only one DMD copy is carried on the X chromosome.

      The reviewer gives the optional suggestion that the generation of these models could have been combined with the removal of one of the copies of the YAC to extend the use of the new models to CRISPR-based therapies. This is correct, but we note that when the data on the removal of a copy of the YAC were published, our new models were already generated and in different stages of QC, colony building and analysis. The procedure described by Chey et al could be used on our new models, but this would require additional time and funding and is therefore outside the scope of this manuscript.

      The labels in figure 2B and 3A would benefit from showing the PCR fragment lengths as well as the sizes of obtained hDMD exon deletions. On could also include an additional figure panel demonstrating the principle of ASO-induced exon skipping

      Reviewer #1 also has a minor comment regarding the exact deletions in figure 2B and 3A. For fig. 2B he/she suggests to include the sizes of the PCR fragments next to the gel. Especially for the gel regarding PCR1, which detects the deleted YAC copy, this will not be very informative as this can be (and is) different for different clones depending on the NHEJ-mediated repair in the specific clone. Adding sizes is only interesting for each specific clone, and adding them all will make a very messy figure. The important message from this gel is the presence of any fragment, as the undeleted copy is not amplified under the conditions used. For the gel of PCR2 the opposite is the case, here the PCR fragment shown is simply the undeleted YAC copy, and here we are only interested in the absence of the PCR fragment.

      We thank the reviewer for the suggestion of adding the deletion sizes to fig 3A. This made us realize that an additional table with the details of the mutant alleles in all models had been omitted, and we apologize for this error. With the revised version we include details on the size of the deletions and their genomic coordinates (in the human genome as it is in the human YAC) of each of the new models (revised Sup. Table 1). We trust that adding these details will clarify this reviewer’s minor comment.

      The reviewer requests to include an additional figure panel demonstrating the principle of ASO-induced exon skipping. We have now added this to the revised version of the manuscript (new fig. 5).

      The study is fairly limited in scope and will be of primary interest to those working in the DMD field.

      We are aware of 9 clinical trials for exon 51 and 53 studies that are ongoing or were recently stopped. For four of these compounds companies have a license to our hDMDdel52/mdx mouse model, and one of these studies has been published. An additional 7 clinical trials are planned or ongoing for exon 44, 45 and 50 skipping for which the newly developed models are being or can be used for preclinical studies.

      Reviewer 2.

      To further strengthen the rigor of the study, it would be valuable to include an analysis of potential off-target effects of CRISPR editing, particularly given that double targeting of two YAC copies was required. This is especially important for germline edits, as off-target mutations could introduce confounding phenotypes in the resulting mice. Demonstrating minimal or absent off-target activity would increase confidence in the specificity and safety of the generated models.

      There has indeed been one major study suggesting a large number of CRISPR-induced off-target mutations in mouse models. However, this publication was rapidly questioned by multiple groups for having used the wrong control animals and the original publication was retracted (https://doi.org/10.1038/nmeth0518-394a). Another study at that time, using the correct controls, did not find mutations that could be attributed to CRISPR-induced off-target mutations. A more recent study analysed founder animals from transgenic projects using 163 different guide RNAs and concluded ‘In total, only 4.9% (8/163) of guides tested have detectable off-target activity, at a rate of 0.2 Cas9 off-target mutations per founder analysed. In comparison, we observe __~1,100 unique variants in each mouse regardless of genome exposure to Cas9 __indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice.’ In short, the background mutation rate in mice is much higher than the Cas9 off-target mutation rate. In addition to this, we only used guide RNAs that did not have any predicted off-target sites (according to the CRISPOR tool; https://crispor.gi.ucsc.edu/crispor.py) on the same chromosome or in protein coding sequences, so that any undetected off-target mutation will rapidly be lost in the subsequent breeding. We also would like to refer the reviewer to the ‘referee cross-commenting remark’ from reviewer #3 on this topic.

      The validation of the dystrophic phenotype is generally convincing. However, the authors should clarify how "human dystrophin" is detected in the deletion models. Since only part of the dystrophin gene in these mice is humanized (the remainder is murine), it is important to specify, also in the results, which antibody was used and which epitope/exon it recognizes. If the antibody targets a deleted exon in a given model, this could lead to misinterpretation of the dystrophin signal. Providing this clarification would ensure the conclusions regarding dystrophin expression are fully supported.

      This question is based on the incorrect assumption that only part of the DMD gene in these models is humanized. As described in the original publication on the YAC transgenics the complete human gene is in the YAC. Here, we deleted a particular exon from this complete human DMD gene. In combination with the mdx allele, these mice lack the full-length mouse and human dystrophin isoforms expressed in muscle. As mentioned in the materials section, the human dystrophin protein was detected with the Mandys 106 antibody (recognizing exon 43; amino acids 2063-2078), which only has reactivity with human dystrophin according to the product specification of Sigma Aldrich. We confirmed this for wild type mouse tissue, showing no dystrophin for this antibody. In fig 4 we confirm lack of human dystrophin in the deletion models using this antibody. The mouse and human dystrophin protein was detected with the AB154168 antibody of Abcam (recognizing the last 100 amino acids of the C-terminal part of the protein), which has reactivity with both mouse and human. So neither antibody did target a deleted exon. For the exon skipping validation, solely the Abcam antibody was used, as none of the deleted or skipped exons was recognized by this antibody. Information regarding the targeted protein region has now been added to the materials section.

      Additionally, to further strengthen the characterization of the muscular dystrophy phenotype, the authors could quantify muscle fibre size and the percentage of centrally nucleated fibres, both of which are widely accepted quantitative markers of ongoing degeneration/regeneration in DMD models.

      and

      The validation of exon skipping in the new hDMD deletion models is convincing at the molecular level. However, since the ASOs were injected into both gastrocnemius and triceps muscles, it would be helpful to include at least a brief characterization of the triceps, even in supplementary data, as different muscles can show slightly different pathology and responses. Additionally, while the molecular readouts (RT-PCR and Western blot) demonstrate restoration of dystrophin expression, including simple histological analysis, such as H&E staining, could further support functional improvement and reinforce the physiological relevance of exon skipping in these models.

      The proof-of-principle nature of the current manuscript is focused on restoration of dystrophin expression shortly after ASO treatment, and the current sample sizes (n=3 mice per strain) are too limited for actual quantification of histopathological improvements. Furthermore, the timespan between the intramuscular injection and tissue collection (2 weeks) does not allow sufficient time for histopathological improvements to develop. Notably, a large natural history analysis of all these new models is currently ongoing, which includes a large variety of in vivo functional outcome measures and provides a full description of the histopathological aspects of these mice. The proposed characterization of the triceps is now included as supplementary data of the manuscript (Sup. Fig 1).

      Reviewer 3.

      This reviewer starts with pointing out some typos, or requested rephrasing to sentences for clarification. We appreciate this and have addressed this in the revised version of the manuscript.

      Generation of the models: it is not clear why the authors generated line 44 in ES cells, then switched to direct gene editing in zygotes. Was this due to advent of electroporation of zygotes at the time? This may need clarification beyond the sentence "Encouraged by the specificity of our new prescreen workflow and the efficiency of correct targeting of human exon 44 in ES cells, we generated additional models ... directly in mouse zygotes".

      The simple answer to this is that we were (pleasantly) surprised ourselves by the efficiency we got in the ES cells (which was based on the previous experience generating the del52 model). For animal welfare reason we prefer to generate models via ES cells if we expect a long and cumbersome quality control process and / or very low efficiency, as ES cells allow us to do this QC before the actual animals are generated, thus reducing the number of animals generated during the model generation phase. Expecting very low efficiency, we originally picked 10 x 96 well plates of clones for this del44 targeting, but after pre-screening the first two plates (192 clones), we realized this was an enormous overkill in clones, and the additional 8 plates were not analysed. With this much higher than expected efficiency, and the power of the two-step pre-screen described in the manuscript, we decided to try the next model (the del45) directly in zygotes. This was found efficient enough to also do the last two models directly in zygotes. We can only speculate on the much higher efficiency than observed for the del52 targeting. Clearly the fact that we knew of the double integration this time allowed us to develop the successful 2-step pre-screen. Another difference is that the del52 model was generated using TALENs as genome editors, whereas now we could use CRISPR/Cas9.

      Antisense oligonucleotide treatment: there is no description of the design of the ASOs beyond their sequence in suppl. Table 4. How were they designed? Moreover, they have been injected at two different doses (i.e., 50ul for Exon 51 & 53; 100ul for Exon 44 & 45). What is the rational for this? There is no justification in the manuscript.

      The requested additional details on ASO design and dosing have been added to the materials section of the revised manuscript. The reviewer also pointed out that fig 4 includes both a protein sample diluted to 10% of protein of both a C57BL/6J and hDMD/mdx control mouse, and requested a justification for this. We included samples of both wildtype strains to confirm species reactivity of the dystrophin antibodies used, with the AB145168 antibody being specific for both mouse and human protein (showing a dystrophin band in both wildtype samples), and the Mandys106 antibody being specific to only human protein (showing a dystrophin band in the hDMD/mdx control only).

      Phenotypic validation of the new models: a description of the mdx line with C57BL/6J mice is mentioned. Is this why Fig.4 includes "10% Bl6" and "10% hDMD/mdx"? If so, this should be clarified in the text (or deleted from the figure). The authors mentioned "As expected, the gastrocnemius of healthy hDMD/mdx mice expressed dystrophin of human origin at wildtype levels". Why would this be expected? If 2 copies of the gene, including the human promoter, are integrated, why would one expect a wildtype level of expression? In fact, in the original paper describing the hDMD/mdx model ('t Hoen et al. 2008), the human transcripts are expressed at 2 to 4-fold higher than their endogenous counterparts (which is in line with the integration of 2 copies).

      It is true, as he/she points out, that qRT-PCR data in the original YAC transgenic publication showed double expression of the human transcript, consistent with the double integration. However, fig. 3b in the same paper shows that at the protein level the expression of human DMD is comparable to the mouse protein. We don’t know the reason for the discrepancy between transcript and protein levels in this model, but in the current manuscript we are referring to this protein expression.

      A quantification of the expression levels on Figure 4 should be done (normalized to actinin) to resolve this. The size of the Marker should also be added on Figure 4.

      We feel that proper quantification can only be done with the utilization of a standard curve. As we expected no, or trace levels of dystrophin in the deletion models, we only included wildtype samples diluted to 10% of wildtype protein. This prevents us from accurate quantification of the trace dystrophin levels observed in the del45 and del51 models. However, as can be appreciated from fig 4, expression is very minimal. We added information on the marker in the materials section, and indicated the size (85 kDa) in the figure legend.

      Finally, the authors observed histological hallmarks of the disease in the new models (i.e., muscle degeneration and fibrosis). Although obvious on the images, it may be useful to add indications (e.g., arrows) on the images for readers non familiar with DMD.

      We added information on the marker in the materials section, and indicated the size (85 kDa) in the figure legend. Lastly, we also added the requested arrows to the pictures of fig. 4B to allow distinction between different histopathological hallmarks, and refer to these in the figure legend.

      Prescreen PCR of hDMD/mdx ES cells (Fig. 2): the authors mentioned that "The PCR conditions were chosen for not being able to amplify the undeleted allele." What does this mean? Was the elongation time reduced? As per the text, the theoretical size of a WT band is around 1.6kb. Yet, on the gel, bands higher than 1kb are visible for some clones.

      This is indeed based on the extension time of the PCR reaction shown in PCR 1 from fig 2B, amplified with primers upstream and downstream of the deleted region (see fig 1 and 2A). However, the approx. 1.6 kb fragment the reviewer refers to is the undeleted-specific amplification shown in Fig 2B PCR 2, which is the result of a primer outside and a primer inside the deleted region (fig 1and 2A). Amplification of the undeleted copy with the primers used in PCR 1 would give a fragment of 3902 nt. The deletion of exon 44 in the final model is 3584 nt, which details will be shown in the excel file that was erroneously omitted (see our response to reviewer #1), with the PCR 1 product of the deleted copy in the clone used for the mouse model being 318 nt. It is straight-forward to select an extension time that would be insufficient for a 3.9 kb fragment, but which can amplify fragments that are shorter due to the deletion. Even in a clone with a single copy of exon 44 deleted, one would not expect to see the 3902 nt fragment due to preferential amplification of the much shorter mutant band. This has now been clarified in the legend of figure 2 of the revised version of the manuscript.

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      Referee #1

      Evidence, reproducibility and clarity

      Summary:

      The study describes the creation and preliminary validation of four humanized mouse DMD models. The authors utilized the pre-existing hDMDmdx mouse line as a platform to generate clinically relevant models that carry the deletion of exons 44, 45, 51 and 52 of the hDMD transgene using CRISPR/Cas9 technology. This proved somewhat challenging as the hDMD YAC transgene present in the original hDMDmdx line is inserted as a tail-to-tail tandem in the mouse genome. The initial mouse line carrying deletion of exon 44 was performed using a combination of CRISPR/Cas9 and ES cell technologies whereas the remaining mouse lines were generated by applying CRISPR technology directly of hDMCmdx zygotes. In order to identify and select ES cell lines and animals carrying the desired deletion patterns, the authors devised a two steps PCR-based selection strategy followed by a copy number PCR for individual hDMD exons. Once the desired mouse lines were obtained, the authors performed Western blots and histological staining to prove the loss of the hDMD protein expression and the appearance of associated DMD muscle phenotypes. Finally, in vivo experiment was carried out where intramuscular injection of exon-specific ASOs lead to exon skipping and partial restoration of the expression of the truncated but potentially functional hDMD protein variants. The experiment was carried out solely as a proof-of-concept and was terminated before any therapeutic effect of the ASOs could be potentially observed. Nevertheless, the authors argue (correctly) that such models can prove useful in future development of treatment strategies for DMD.

      Major comments:

      The study has clear aims and is well described. The performed experiments support the final conclusions presented in the paper. OPTIONAL - From the point of view of the reviewer, it seems plausible to use CRISP/Cas9 to "clean up" the original hDMDmdx mouse line by selectively removing one of the YACs forming the tail-to-tail tandem in the mouse genome. Once such single copy mouse line is generated (and proven viable?) any subsequent rearrangement of the hDMD transgene would prove much less challenging. Such mouse line would also better represent human model where only one DMD copy is carried on the X chromosome.

      Minor comments:

      The labels in figure 2B and 3A would benefit from showing the PCR fragment lengths as well as the sizes of obtained hDMD exon deletions. On could also include an additional figure panel demonstrating the principle of ASO-induced exon skipping

      Significance

      General Assessment:

      The study is fairly limited in scope and will be of primary interest to those working in the DMD field. The new patient-derived hDMD exon deletions will allow testing and validation of human therapeutic moieties in mouse models but as such the study does not advance our knowledge about DMD or transgenic mouse model generation.

      Advance:

      Perhaps the only novelty is a very diligent genotyping approach aimed at identifying lines where both exons in the tail-to-tail hDMD tandem have been deleted. Given the extensive work put into this approach, the author may have missed an opportunity to reengineer the original hDMDmdx mouse line (see OPTIONAL) to generate a mouse line where any future modifications of the hDMD allele would be much more accessible to both CRISPR-mediate NHEJ and HDR approaches.

      Audience:

      The study is fairly limited in scope and will be of primary interest to those working in the DMD field.

      Reviewer's expertise focuses on CRISPR/Cas9 technologies and transgenic mouse model generation.

    1. Assim, o feedback deve ser construtivo, específicoe orientado para a ação

      Em ambientes digitais, o feedback acaba sendo o que mais se aproxima daquela presença natural que acontece presencial. E portanto se o feedback for vago, como só dizer bom trabalho, não ajuda ninguém a melhorar de verdade. Já se for só corretivo, apontando o que deu errado, isso pode desanimar bastante os alunos.

    2. As e-atividades podem ser realizadas deforma síncrona, em que os participantes estão conectados ao mesmotempo, interagindo em tempo real, ou de forma assíncrona, em que osparticipantes podem realizar as atividades em momentos diferentes, masainda assim interagir por meio de ferramentas digitais

      Este trecho ajuda a tirar a discussão do "gosto mais de X ou Y" e a pô-la no terreno do design: síncrono e assíncrono servem propósitos diferentes. O assíncrono dá tempo para pensar, escrever melhor e incluir quem tem horários complicados; mas pode gerar isolamento se não houver rotinas e acompanhamento. O síncrono aumenta presença e ritmo, mas não pode ser só exposição prolongada, porque isso reduz participação e tende a favorecer sempre os mesmos. Na prática, eu desenharia o assíncrono como ponto de central (reflexão + resposta entre pares + síntese semanal) e o síncrono como momento de alinhamento/feedback: discutir dúvidas reais, comparar perspetivas e fechar com orientações para a fase seguinte.

    3. mos assim que, ao

      Um ponto crucial neste capítulo é a função não-cognitiva das e-atividades. Observando o modelo de Salmon (Fig 3.2), vemos que a Socialização Online é um degrau fundamental antes da construção do conhecimento. Muitas vezes desenhamos atividades focadas apenas no conteúdo, mas o texto lembra-nos que as e-atividades devem também promover a motivação e a socialização para combater o isolamento no ensino online

    4. As etapas da atividade devem ser claras esequenciais, com orientações claras e precisas para os alunos.

      O desenho de e-atividades exige alinhamento: objetivo claro, etapas sequenciadas com tempos realistas, orientações e critérios explícitos. No fim, a classificação deve vir acompanhada de feedback construtivo, para promover autorregulação.

    1. Kleine molecuul transmitters: de belangrijkste kleine-molecuul neurotransmitters in het centrale zenuwstelsel zijn: acetylcholine (ACh), dopamine (DA), noradrenaline (NE) en serotonine (SE). Deze stoffen worden in de axonterminal gesynthetiseerd uit voedingsstoffen en zijn direct beschikbaar voor afgifte. Na vrijgave in de synaptische spleet kunnen zij snel worden aangevuld door heropname en hergebruik.

      Belangrijk! acetylcholine --> acetaat en choline serotonine--> tryptofaan--> depressie GABA--> glutamaat exitatie en inhibitie tyrosine

    1. Reviewer #3 (Public review):

      Summary:

      The manuscript presents an elegant and cost-effective approach for generating a tunable Bessel beam on a conventional two-photon microscope. The authors assemble a compact optical module comprising three axicons and a series of lenses that permits rapid adjustment of both lateral resolution and axial extent without modifying the focal plane. This flexibility enables the system to be readily adapted to a variety of biological preparations. As a proof of concept, the authors employ the device to record blood flow velocities in cortical microcapillaries, arterioles, and venules, thereby directly visualizing vasodilatation and vasoconstriction dynamics and permitting quantitative analysis of neurovascular coupling across cortical layers in awake mice.

      The authors demonstrate that the tunability of the Bessel beam can be exploited to match the numerical aperture to the vessel type: a high NA configuration, albeit slower scan, is optimal for resolving flow in capillaries, whereas a low NA setting provides faster acquisition suitable for arterioles and venules. By implementing a one-dimensional line scan with the Bessel beam, they achieve an imaging speed that is twentyfold faster than conventional frame-by-frame scanning, which proves sufficient to capture hemodynamic transients before and after an induced ischemic stroke.

      In addition to pure observation, the authors integrate a co-propagating Gaussian line to the system, allowing simultaneous imaging and photostimulation within the same focal plane. This capability addresses a common limitation of other Bessel beam implementations, in which the observation and perturbation planes often become misaligned when the Bessel beam is altered. The manuscript also emphasizes the advantage of Bessel beam excitation for calcium imaging after a perturbation, because it captures neuronal activity in planes both above and below the nominal focal plane, signals that would be missed with a standard Gaussian focus. Finally, the authors apply the technique to investigate the neuroimmune response following targeted microglial ablation; they report that adjacent microglia extend processes toward the injury site while retracting processes in the opposite direction.

      Overall, the work offers a technically straightforward yet powerful extension to existing two-photon platforms, providing high-speed, volumetric imaging and stimulation capabilities that are well-suited to a broad range of neurovascular and neuroimmune studies. The experimental validation is quite thorough, and the presented data convincingly illustrates the benefits of the approach.

      Strengths:

      The authors present a truly clever and inexpensive optical module that can be integrated into almost any two-photon microscope, providing a tunable Bessel beam with a minimal modification of the existing system. The experimental data and accompanying quantitative analysis convincingly demonstrate that the system can reveal physiological events, such as capillary flow, calcium transients across multiple axial planes, and microglial process dynamics, that are difficult or impossible to capture with a conventional Gaussian beam. The breadth of experiments chosen for the manuscript illustrates the practical utility of the device and supports the authors' conclusions that it extends the functional repertoire of standard two-photon microscopy.

      Weaknesses:

      The manuscript would benefit from a more detailed contextualisation of the claimed speed advantage. Although the authors mention other techniques in the introduction, they do not provide any direct comparison with other state-of-the-art high-speed two-photon approaches such as light beads microscopy (Demas et al., Nat. Methods 2021), temporal multiplexing schemes (Weisenburger et al., Cell 2019), or random access microscopy (Villette et al., Cell 2019). A brief comparison of imaging speed, spatial resolution, and instrumental complexity would enable readers to assess the relative merits of the present method.

      A second limitation that warrants discussion is the inherent trade off between volumetric coverage and image specificity. Because the Bessel beam excites fluorescence throughout an extended axial range, the detector inevitably integrates signal from a three dimensional volume into a two dimensional image. In densely labelled tissue, this can lead to significant signal crosstalk, reducing contrast and complicating quantitative interpretation. A brief analysis of how labeling density affects the fidelity of flow or calcium measurements, or suggestions for mitigating crosstalk (e.g., computational deconvolution, adaptive excitation shaping, or combinatorial sparse labeling), would broaden the applicability of the technique.

    1. Reviewer #3 (Public review):

      Summary:

      While our knowledge regarding visual opsins is largely very good, a lot more uncertainty exists around the role of non-visual opsins. Using the power of the Drosophila melanogaster model system, Kirsh et al. investigate the role of the non-visual opsin Rhodopsin7 (Rh7). Expression analysis, based on Rh7-Gal4>UAS-GFP and HRC in situ staining, reveals strong expression in the optic lobes and somewhat weaker, but nevertheless extensive expression in the brain. An investigation of motor activity reveals that loss of function leads to an altered day and night rhythm, specifically decreasing activity during the dark phase. These flies were also less sensitive, but still responsive to a light-induced startle response and showed deficiencies in the optomotor response. To further investigate how Rh7 may modulate these responses, inspired by the Dark line of flies (which were kept in the dark for ~1400 generations) and which has accumulated C-terminal related losses, the authors conducted rescues with an intact and a C-terminal-deficient Rh7 and were able to pinpoint that region as an important driver of related behavioral shifts. These findings are particularly intriguing as Rh7 represents an ancient opsin with phylogenetic and mechanistic parallels to mammalian melanopsin.

      Strengths:

      The paper is well-written and contains high-quality data with appropriate sample sizes, and the conclusions are well supported.

      Weaknesses:

      No weaknesses were identified by this reviewer, but the following recommendations are made:

      (1) The authors should clarify exactly what tissues were taken for the comparative qPCR. This is particularly interesting in terms of the retina. Since Rh7 appears not to be expressed within the photoreceptor cells of the retina, this raises the important question as to which cells it is expressed in. To address this important question, it would also be helpful to include an expression analysis of the retina itself (by extending the RH7-GFP expression patterns and/or adding HCR in situ of the ommatidia array). The cell types of the retina are very well classified, and some evidence already exists for Rh7 expression in support cells (e.g., Charlton-Perkins et al., (2017); PMID: 28562601). This study has a unique opportunity to investigate this further by adding these critical data for a more complete picture of Rh7.

      (2) Mammalian opsins should be included in the phylogenetic analysis illustrated in Figure 2A and indicate their position on the tree. This will allow readers to better put the authors' statements regarding the intermediate position of Rh7 into perspective. In addition, note that the distinction between red and deep red is easy to miss regarding the Rh7 cluster. Perhaps the authors could use a more distinct colour scheme, for example, orange and deep red.

      (3) More details should be provided on the optomotor response experiments. Specifically, specifications of the frequencies used for the optomotor response are needed. Results show a relatively large level of variation, which may be due to different angular perspectives that flies may have had while viewing the stimulus. If possible, provide videos as examples, as they will make it clearer to viewers how much flies could move around in the setup (from the methods, it seems they could move within the 2.2 of the 3 cm diameter of the arena, which would lead to substantial differences in the visual angle of the viewed grating.

    1. The Examined Life is Wise Living: The Relationship Between Mindfulness, Wisdom, and the Moral Foundations.Published in:Journal of Adult Development, Dec2020,Academic Search CompleteBy:Verhaeghen, PaulVerhaeghen, Paul The Examined Life is Wise Living: The Relationship Between Mindfulness, Wisdom, and the Moral Foundations  This correlational study of two independent samples (260 college students and 173 Mechanical Turk workers aged 21–74) examined whether and how mindfulness (broadly construed as a manifold of self-awareness, self-regulation, and self-transcendence), influences wisdom about the self (Adult Self-Transcendence Inventory and Self-Assessed Wisdom Scale) and wisdom about the (social) world (Three-Dimensional Wisdom Scale), and how mindfulness and wisdom impact ethical sensitivities (the five moral foundations). Mindfulness predicted wisdom about the self, and wisdom about the self was linked to an emphasis on the individualizing moral foundations of care/harm avoidance and fairness and, to a lesser degree, on the binding moral foundations of loyalty, authority, and purity. Wisdom about the (social) world was not associated with either mindfulness or the moral foundations. Age was a significant positive predictor for wisdom about the self once the self-awareness component of mindfulness was taken into account. Keywords: Wisdom; Mindfulness; Moral foundations; Ethics This paper investigates the links between trait mindfulness, wisdom, and ethical sensitivities (operationalized as sensitivity to the five moral foundations) in two independent samples, one of college students and one of adults spanning ages 21–74. Two principal ideas guided the study. The first idea is that wisdom, whether one conceptualizes it as a form of expertise or as a virtue or personality characteristic, might be well served by the specific quality or qualities of attention the individual brings to their experiences. It makes sense to expect that a habitual mindful attitude (i.e., taking an open, non-judgmental, reflective, self-regulatory, and sometimes self-transcendent stance towards life) might be a good indicator or exemplifier of such qualities. The second idea is that most, if not all, current adult-developmental theories consider wisdom to be of practical consequence, in the sense that wise people are expected to generally display prosocial attitudes and behavior (for a review, see Bangen et al. [10]). Consequentially, one might expect this wise stance to give rise to ethical sensitivities that are compatible with the characteristics of wisdom (as defined within these theories). Wisdom It is probably fair to say that within the field of psychology the study of wisdom started from an adult development perspective (e.g., Clayton and Birren [20]; Erikson [26]; Kramer [44]; Pascual-Leone [54]). Initial conceptualizations tended to view wisdom primarily from a cognitive angle, that is, as an advanced form of postformal thought. For instance, Baltes and Staudinger ([ 9 ]) define wisdom as 'expertise in the conduct and meaning of life' (p. 124). In this approach, wisdom is conceptualized as a form of crystallized intelligence, more specifically 'expert knowledge in the fundamental pragmatics of life that permits exceptional insight, judgment, and advice about complex and uncertain matters' (Pasupathi et al. [56], p. 351). Other approaches—Glück and Bluck ([31]) label these 'integrative views'—have supplemented this cognitive view by additionally emphasizing the reflective, affective, and conative qualities of the wise person, making wisdom more akin to a personality characteristic or a virtue (e.g., Ardelt [ 3 ]; Mitchell et al. [52])—wisdom as 'personal, concrete, applied, and involved' (Ardelt [ 3 ], p. 262). The different conceptualizations of wisdom do have a common core. From a review of 24 different key theories or definitions of wisdom, Bangen et al. ([10]) concluded that five subcomponents were present in at least half of the papers: (a) social decision making and pragmatic knowledge of life; (b) prosocial attitudes and values; (c) reflection and self-understanding (including a desire to learn); (d) acknowledgement of and coping with uncertainty; and (e) emotional homeostasis. Although there are qualitative, performance-based measures of wisdom, such as the Berlin wisdom paradigm (Baltes and Smith [ 8 ]), where participants describe how they would solve a particular life problem and answers are scored along a series of dimensions, self-report measures were used here, simply because quantitative measures allow for more efficient data collection and scoring, which in turn allows to query a larger sample of respondents. Specifically, I used the three quantitative self-report measures for wisdom recommended by Glück ([30]), Glück et al. ([34]), and Staudinger and Glück ([64])—Ardelt's Three-Dimensional Wisdom Scale (3D-WS; [ 2 ]), Levenson's Adult Self-Transcendence Inventory (ASTI; Levenson et al. [47]), and Webster's Self-Assessed Wisdom Scale (SAWS; [71], [72]). These three scales have different emphases. The 3D-WS measures wisdom as the integration of cognitive, reflective, and affective/compassionate personal characteristics; the SAWS gauges five dimensions, namely critical life experience, emotional regulation, reminiscence and reflectiveness, humor, and openness; the ASTI taps into self-transcendent wisdom, defined as a self-expansive process entailing decreased self-concern and increased empathy, understanding, spirituality, and feelings of connectedness with past and future generations. Not all of these scales cover all five subcomponents mentioned above: Arguably, the 3D-WS does; the SAWS covers social decision making, self-reflection, and emotional homeostasis; and the ASTI includes items about prosocial attitudes, self-reflection, and emotional homeostasis. Glück et al. ([34]) and Staudinger and Glück ([64]) additionally make a distinction between personal and general wisdom. The former refers to a person's insight into themselves and their own lives; the latter to insights into life and the world in general. The assumption is that personal wisdom is obtained through actual personal experience, whereas general wisdom does not have personal experience as a necessary condition. In Glück's conceptualization, all three scales mentioned above measure personal wisdom; only performance-based measures tap into general wisdom. Glück et al. ([34]) also posit a third, often underappreciated facet of wisdom, namely other-related wisdom, which they define as 'an empathy-based caring concern for both concrete other people and humankind at large' (p. 5); it is most evident in two of the three 3D-WS scales, namely the cognitive and reflective scales, and is possibly a subcomponent of personal wisdom. In (partial) confirmation of this view, Glück et al. found that all three 3D-WS scales loaded on a different factor than the two other quantitative scales. Given that the cognitive scale of the 3D-WS contains items that are indeed about the other (e.g., 'People are either good or bad' and 'You can classify almost all people as either honest or crooked'—both items are reverse-scored), but also items that are often general and external (e.g., 'ignorance is bliss' and 'It is better not to know too much about things that cannot be changed'—both items are reverse-scored), it seems to us that this dimension could be labeled more accurately as 'wisdom about the (social) world', in contrast with the 'wisdom about the self' tapped in personal-wisdom scales. Mindfulness Mindfulness is often defined as a particular way of paying attention—the ability or propensity to engage in "nonelaborative, non-judgmental, present-centered awareness in which each thought, feeling, or sensation that arises in the attentional field is acknowledged" (Bishop et al. [12], p. 232); this awareness requires cultivation (Nilsson and Kazemi [53]). One corollary is that "thought or events are observed as events in the mind without over-identifying with them and without reacting to them in an automatic, habitual pattern of reactivity", thus "introducing a 'space' between one's perception and response" and allowing one "to respond to situations more reflectively (as opposed to reflexively)" (Bishop et al. [12], p. 232). Mindfulness has been found to be broadly beneficial to the individual—mindfulness interventions lead to positive outcomes regarding stress, well-being, anxiety, depression, negative emotions, emotion regulation, rumination, self-compassion, and empathy (Eberth and Sedlmeier [25]; Verhaeghen [68]). These relationships are at least partially causal: changes in dispositional mindfulness after meditation training correlate with changes in self-perceived stress, anxiety, depressed mood, positive affect, negative affect, rumination, and general well-being (Gu et al. [40]; Khoury et al. [43]). Recent theoretical work within the field has converged on the conclusion that mindfulness is a complex concept, more akin to a manifold (or even a cascade of processes) than to a singular construct. The starting point of this work has been an examination of the reasons why mindfulness interventions lead to such a wide array of positive outcomes. Many models have been advanced to explain the translation of mindfulness into positive outcomes (e.g., Baer [ 5 ]; Brown et al. [16]; Chiesa et al. [19]; Creswell and Lindsay [21]; Grabovac et al. [35]; Hölzel et al. [42]; Segal et al. [59]; Shapiro et al. [60]; Vago and Silbersweig [67]), each with their own emphases and levels of complexity. Although details of the different proposed models vary, the list of proposed mechanisms generally contains three categories, as Vago and Silbersweig ([67]) point out. A first proposed mechanism is a change in self-awareness. This involves recognizing automatic habits and automatic patterns of reactivity, as well as an increased awareness of momentary states of body and mind—what is typically meant by mindfulness. A second proposed mechanism is a change in self-regulation. This includes better regulation of emotions, heightened self-compassion, increased emotional and cognitive flexibility, decreased rumination and worry, and increased nonattachment and acceptance. A final proposed mechanism is increased self-transcendence . This implies increased decentering, a stronger awareness of interdependence between self and others, and heightened compassion. Vago and Silbersweig label this common-denominator model the S-ART model, after its three components: self-awareness, self-regulation, and self-transcendence. Our own empirical work on the subject (Verhaeghen [69]; Verhaeghen and Aikman [70]), based on exploratory and confirmatory factor analysis as well as structural equation modeling on 3 independent samples of about 300 subjects each has indeed confirmed the plausibility of this S-ART mindfulness manifold, suggesting a flow of influence from self-awareness over self-regulation to self-transcendence, and then outward to well-being and other aspects of psychological health (for a schematic representation, see Fig. 1). Factor analysis showed that additional subdivisions were present within the components of self-awareness and self-regulation: self-awareness incorporated reflective awareness (the more active, deliberate, probing aspect of mindfulness) and controlled sense-of-self in the moment (the more passive, equanimous, non-judgmental aspect of mindfulness) (for more details on these components and how they are measured, see the "Methods" section below); self-regulation was tapped by (the opposite of) self-preoccupation and by self-compassion. Graph: Fig. 1 The S-ART mindfulness manifold as obtained in Verhaeghen ([69]) Mindfulness and Wisdom There are obvious points of contact between this conceptualization of mindfulness and those of wisdom, suggesting they operate in the same nomological space. First, some of the common-core wisdom subcomponents align with the mindfulness manifold. Clearly, the reflection and self-understanding subcomponent of common-core wisdom has a natural affinity (if not identity) with the reflective awareness component in the mindfulness manifold. A few examples from specific theories illustrate this quite nicely. For instance, Ardelt ([ 3 ]) explicitly claims that '[t]he development of wisdom requires the transcendence of one's subjectivity and projections, which can be accomplished through self-examination, self-awareness, and a reflection on one's own behavior and one's interactions with others' (p. 269). Likewise, Glück and Bluck's ([32]) MORE (mastery, openness, reflectivity, and emotion regulation) model of wisdom posits that wisdom-related knowledge develops through an interaction of life experiences with the four MORE resources, and that therefore wisdom should manifest itself in how people reflect upon past experiences. As a third example, Brown and Greene's model of Wisdom Development ([14]) states that wisdom ripens when individuals go through a core 'learning-from-life' process, comprised of reflection, integration, and application. Pascual-Leone ([55]), as a final example, considers meditation (one possible cultivator of mindfulness) as a path towards wisdom, through its fostering of insight, self-insight, and self-transcendence. Second, emotional homeostasis can be understood as an aspect or outcome of self-regulation. Third, some wisdom researchers explicitly view self-transcendence as a critical component of wisdom (see the Ardelt quote above; also Curnow [22]; Levenson [46]). There are a few empirical indications of a mindfulness-wisdom link as well. One study (Brienza et al. [13]) used its own process-based measure of wisdom, and found correlations with mindfulness scales, especially observing and orienting. Two studies used a training approach to foster wisdom by incorporating mindfulness either explicitly (Sharma and Dewangan [61]) or implicitly (as reflective awareness through a self-reflection journal and a life experience journal; Bruya and Ardelt [17]). The former study did not find intervention effects on either mindfulness or wisdom, but did find significant correlations at pretest between mindfulness (measured by the Mindful Attention Awareness Scale, MAAS; Brown and Ryan [15]) and the affective and reflective components of wisdom. The latter study obtained an intervention effect of the reflective exercises over and beyond those of attending a cognitively oriented class on wisdom, but did not include a measure of mindfulness to verify the proximal cause of the effect. These intervention studies, then, are somewhat suggestive of (but far from definitive about) a positive relationship between mindfulness and wisdom. Wisdom and Ethical Sensitivities The psychological study of ethical sensitivities and attitudes (e.g., Greene [37]; Haidt [41]) has converged on the conclusion that ethical actions are not always the product of the careful application of rational thought, but instead tend to be largely (although not exclusively) based on intuitions—evolved, automatic responses, inaccessible to awareness, which sometimes operate in contradiction with logical constraints. Researchers in this field often consider the vessels for these intuitions to be innate—for instance, Haidt's Moral Foundations Theory (MFT; Graham et al. [36]) posits that ethical sensitivities ultimately boil down to the five dimensions of promoting care/avoiding harm, fairness, ingroup loyalty, (respect for) authority, and purity (or sanctity). The former two are often combined into an 'individualizing' foundation, because they focus on the provision and protection of individual rights; the remaining three into a 'binding' foundation, because they focus on ingroup cohesion. The idea is that every individual is sensitive to these five aspects, but that the intuitions themselves are built through experience, and are thus open to individual and cultural differences through a tuning up or down of the emotional responses due to experiences that fit into these vessels (Flanagan and Williams [28]). In our previous study (Verhaeghen and Aikman [70]), where we adopted the Moral Foundations framework, we found clear links between the mindfulness manifold and ethical sensitivities, which possibly might be mediated through wisdom. Specifically, we found that reflective awareness and self-transcendence were directly related to the individualizing aspects of morality (i.e., an emphasis on care and fairness); only self-transcendence was related to the binding aspects of morality (i.e., an emphasis on loyalty, authority, and sanctity). One reason to suspect that wisdom might play a role in the individualizing foundation stems from its very definition—prosocial attitudes and values are the second most cited key component in Bangen et al.'s ([10]) literature review (21 out of 24 theories or models incorporated this component). A key mechanism may be the self-transcendental character of wisdom, which it has in common with mindfulness. There are empirical reasons to suspect that wisdom is implicated in moral attitudes (for a review of empirical and theoretical links between wisdom and ethics, see Sternberg and Glück [65]). For instance, wisdom has been found to correlate positively with other-oriented values such as well-being of friends, societal engagement, and ecological protection (Kunzmann and Baltes [45]; Webster [73]). Implicit lay theories of wisdom also include value orientations that align, in Haidt's model, with care and fairness (Glück et al. submitted). The Present Study The literature reviewed suggests that mindfulness, wisdom, and ethical sensitivities are related, but the pieces of this puzzle have not yet been fit together. One wide-open question is how the different components of mindfulness, broadly defined as self-awareness, self-regulation, and self-transcendence relate to wisdom; another whether (or how) wisdom might be a mediator translating, and perhaps crystalizing, mindfully experienced events into ethical attitudes. From the literature reviewed above, I expect that all three aspects of mindfulness would be positively related to wisdom. To assess wisdom, I used the three scales most commonly used in quantitative research—the 3D-WS, the ASTI, and the SAWS. After Glück et al. ([34]), I expect that a factor analysis of these measures will yield two dimensions: wisdom about the self (ASTI and SAWS) and wisdom about the (social) world (3D-WS). Given that mindfulness is primarily associated with knowledge of the self, I would expect that the mindfulness-wisdom connection would be stronger for wisdom about the self than for wisdom about the (social) world. Extending our prior work on mindfulness and ethical sensitivities, as well as building on Glück et al. (submitted), I expect that wisdom will be positively connected to the individualizing moral foundations—care and fairness. For the binding foundations—authority, loyalty, and sanctity/purity—the connection is likely less strong. Because wisdom is very often considered an aspect of adult development, I included a group of adults sampled across a large sweep of the adult life span (Sample B, age 25–74), aside from the more usual sample of college students (Sample A). Adding the former sample allows me, first, to check if the results from the first sample replicate, and second, to test whether or not any of the wisdom or ethical components are age-sensitive, as has sometimes been claimed (e.g., Ardelt [ 1 ]; Baltes and Kunzmann [ 7 ]; but see, e.g., Grossmann and Kross [39]; Mickler and Staudinger [51]). Methods Participants Sample A consisted of 260 undergraduate students from the Georgia Institute of Technology, who received course credit in return for their participation. They were invited to participate in a study on 'mindfulness, acceptance, and psychology'. They were aged 18–26 (mean = 19.7, SD = 1.5); 54% were women. Sample B consisted of 173 participants recruited from Mechanical Turk. They were invited to participate in a study on 'mindfulness, acceptance, and psychology', and offered $4 in return for their time. Workers needed to be highly qualified in order to participate—more than 5000 Human Intelligence Tasks (HIT; i.e., surveys or other online tasks) completed to the requesters' satisfaction, and at least 98% of all lifetime HITs approved by the requester. They were aged 21–74 (mean = 39.8, SD = 11.7); 44% were women. The age distribution was as follows: age 21–30: 38 participants; age 31–40: 69 participants; age 41–50: 33 participants; age 51–60: 18 participants; age 61–74: 12 participants. On average, participants had completed 14.9 years of education (SD = 1.9). Although Mechanical Turk is generally considered to be a useful, valid, and reliable tool for behavioral researchers (e.g., Mason and Suri [49]), we found it prudent to assess potential differences in data quality between the two samples. We did this by comparing Cronbach's α values for all subscales (see the "Measures and Procedure" section below for all α values). Sample B (Mechanical Turk) tended to have higher reliability values (median = 0.84, ranging from 0.41 to 0.93) than Sample A (students) (median = 0.71, ranging from 0.48 to 0.90). The correlation between Fisher z -transformed reliability values between the samples was 0.78 (this transformation was applied to linearize the measurement scale), suggesting that both groups were about equally sensitive to differences in the item characteristics that drive reliability. Measures and Procedure Participants filled out all questionnaires online; they took about 45–60 min to complete. Below, questionnaires are grouped thematically; the mindfulness measures (i.e., self-awareness, self-regulation, and self-transcendence) are presented as they resulted from the set of factor analyses (an exploratory analysis on 488 participants, and a confirmatory analysis on an independent sample of 222 participants) in Verhaeghen ([69]); this structure was replicated in Verhaeghen and Aikman ([70]). All measures were collected from both samples. Cronbach's α values reported are the values obtained in the present study, reported separately for Samples A and B, respectively. Note that some scales (notably the subscales of the Self-Compassion Scale) contain a very small number of items, possibly depressing the α values. Control Variables The Mini-IPIP (Donnellan et al. [23]) is a 20-item measurement of the Big Five personality factors , 4 items for each factor: Extraversion (sample item: 'I am the life of the party', Cronbach's α = 0.83 and 0.87), Agreeableness (sample item: 'I sympathize with others' feelings', Cronbach's α = 0.77 and 0.85), Conscientiousness (sample item: 'I get chores done right away', Cronbach's α = 0.68 and 0.78), Openness (which the IPIP labels Intellect/Imagination; sample item: 'I have a vivid imagination', Cronbach's α = 0.71 and 0.84), and Neuroticism (sample item: 'I have frequent mood swings', Cronbach's α = 0.74 and 0.78). Additionally, participants were asked for their age and gender . Social Conservatism Social conservatism was measured via the Social Conservatism subscale (6 items; sample item: 'Please indicate the extent to which you feel positive or negative towards each issue: ... Abortion'; Cronbach's α = 0.62 and 0.69) of the Social and Economic Conservatism Scale (SECS; Everett [27]). Self-awareness Two constructs were assessed within self-awareness. The first, reflective awareness , is the unit-weighted composite of the z -scores of three scales: (a) the Observing subscale of the Five Facets Mindfulness Questionnaire (FFMQ; Baer et al. [ 6 ]) (8 items; sample item: 'When I'm walking, I deliberately notice the sensations of my body moving', Cronbach's α = 0.73 and 0.87); (b) the Reflectiveness subscale of the Broad Rumination Scale (BRS; Trani et al. in preparation) (4 items; sample item: 'It is important for me to understand why I feel a certain way', Cronbach's α = 0.81 and 0.81); and (c) Search for Insight/Wisdom of the Aspects of Spirituality scale (ASP; Büssing et al. [18]) (7 items; sample item: 'I strive for insight and truth', Cronbach's α = 0.84 and. 90). In both samples, the composite was normally distributed, as ascertained via a Kolmogorov–Smirnov test ( p > 0.2). The second construct, controlled sense-of-self in the moment , is the unit-weighted composite of the z -scores of three scales: (a) the Acting with Awareness subscale from the FFMQ (8 items, sample item: the reverse of 'When I'm doing things, my mind wanders off and I'm easily distracted', Cronbach's α = 0.87 and 0.91); (b) the Sense-of-self Scale (SOSS; Flury and Ickes [29]) (12 items, sample item: 'I have a clear and definite sense of who I am and what I'm all about'; Cronbach's α = 0.86 and 0.88); and (c) the Non-judging of inner experience subscale of the FFMQ (8 items, sample item: the reverse of 'I criticize myself for having irrational or inappropriate emotions', Cronbach's α = 0.90 and 0.93). In both samples, the composite was normally distributed, as ascertained via a Kolmogorov–Smirnov test ( p > 0.2). Self-regulation Two constructs were assessed within self-regulation. The first, self-preoccupation , is the unit-weighted composite of the z -scores of two subscales from the BRS, namely Compulsivity (5 items; sample item: 'When I start to worry, it's very hard for me to stop', Cronbach's α = 0.79 and 0.87) and Worrying (3 items; sample item: 'Uncertainty about the future bothers me', Cronbach's α = 0.58 and 0.68), as well as two subscales from the Self-Compassion Scale, Short Form (SCS; Raes et al. [57]), namely Isolation (2 items; sample item: 'When I'm feeling down, I tend to feel like most other people are probably happier than I am', Cronbach's α = 0.56 and 0.63) and Over-Identified (2 items; sample item: 'When I fail at something important to me I become consumed by feelings of inadequacy', Cronbach's α = 0.66 and 0.58). In both samples, the composite was normally distributed, as ascertained via a Kolmogorov–Smirnov test ( p > 0.2). In our previous work, as here, self-preoccupation correlated negatively with other aspects of mindfulness, as one would expect—better self-regulation implies lower, not higher, levels of self-preoccupation. This may be confusing for some readers. Because the construct is, however, measured by scales that tap explicitly into the self-preoccupation aspect, and not its absence or opposite, we preferred to keep the self-preoccupation label. The second, self-compassion , was measured as the unit-weighted composite of the z -scores of three subscales from the SCS, namely Self-Kindness (2 items; sample item: 'I try to be understanding and patient towards those aspects of my personality I don't like', Cronbach's α = 0.61 and 0.60), Common humanity (2 items; sample item: 'I try to see my failings as part of the human condition', Cronbach's α = 0.49 and 0.57), and Mindfulness (2 items; sample item: 'When something painful happens I try to take a balanced view of the situation', Cronbach's α = 0.66 and 0.68), as well as the Decentering subscale of the Experiences Questionnaire (EQ; Fresco et al. 2007) (13 items, sample item: 'I am better able to accept myself as I am'; Cronbach's α = 0.84 and 0.93). The composite was normally distributed in Sample A, Kolmogorov–Smirnov = 0.042, p > 0.2, but not Sample B, Kolmogorov–Smirnov = 0.075, p = 0.034. Self-transcendence Self-transcendence was measured as the unit-weighted composite of the z -scores of 2 subscales from the Dispositional Positive Emotion Scale (DPES; Shiota et al. [62]), namely Joy (6 items; sample item: 'I am an intensely cheerful person', Cronbach's α = 0.84 and 0.90), and Love (6 items; sample item: 'I develop strong feelings of closeness to people easily', Cronbach's α = 0.82 and 0.90), and 1 subscale from the Resilience Scale (RS; Lundman et al. [48]), namely Meaningfulness (7 items, sample item: 'My life has meaning', Cronbach's α = 0.81 and 0.91). The composite was normally distributed in Sample A, Kolmogorov–Smirnov = 0.042, p > 0.2, but not Sample B, Kolmogorov–Smirnov = 0.072, p = 0.046. Moral Foundations This construct was measured using the 5 subscales of the Moral Foundations Questionnaire (Graham et al. [36]): (a) Care/harm (6 items; sample item: 'When you decide whether something is right or wrong, to what extent are the following considerations relevant to your thinking? – Whether or not someone suffered emotionally'; Cronbach's α = 0.52 and 0.76); (b) Fairness (6 items; sample item: '... Whether or not some people were treated differently than others'; Cronbach's α = 0.56 and 0.64); (c) Ingroup loyalty (6 items; sample item: '... Whether or not someone's action showed love for his or her country'; Cronbach's α = 0.48 and 0.84); (d) Authority (6 items; sample item: '... Whether or not someone showed a lack of respect for authority'; Cronbach's α = 0.61 and 0.85); and (e) Purity (6 items; sample item: '... Whether or not someone violated standards of purity and decency'; Cronbach's α = 0.69 and 0.92). Wisdom Scales Participants filled out three self-report wisdom surveys. The Adult Self-Transcendence Inventory (ASTI; Levenson et al. [47]) measures, in the words of the authors, "a decreasing reliance on externals for definition of the self, increasing interiority and spirituality, and a greater sense of connectedness with past and future generations" (p. 127). After factor analysis, Levenson et al. derived a more focused self-transcendence scale, which is used here (Factor 1 of their Table 1; 10 items; sample item: 'My peace of mind is not so easily upset as it used to be'; Cronbach's α = 0.67 and 0.79). The Self-Assessed Wisdom Scale (SAWS; Webster [71]) measures 5 interrelated dimensions of wisdom: experience (8 items; sample item: 'I have experienced many painful events in my life'; Cronbach's α = 0.81 and 0.84), emotions (8 items; sample item: 'I am good at identifying subtle emotions within myself'; Cronbach's α = 0.83 and 0.86), reminiscence (8 items; sample item: 'Reviewing my past helps gain perspective on current concerns'; Cronbach's α = 0.86 and 0.91), openness (8 items; sample item: 'I like to read books which challenge me to think differently about issues'; Cronbach's α = 0.71 and 0.80), and humor (8 items; sample item: 'I can chuckle at personal embarrassments'; Cronbach's α = 0.86 and 0.91). The Three-Dimensional Wisdom Scale (3D-WS; Ardelt [ 2 ]) consists of 3 subscales, tapping the cognitive (14 items, sample item: 'It is better not to know too much about things that cannot be changed'; Cronbach's α = 0.78 and 0.86), reflective (12 items, sample item: 'When I'm upset at someone, I usually try to "put myself in his or her shoes" for a while'; Cronbach's α = 0.55 and 0.54), and affective (13 items, sample item: 'I can be comfortable with all kinds of people'; Cronbach's α = 0.49 and 0.41) components of wisdom. Factor analysis of the nine wisdom scales in both samples; principal axis analysis with oblimin rotation Sample ASample BFactor 1 wisdom about the selfFactor 2 wisdom about the social worldFactor 1 wisdom about the selfFactor 2 wisdom about the social worldASTI (total).67.80SAWS-emotion regulation.72.78SAWS-experience.79.75SAWS-humor.71.77SAWS-openness.65.74SAWS-reminisce-reflect.80.733D-WS-affective.71.803D-WS-cognitive.57.683D-WS-reflective.76.68 N = 260 for Sample A and 173 for Sample B. For legibility reasons, factor loadings below.30 are not represented Measures Collected but Not Included in the Analyses Additionally, participants filled out the Nonattachment Scale (NAS; Sahdra et al. [58]), the Emotional Resilience Scale (ERS; Gross and John [38]); the QUEST scale (Batson and Schoenrade [11]), the Varieties of Inner Speech Questionnaire (VISQ; McCarthy-Jones and Fernyhough [50]), and the Self-Verbalization Scale (SVS; Duncan and Cheyne [24]). Some of those measures were remnants of an earlier (Verhaeghen [69]) attempt at casting a wide net of mindfulness measures; these measures failed to make the final cut after the factor analysis described in that paper (NAS, ERS, and QUEST); others were are not relevant to the present project (VISQ and SVS). Results Factor Analysis of the Wisdom Scales Two exploratory factor analyses (principal axis analysis with oblimin rotation), one for each sample, were conducted on the nine wisdom scales (i.e., the ASTI scale, the three 3D-WS scales and the five SAWS scales). Scale or subscale scores (i.e., not item scores) were the unit of analysis. Eigenvalues and the scree plot suggested a 2-factor solution in both samples. This solution is presented in Table 1; it explains 55% of the variance in Sample A, and 57% of the variance in Sample B. Both analyses converged on the same solution: the ASTI and all the SAWS scales loaded on one factor, and all three 3D-WS scales loaded on another. As mentioned in the introduction, the ASTI and the SAWS scale have in common that they survey wisdom from an intrapersonal perspective, that is, they appear to tap self-knowledge and self-acceptance; the 3D-WS arguably captures skills and wisdom about how to deal with the social world and with external circumstances. Consequently, I will label the first factor wisdom about the self , and the second wisdom about the ( social ) world . The two factors are relatively independent: Their intercorrelation was 0.18 in Sample A and 0.07 in Sample B. Wisdom and the Mindfulness Manifold To examine how the mindfulness manifold is related to self-assessed wisdom, as well as to control for the effects of the set of background variables (personality, age, and gender), hierarchical multiple regression analysis was applied to the data, separated by sample, with the two types of wisdom (wisdom about the self and wisdom about the [social] world) as the final outcome. For these analyses, a unit-weighted composite was constructed from the z -scores for the ASTI and the different SAWS scales to represent wisdom about the self. The unit-weighted composite of the z -scores of the three 3D-WS scales represented wisdom about the (social) world. Both unit-weighted wisdom composites were normally distributed in both samples; highest Kolmogorov–Smirnov = 0.057, p > 0.200. In the first step, the background variables—the five IPIP scales, age, and gender—were entered. The next step added the two self-awareness composites (reflective awareness and controlled sense-of-self in the moment); the step after that the two self-regulation composites (self-preoccupation and self-compassion); the final step added self-transcendence. Pearson correlations between all variables are reported in Table 2; results from the regression analyses in Table 3. Note that in these analyses, self-preoccupation is scored as defined above, that is, higher values indicate higher levels of self-preoccupation, which indicates a low level of self-regulation. Because of the potential conceptual overlap between the mindfulness concept of self-transcendence and wisdom as defined through the ASTI, analyses were rerun after removing the ASTI from the composite measuring wisdom about the self. The wisdom about the self variable and the wisdom about the self variable with the ASTI removed were virtually identical ( r = 0.98 in Sample A and 0.99 in Sample B); the pattern of the regression results was identical (i.e., variables that were significant remained significant and variables that were not remained non-significant). Correlation matrix for the background variables, mindfulness variables, and wisdom factors; Sample A data presented above the diagonal, Sample B below 12345678910111213141516171 IPIP extraversion1.00.29**.01 −.12*.13*.09.10.03.12.22** −.22**.13*.40**.31**.19**.06.062 IPIP agreeableness.25**1.00.17** −.02.25**.18**.03.28**.36**.19**.00.20**.51**.38**.23**.31**.063 IPIP conscientiousness.12.30**1.00 −.16**.05.18**.03.11.09.34** −.11.18**.27**.10 −.02.05.19**4 IPIP neuroticism −.43** −.34** −.36**1.00 −.09 −.04 −.03.24**.08 −.53**.60** −.48** −.34** −.18** −.11.06 −.045 IPIP intellect/imagination.29**.18* −.02 −.20**1.00.07.04 −.15*.35**.08 −.08.07.20**.36**.03.04 −.116 Social conservatism −.04.14.23** −.19* −.111.00 −.05.07.16*.15* −.02.14*.24**.18*.03.11.54**7 Age −.05.13.07 −.08 −.08.30**1.00 −.07.05.03.03 −.02 −.03.03.07 −.03.088 Gender.05 −.31** −.17* −.02.03 −.07 −.21**1.00.04 −.03.21** −.05.13*.05.13*.30**.009 Reflective awareness.22**.34**.26** −.18*.43** −.02 −.12 −.141.00 −.08.22**.23**.35**.60**.15*.37**.23**10 Controlled sense-of-self in the moment.33**.40**.37** −.62**.21**.05.17* −.10.17*1.00 −.54**.42**.43**.22**.14* −.03.0111 Self-preoccupation −.37** −.22** −.23**.57** −.19* −.08 −.17* −.08 −.02 −.56**1.00 −.44** −.27** −.08 −.14*.30**.1112 Self-compassion.06.16* −.07 −.20**.03.05.04 −.04.17* −.01.17*1.00.48**.41**.21**.14*.17**13 Self-transcendence.52**.59**.34** −.66**.16*.26**.04 −.12.43**.54** −.47**.21**1.00.57**.27**.35**.24**14 Wisdom about the self.34**.51**.32** −.47**.40**.10.11 −.14.66**.45** −.28**.22**.68**1.00.28**.41**.26**15 Wisdom about the (social) world.11.06.08 −.08.08 −.05.05 −.06.10.05 −.06.00.11.101.00.18**.1016 Individualizing foundation.09.38**.09 −.13.17* −.08.06 −.15.31**.13 −.02.03.29**.43**.111.00.33**17 Binding foundation −.04.20**.20* −.12 −.20*.77**.13 −.10 −.01 −.02.09.07.31**.16*.01.071.00 N = 260 for Sample A and 173 for Sample B IPIP International Personality Item Pool (https://ipip.ori.org/) * p <.05 Results from hierarchical regression analyses to predict the wisdom factors Step 1Step 2Step 3Step 4Sample ASample BSample ASample BSample ASample BSample ASample BWisdom about the self IPIP extraversion0.19**0.080.16**0.020.17**0.030.11* − 0.06 IPIP agreeableness0.24**0.26**0.080.17**0.060.17** − 0.010.05 IPIP conscientiousness0.010.07* − 0.060.01 − 0.060.03 − 0.080.02 IPIP neuroticism − 0.16** − 0.21** − 0.15** − 0.19** − 0.10 − 0.17* − 0.06 − 0.05 IPIP intellect/imagination0.28**0.31**0.13**0.110.16**0.110.14*0.18** Age − 0.010.08 − 0.020.13* − 0.010.12*0.010.13* Gender0.07 − 0.060.080.010.070.020.050.02 Reflective awareness0.52**0.50**0.46**0.49**0.40**0.38** Controlled sense-of-self in the moment0.15*0.120.120.130.070.09 Self-preoccupation0.04 − 0.010.050.05 Self-compassion0.19**0.060.14*0.03 Self-transcendence0.28**0.41**R2.296.455.506.622.526.625.561.673R2 change.296**.455**.210**.167**.020**.003.035**.048**Wisdom about the (social) world IPIP extraversion0.130.120.100.130.090.130.060.12 IPIP agreeableness0.21** − 0.010.16*0.000.16*0.000.16 − 0.01 IPIP conscientiousness − 0.090.03 − 0.130.04 − 0.120.04 − 0.13*0.04 IPIP neuroticism − 0.17** − 0.02 − 0.13 − 0.08 − 0.07 − 0.09 − 0.05 − 0.08 IPIP intellect/imagination − 0.050.06 − 0.080.06 − 0.080.06 − 0.080.07 Age0.050.040.050.040.060.050.070.05 Gender0.11 − 0.070.10 − 0.070.11 − 0.070.10 − 0.07 Reflective awareness0.110.040.130.040.100.02 Controlled sense-of-self in the moment0.12 − 0.120.07 − 0.110.05 − 0.12 Self-preoccupation − 0.120.03 − 0.110.04 Self-compassion0.03 − 0.000.01 − 0.08 Self-transcendence0.130.06R2.116.033.132.043.140.043.148.044R2 change.116*.033.016.009.008.000.008.001 N = 260 for Sample A and 173 for Sample B IPIP International Personality Item Pool (ipip.ori.org) * p <.05, ** p <.01 Ethical Sensitivity as Consequence of Mindfulness and Wisdom Hierarchical regression was applied to investigate how wisdom and the mindfulness manifold potentially shape ethical sensitivity, operationalized here as the moral foundations. To keep the number of analyses manageable, the two individualizing foundations were collapsed into a single construct by taking the average of the z -scores of the Care/Harm and Fairness scales (the correlation between the two individualizing foundations was 0.50 in Sample A, and 0.57 in Sample B); likewise, a unit-weighted z -score composite was built from the three binding foundations, namely Ingroup loyalty, Authority, and Purity (intercorrelations between the three binding foundations ranged from 0.59 to 0.64 in Sample A, and from 0.63 to 0.78 in Sample B). As is usual (because individuals generally tend to skew towards the ethical side of the distribution), these composites were not normally distributed, Kolmogorov–Smirnov = 0.109, 0.112, 0.139, and 0.073, for individualizing in Samples A and B and binding in sample A and B, respectively, p = 0.000, 0.000, 0.000, and 0.040, respectively. Pearson correlations are reported in Table 2; results from the regression analyses in Table 4. Rerunning the regression analyses with the alternate measure of wisdom about the self, that is, with the ASTI removed, yielded an identical pattern as obtained for the original wisdom about the self concept (i.e., variables that were significant remained significant and variables that were not remained non-significant). Results from hierarchical regression analyses to predict the moral foundations Step 1Step 2Step 3Step 4Step 5Sample ASample BSample ASample BSample ASample BSample ASample BSample ASample BIndividualizing foundation IPIP extraversion − 0.06 − 0.02 − 0.04 − 0.03 − 0.01 − 0.03 − 0.06 − 0.11 − 0.10 − 0.09 IPIP agreeableness0.23**0.34**0.110.33**0.100.34**0.050.25*0.030.23* IPIP conscientiousness0.060.010.01 − 0.02 − 0.00 − 0.04 − 0.03 − 0.040.01 − 0.05 IPIP neuroticism − 0.04 − 0.03 − 0.10 − 0.10 − 0.21* −.16 − 0.17 − 0.07 − 0.17* − 0.05 IPIP intellect/imagination0.15*0.080.040.020.070.020.040.08 − 0.030.03 Social conservatism0.01 − 0.15 − 0.00 − 0.16 − 0.01 − 0.16 − 0.03 − 0.22* − 0.02 − 0.20* Age − 0.060.05 − 0.050.09 − 0.080.11 − 0.060.13 − 0.070.07 Gender0.21** − 0.060.25** − 0.030.21** − 0.030.18* − 0.020.17* − 0.02 Reflective awareness0.33**0.190.22**0.20*0.17*0.110.03 − 0.05 Controlled sense-of-self in the moment − 0.05 − 0.120.05 − 0.110.02 − 0.15 − 0.00 − 0.17 Self-preoccupation0.38**0.100.39**0.170.39**0.13 Self-compassion0.10 − 0.110.04 − 0.15 − 0.01 − 0.15 Self-transcendence0.27**0.35*0.160.17 Wisdom about the self0.42**0.41** Wisdom about the self (ASTI excluded)(NA)(NA) Wisdom about the (social) world0.010.04R2.158.160.233.191.300.202.329.232.404.285R2 stepwise change.158**.160**01,075**.033.067**.011.029**.031*.075**.053**Binding foundation IPIP extraversion − 0.020.030.000.040.030.050.00 − 0.02 − 0.01 − 0.02 IPIP agreeableness − 0.080.09 − 0.120.10 − 0.130.11 − 0.15*0.04 − 0.15*0.03 IPIP conscientiousness0.21**0.030.22**0.040.21**0.020.20**0.030.21**0.02 IPIP neuroticism0.070.07 − 0.020.02 − 0.05 − 0.06 − 0.030.00 − 0.030.02 IPIP intellect/imagination0.02 − 0.10 − 0.03 − 0.10 − 0.01 − 0.11 − 0.02 − 0.06 − 0.06 − 0.09 Social conservatism0.54**0.80**0.54**0.80**0.54**0.80**0.53**0.74**0.53**0.75** Age0.02 − 0.100.02 − 0.110.00 − 0.090.01 − 0.060.01 − 0.09 Gender − 0.13 − 0.04 − 0.10 − 0.05 − 0.13* − 0.03 − 0.14* − 0.02 − 0.14* − 0.02 Reflective awareness0.130.000.040.010.02 − 0.06 − 0.06 − 0.13 Controlled sense-of-self in the moment − 0.15* − 0.08 − 0.12 − 0.06 − 0.13 − 0.09 − 0.15 − 0.10 Self-preoccupation0.21*0.15*0.22**0.20**0.21*0.19** Self-compassion0.14 − 0.090.12 − 0.11*0.09 − 0.12* Self-transcendence0.100.28**0.050.22* Wisdom about the self0.23**0.15 Wisdom about the self (ASTI excluded)(NA)(NA) Wisdom about the (social) world − 0.040.04R2.361.651.391.655.419.668.423.690.447.698R2 stepwise change.361**.651**.030*.004.029*.013.004.024**.023*.008 N = 260 for Sample A and 173 for Sample B IPIP International Personality Item Pool (https://ipip.ori.org/) * p <.05, ** p <.01 Discussion In the present study, I investigated if and how wisdom might be related to dispositional mindfulness, broadly construed as a manifold of self-awareness, self-regulation, and self-transcendence, and if and how it might promote ethical sensitivities. Wisdom was measured using the three self-report surveys most often used in quantitative research on the topic—the 3D-WS, the ASTI, and the SAWS. Two independent samples were included: A sample of college students (Sample A), and one of adult workers on Mechanical Turk with a much wider age range (viz., 21–74; Sample B). The Structure of Wisdom A first expectation (after Glück et al. [34]) was that factor analysis on the subscales of the three surveys would reveal a bifurcation between wisdom about the self (ASTI and SAWS) and wisdom about the (social) world (3D-WS). Factor analysis indeed confirmed this divergence, in both samples. The correlation between the two dimensions was small, 0.18 in Sample A and 0.07 in Sample B, underscoring the relative independence of these two aspects of wisdom. This result replicates that of Glück et al., who obtained a correlation of 0.11. The present study is the first to also show functional independence between the two constructs, in that both types of wisdom have different correlates, as explicated in the next two sections. Predicting Wisdom About the Self From the literature reviewed in the Introduction, I expected that all three aspects of mindfulness—self-awareness, self-regulation, and self-transcendence—would be positively related to wisdom. Regression analysis suggested that this is (partially) true, but only for wisdom about the self. Before I detail these results, note that the background variables explained a fair amount of variance in wisdom about the self: it was negatively related to neuroticism, and positively related to agreeableness and intellect/imagination in both samples, and additionally to extraversion in the college sample and conscientiousness in the Mechanical Turk sample. After taking mindfulness into account, only the influence of intellect/imagination (in both groups) and extraversion (in the college sample) remained significant, but the coefficients were substantially reduced (with β s roughly half of those in Step 1). This suggests that the effects of agreeableness and neuroticism are wholly mediated through the effects of mindfulness, and those of extraversion and intellect/imagination are partially mediated. Levenson et al. ([47]) obtained a negative effect of neuroticism, and a positive effect of openness (i.e., imagination/intellect in this sample), agreeableness, and conscientiousness on the ASTI, a measure of wisdom about the self; only the latter correlation was absent from the present results. Within the Berlin wisdom paradigm, openness to experience is likewise a strong predictor of wisdom scores (e.g., Pasupathi et al. [56]; Staudinger and Glück [64]). This makes sense: if wisdom is at least partially based on experience, an openness to new experiences would be key for its development or flourishing. Crucially, the mindfulness manifold explained an additional 21% to 26% of the variance in wisdom about the self, over and beyond the variance explained by personality, age, and gender. In both samples, one aspect of self-awareness—reflective awareness—was a significant and strong predictor of wisdom about the self, with β values around 0.40 for the final step. The other aspect of self-awareness, however—controlled sense-of-self in the moment—was not a significant predictor (except in Step 2 in the college sample). It appears, then, that wisdom about the self is associated with a reflective stance about one's experiences (i.e., reflective awareness), but not with the experience of being present in the moment (i.e., controlled sense-of-self in the moment)—in other words, it is the examination of or the investigation into one's experiences rather than the mere witnessing of those experiences that is important for this type of wisdom, as many models of wisdom (e.g., Ardelt [ 3 ]; Brown and Greene [14]; Glück and Bluck [31]) indeed explicitly predict. It is interesting to note that self-compassion (at least in the college sample) was an additional predictor for wisdom about the self. The reasons might be that self-compassion allows one to step back from the immediacy of the experience, and consider oneself the way one would consider a friend—this friendly distancing, like the reflection/examination component, might possibly help to foster the transcendence Ardelt ([ 3 ]) considers so necessary for the development of wisdom. Self-preoccupation was not related to wisdom in either sample. One additional link found here was that between self-transcendence and wisdom about the self (with β values on par with or a little lower than those for reflective awareness). This association is almost self-evident, given that quite a few theorists consider self-transcendence to be a critical component of wisdom (Ardelt [ 3 ]; Curnow [22]; Levenson [46]). Note that this relationship remained unchanged when the ASTI, a measure of wisdom the conceptually relies on self-transcendence, was removed from the composite that tapped wisdom about the self, suggesting that the relationship cannot be explained merely by conceptual overlap between the measure of self-transcendence and the ASTI. The role of reflective awareness and self-compassion in wisdom about the self, however, is not merely to foster self-transcendence: the final step in the regression analyses clearly shows that the effects of reflective awareness (both samples) and self-compassion (college sample) are far from completely mediated by self-transcendence. It is also important to stress that the three background variables and the mindfulness manifold provide us with a very good handle on the individual differences in wisdom about the self: they explain a little more than half to two thirds of the variance (between 56 and 67%, to be precise), indicating that these constructs probably should be important components in any realistic theory of wisdom about the self. Predicting Wisdom About the (Social) World Wisdom about the (social) world, in contrast, was not predicted by the mindfulness manifold at all. There is some indication that wisdom about the (social) world might have roots in individual differences in personality instead: individuals scoring higher on agreeableness and lower on neuroticism scored higher on wisdom about the (social) world; however, this was only true in the student sample. As in wisdom about the self, the effects of agreeableness and neuroticism were wholly mediated through the effects of mindfulness, even though the latter effects did not rise to the level of significance. These personality correlates have some face validity in their predictive value. That is, it makes sense that people who are (or want to appear) more friendly, warm, and helpful might be better at picking up on social cues or be more interested in understanding how the social world and the world in general works. Neuroticism, in general, is related to overreactivity, negative emotions, and feeling easily threatened by social situations; none of these qualities would likely be conducive to acquire the type of equanimity associated with wisdom in general (see Wink and Staudinger [74], for a similar argument). Note that Ardelt et al. ([ 4 ]) found that openness and extraversion correlated with the 3D-WS (in a sample of 98 males who were approximately 80 years old); we found such correlations for wisdom about the self, not for wisdom about the (social) world. The reason for the discrepancy is unclear. The reason why the influence of personality variables on wisdom about the (social) world is constrained to the college group is likewise unclear. One potential reason could be adult development: perhaps as people grow older the grip of personality on their outlook on the world loosens. There is a hint of this in the present data: after a median split on the Mechanical Turk sample, the relevant correlations were nominally higher in the younger sample (correlation of wisdom about the [social] world with agreeableness was 0.11, with neuroticism − 0.12) than the older subsample (0.01 and − 0.04, resp.). None of these correlations, however, reached significance. This, then, remains an area for further research. Note that the Mechanical Turk sample was highly educated (about 3 years of college), so educational differences are unlikely to explain the cross-sample differences. Also note that the relationship with personality is much smaller than that observed in wisdom about the self: the background variables (personality, age, and gender) explained 30–46% of the variance in wisdom about the self, versus only 3–12% in wisdom about the (social) world. Wisdom about the (social) world is not only distinct from wisdom about the self; it also seems, with the present measures, much harder to explain. Wisdom and the Moral Foundations Turning now to ethical sensitivity as a potential consequence of mindfulness and wisdom, I found, first, a conceptual (partial) replication of our earlier paper (Verhaeghen and Aikman [70]) on the effects of mindfulness on the moral foundations. In that paper, we found, in two independent samples, that reflective awareness, self-preoccupation, and self-transcendence were related to the individualizing aspects of morality (i.e., an emphasis on care and fairness) (note that the relationship with self-preoccupation was only significant in Sample A in the present study). Self-compassion and self-transcendence were positively related to the binding aspects of morality (i.e., an emphasis on loyalty, authority, and sanctity). In the present data, an additional effect of self-preoccupation on binding was obtained, and the effect of self-compassion on binding was not significantly different from zero in one sample, and, surprisingly, negative in the other. Wisdom about the self turned out to be a strong predictor for the individualizing foundation, that is, one's sensitivity to the ethical dimensions of care and fairness ( β for the final step = 0.42 and 0.41, resp.). In contrast, wisdom about the (social) world had only a negligible and non-significant influence on the individualizing foundation ( β = 0.01 and 0.04). While most theories about wisdom posit an effect on ethics, notably "prosocial attitudes and behaviors, which include empathy, compassion, warmth, altruism, and a sense of fairness" (Bangen et al. [10], p. 1257), the present data suggest that this effect remains restricted to wisdom about the self, and does not extend to wisdom about the (social) world. Within the group of mindfulness variables, the effects of self-awareness on the individualizing foundation were partially mediated through self-transcendence (i.e., the coefficients associated with self-awareness become smaller once self-transcendence enters the equation) and wholly mediated through wisdom about the self (i.e., the coefficients associated with self-awareness became non-significant once the wisdom variables enter the equation, but only wisdom about the self had a reliable effect). The effects of self-transcendence on individualizing, in turn, were fully mediated through wisdom, and particularly wisdom about the self. One possible interpretation of the latter finding is that self-transcendence is a precursor for wisdom about the self; another that self-transcendence as defined here is subsumed under or maybe even synonymous with wisdom about the self. The latter interpretation is certainly compatible with views about wisdom as a form of self-transcendence (Ardelt [ 3 ]; Curnow [22]; Levenson [46]). Whatever the mechanism, wisdom about the self thus appears to foster an increased emphasis on the ethical dimensions of care and fairness, and this is partially due to the influence of reflective awareness and self-transcendence on wisdom about the self. The effects of wisdom on the binding foundations (i.e., an emphasis on authority, ingroup loyalty, and purity) were rather small. The strongest predictor for the binding foundation remained social conservatism, with people who are more conservative showing larger interest in the binding foundation ( β for the final step = 0.53 and 0.75). Wisdom about the self had a much smaller effect ( β for the final step = 0.23 and 0.15; the latter value was ns ); the contribution of wisdom about the (social) world was essentially nil ( β for the final step = − 0.04 and 0.04, ns ). In the college sample, participants who were less agreeable, more conscientious, male, and more self-preoccupied showed a larger interest in the binding foundation. The latter effect replicated for the Mechanical Turk sample, where lower levels of self-compassion and higher levels of self-transcendence were additionally related to a higher interest in binding. If we look at the results that replicate across both samples, the take-away message is that an interest in the binding foundation is determined mostly by social conservatism, and maybe, but to a much smaller extent, by wisdom about the self. This implies a second amendment to the Bangen et al. ([10]) quotation above, to the effect that wisdom's fostering of prosocial attitudes applies mostly to attitudes that make the rights and concerns of others visible (i.e., treating individuals with care and fairness), and less so to attitudes pertaining to ingroup cohesion (i.e., a focus on loyalty, authority, and purity).
    1. Philosophy for Children, Values Education and the Inquiring Society.Published in:Educational Philosophy & Theory,Oct2014,Professional Development CollectionBy:Cam, Philip Philosophy for Children, Values Education and the Inquiring Society.  How can school education best bring about moral improvement? Socrates believed that the unexamined life was not worth living and that the philosophical examination of life required a collaborative inquiry. Today, our society relegates responsibility for values to the personal sphere rather than the social one. I will argue that, overall, we need to give more emphasis to collaboration and inquiry rather than pitting students against each other and focusing too much attention on 'teaching that' instead of 'teaching how'. I will argue that we need to include philosophy in the curriculum throughout the school years, and teach it through a collaborative inquiry which enables children to participate in an open society subject to reason. Such collaborative inquiry integrates personal responsibility with social values more effectively than sectarian and didactic religious education. Keywords: religion; ethics; community of inquiry; spiral curriculum Introduction [ 1 ]As Socrates would have it, the philosophical examination of life is a collaborative inquiry. The social nature of the enterprise goes with its spirit of inquiry to form his bifocal vision of the examined life. These days, insofar as our society teaches us to think about values, it tends to inculcate a private rather than a public conception of them. This makes reflection a personal and inward journey rather than a social and collaborative one, and a person's values a matter of parental guidance in childhood and individual decision in maturity. The relegation of responsibility for values to the personal sphere also militates against societal self-examination. On the other hand, the traditional pontifical alternative is equally presumptive and debilitating in ignoring the possibility of personal judgement. How can education steer a course between the tyranny of unquestionable moral codes and the bankruptcy of individualistic moral relativism? It remains to be seen whether there is a way in which education could teach children to engage productively across their differences rather than responding to difference with suspicion or prejudice. Gilbert Ryle (in Cahn, 1970) made a clear distinction between 'teaching how' and 'teaching that', arguing from a behaviourist perspective that teaching how had a much more lasting impact than simply teaching the facts. However, too much emphasis on 'teaching how' can result in conditioning, training, teaching to conform to habit, teaching obedience with the threat of hellfire if the rules are broken. There is a third way, the way of philosophy espoused by Matthew Lipman ([ 8 ]) in his Philosophy for Children, which involves giving more emphasis to collaboration and inquiry rather than pitting students against each other and focusing too much attention on 'teaching that' instead of 'teaching how'. Philosophy as it is traditionally taught may well involve teaching how to follow the rules of formal logic correctly, or learning facts about the life and death of Socrates, but it also requires a capacity for critical reflection, consideration of alternative possibilities, and a genuine concern for truth and clarity. I argue that we need to include philosophy in the curriculum throughout the school years, but it needs to be a philosophy taught in the spirit of Socrates which balances individual and social values. Religious instruction tends to inculcate values through adult imposition and denies space to critical judgement. Ryle's distinction between 'learning that' and 'learning how' implied that these were discrete and exclusive ways of learning. However, learning how to do things is more than a matter of memorizing facts or following procedural instructions. Being able to cook is more than being able to follow a recipe book. Again, while some instruction is useful in learning to ride a bike, it is mostly a matter of trying to ride, and then, under guidance, trying again. It is a case of learning by doing, and doing it under different circumstances, in order to apply it in different circumstances. This is working out for oneself how to exercise individual judgement, rather than first learning a set of instructions and then carrying them out (Ryle, in Cahn, 1970, pp. 413–424). Whatever the rules are, they are heuristic and strategic, depending on different contexts, rather than algorithmic and learnable by rote. 'Learning how' can be important in many areas of the curriculum where training in skills is an important feature, especially in physical education and the arts, However, learning the art of inquiry requires a slightly different type of 'learning how' from training, rehearsal, repetition. A curriculum that is based on inquiry is one that is centred on thinking. There is a world of difference in the outcome to be expected from an education that treats knowledge as material with which to think and one that emphasizes memorization of knowledge. It is the difference between an inquiring society and one in which those few who have developed an inquiring mind have done so in spite of their education rather than because of it (Dewey, 1916/1966, chap. 12; Lipman, [ 8 ]). The concept of a community of inquiry owes much to Dewey who, in Democracy and education (1916/1966), described the healthy relation between an individual and his or her environment as functional. Dewey insisted that because the relationship between the individual and his or her environment must be based on mutual adjustment, fitting into society might well involve radically changing it. Dewey believed in the importance of preparing students for democratic citizenship. He stressed that consciously guided education aimed at developing the 'mental equipment' and moral character of students was essential to the development of civic character. Is this not what religious instruction tries to do? The relationship between the individual and society was far more important for Dewey than the child's relationship with an abstract God. It was organic and continually evolving in mutual adaptation. It differs from religious instruction in that its aim is to develop a model of free inquiry, which requires tolerance of alternative viewpoints, and free communication. He also believed that children's capacity for the exercise of deliberative, practical reason in moral situations could be cultivated not by ready-made knowledge but by 'a mode of associated living' characteristic of democracy. Lipman ([ 7 ]) was to elaborate on this idea of schools as a model of a participatory democracy and his classroom community of inquiry provided close analogies with the democratic school, a microcosm of the wider society. Thinking Together When we move away from the traditional classroom to the inquiring one and the teacher becomes less occupied with conveying information—with teaching 'that'— it becomes educationally desirable for students to engage with one another. When human conduct stimulates moral inquiry it is usually because that conduct is controversial, which is to say that there are different points of view as to how it should be judged. If you and I have different opinions in regard to someone's character or conduct, then we are both in need of justification and our views are subject to each other's objections. When we make a proposal to solve a practical problem of any complexity, we rely upon others who are reasonably well placed for constructive criticism or a better suggestion. If we want students to grow out of the habit of going with their own first thoughts, to become used to considering a range of possibilities, and to be on the lookout for better alternatives, then we could not do better than to have them learn by exploring issues, problems and ideas together. If we want them to become used to giving reasons for what they think, to expect the same of others, and to make productive use of criticism, then we could not go past giving them plenty of practice with their peers. And if we want them to grow up so that they consider other people's points of view, and not to be so closed minded as to think that those who disagree with them must be either ignorant or vicious, then the combination of intellectual and social engagement to be found in collaborative inquiry is just the thing. These are all good reasons for having our students learn to inquire together. Philosophy for Children More than any other discipline, philosophy is an inquiry into fundamental human problems and issues, where all the general conceptions that animate society come under scrutiny. Philosophy as a formal discipline played an important part in its place as a matriculation subject in some Australian states, because there were rigorous rules by which its standards could be maintained. This would involve, say, learning that ignoratio elenchi was an informal fallacy, or that modus tollens is an illegitimate move in deductive logic, or learning how to mount a reasoned argument in defence of a position. When, however, we are talking abut philosophy for children, its subject matter needs to be adapted to the interests and experience of students of various ages and its tools and procedures adjusted to their stage of development. There are models to work from, particularly the series of novels and manuals from Matthew Lipman, and in recent years we have begun to find our way forward.[ 2 ] If part of the difficulty is also that some philosophers think of philosophy as being above all that, it is salutary to remember that other disciplines have long since discovered how to recast themselves in educational form. Just as mathematics was forced to become more practical and relevant to the growing range of children who were staying on at school through the New Maths, so philosophy has been forced to become more real and relevant to children. The move towards an integrated curriculum away from discrete learning areas also required philosophy to make the connections across and through disciplines, raising the larger questions of epistemology, ontology, aesthetics and, for the purpose of this article, the important area of axiology or values. For philosophy to have a formative influence, and thereby to significantly affect both the way people think and the character of their concerns, it needs to be part of the regular fare throughout the school years. Only by this means can it effectively supply its nutrients to the developing roots of thought or knowing that and action or knowing how. We need to counter the view that philosophy is an advanced discipline, suitable only for the academically gifted and intellectually mature. Jerome Bruner made famous the startling claim that 'the foundations of any subject may be taught to anybody at any age in some form' (1960, p. 12), and he suggested that the prevailing view of certain disciplines being too difficult for younger students results in our missing important educational opportunities. Bruner called this structure a spiral curriculum : one that begins with the child's intuitive understanding of the fundamentals, and then returns to the same basic concepts, themes, issues and problems at increasingly elaborate and more abstract or formal levels over the years. A spiral curriculum is vital for developing the kind of deep understanding that belongs to philosophy and the humanities. What else is to be gained from building philosophy into the curriculum throughout the school years? It seems to me that an education in philosophical inquiry will assist students to achieve a rich understanding of a wide array of issues and ideas that inform life and society through an increasingly deep inquiry into them. It will help students to think more carefully about issues and problems that do not have a unique solution or a settled decision procedure, but where judgements and decisions can be better or worse in all kinds of ways. Since most of the problems that we face in life and in our society are of that character, the general-purpose tools that students acquire through philosophy will ensure that they are better prepared to face those problems. If philosophy is carried out in the collaborative style envisaged above, then its recipients will also be more likely to tackle such problems collaboratively, and thereby to be more constructive and accommodating with one another. Let me spell all this out a little under the headings of 'thinking', 'understanding' and 'community'. Thinking Philosophy is a discipline with a particular focus on thinking. It involves thinkers in the cognitive surveillance of their own thought. It is a reflective practice, in the sense that it involves not only careful thinking about some subject matter, but thinking about that thinking, in an effort to guide and improve it. Since philosophical thinking tends to keep one eye on the thinking process, philosophy can supply the tools that assist the thinker in such tasks as asking probing questions, making needful distinctions, constructing fruitful connections, reasoning about complex problems, evaluating propositions, elaborating concepts, and honing the criteria that are used to make judgements and decisions. Dewey's (2010) five-step model of identifying the problem and placing it in context, making creative and testable hypotheses that move towards a possible solution, analysing the hypotheses in terms of past experience, considering alternative hypotheses that may be more suitable, and checking possible solutions against actual experiences was picked up as a model of individual thinking, especially in science and design work. But in a community of inquiry each of these steps is done from the multiple perspectives of the group at any age, allowing not only the falsifiability of any conservative position to truth but also their complete contingency. The skills, abilities and habits of skills, abilities and habits of thinking—acquiring the habit of reflecting carefully upon your own thoughts, as well as what others think; developing the ability to imagine and evaluate new possibilities; developing the habit of changing your mind on the basis of good reasons; and acquiring skill in the establishment and use of appropriate criteria to form sound judgements—provide the methodology of Lipman's community of inquiry. Understanding Philosophy deals with ethical questions about how we should behave, social questions about the good community, epistemological questions about the justification of people's opinions, metaphysical questions about our spiritual lives, or logical questions about what we may reasonably infer, and is therefore a rich source of our cultural heritage and of contemporary thought and debate. In terms of both its history and ways of thinking, philosophy also helps to deepen our understanding of the big ideas and key concepts that have helped to shape civilization and continue to inform the way we live. Our conceptions of what makes something right or wrong, of justice, freedom and responsibility, of our personal, cultural and national identity, of sources of knowledge, of the nature of truth, beauty and goodness, are all central to what we value and how we conduct our affairs. Since such concepts so deeply inform life and society, it is important for students to develop their understanding of them. While we may attempt to deal with these matters elsewhere in the curriculum, philosophical inquiry gives students the tools that they need in order to explore these ideas in depth. Community With regard to cooperative thinking and the importance of community, I would stress the virtues of dialogue. As we work to resolve differences in our understandings, or to subject our reasons to each other's judgement, or try to follow an argument where it leads, we are like detectives whose clues are the experience, inferences, judgements and other intellectual considerations that each thinker brings into the dialogue with others. On this view, philosophical inquiry provides a model of the inquiring community: one that is engaged in thoughtful deliberation and decision making, is driven by a desire to make advance through cooperation and dialogue, and values the kinds of regard and reciprocity that grow under its influence. Just because it has these characteristics, philosophical inquiry can provide a training-ground for people who are being brought up to live together in such a community. Dewey's five steps require the philosophical disposition to give reasons when that is appropriate; and, generally, to cooperate with others and respect different points of view. Values Education The vital significance of educating for judgement in regard to values is nowhere more clearly recognized than in the writings of John Dewey: 'The formation of a cultivated and effectively operative good judgment or taste with respect to what is aesthetically admirable, intellectually acceptable and morally approvable is the supreme task set to human beings by the incidents of experience' (Dewey, 1929/1980, p. 262). This makes the cultivation of judgement the ultimate educational task and the development of good judgement central to values education in particular. Values education therefore cannot be simply a matter of instructing students as to what they should value—just so much 'teaching that'—as if students did not need to inquire into values or learn to exercise their judgement. In any case, it is an intellectual mistake to think that values constitute a subject matter to be learned by heart. They are not that kind of thing. Values are embodied in commitments and actions and not merely in propositions that are verbally affirmed. Nor can values education be reduced to an effort to directly mould the character of students so that they will make the right moral choices—as if in all the contingencies of life there was never really any doubt about what one ought to do, and having the right kind of character would ensure that one did it. Being what is conventionally called 'of good character' will not prevent you from acting out of ignorance, from being blind to the limitations of your own perspective, from being overly sure that you have right on your side, or even from committing atrocities with a good conscience in the name of such things as nation or faith. History is littered with barbarities committed by men reputedly of good character who acted out of self-righteous and bigoted certainty. Far from being on solid moral ground, the ancient tradition that places emphasis upon being made of the right stuff has encouraged moral blindness towards those of different ethnicity, religion, politics, and the like. Whatever else we do by way of values education, we must make strenuous efforts to cultivate good judgement. When it comes to deciding what to do in a morally troubling situation, good judgement involves distinguishing more from less acceptable decisions and conduct. Such discernment needs to be made by comparing our options in the circumstances in which they occur. Any such comparison requires us to ensure that, insofar as possible, we have hold of all the relevant facts. It involves us doing our best to make sure that we have not overlooked any reasonable course of action. It requires us to think about the consequences of making one decision, or taking one course of action, by comparison with another, and to be mindful of the criteria against which we evaluate them. It requires us to monitor the consequences of our actions in order to adjust our subsequent thinking to actuality. In short, good moral judgement requires us to follow the ways of inquiry. Dewey (1920/1957, pp. 163–164) says: A moral situation is one in which judgment and choice are required antecedently to overt action. The practical meaning of the situation—that is to say the action needed to satisfy it—is not self-evident. It has to be searched for. There are conflicting desires and alternative apparent goods. What is needed is to find the right course of action, the right good. Hence, inquiry is exacted: observation of the detailed make-up of the situation; analysis into its diverse factors; clarification of what is obscure; discounting of the more insistent and vivid traits; tracing of the consequences of the various modes of action that suggest themselves; regarding the decision reached as hypothetical and tentative until the anticipated or supposed consequences which led to its adoption have been squared with the actual consequences. The lack of integration of our advanced empirical and scientific knowledge with the remnants of value systems of much earlier times is already a problem of considerable proportions. We should not be adding to this burden when we teach science and technology, or history, or about society and the environment. Instead, we need to introduce our students to ways of thinking that develop their values in conjunction with their other understandings. This approach to values education fits with the emphasis to be placed upon collaborative inquiry for several reasons. First, the idea that values are to be cultivated by student reflection rather than impressed upon the student from without by moral authority does not imply that the pursuit of values is a purely personal affair. That would be a pendulum swing to individualistic relativism. Collaborative inquiry supplies a middle road—a way forward between an unquestioningly traditional attitude towards values and an individualism that makes each person their own moral authority. The development of good judgement through collaborative inquiry is the path towards a truly social intelligence. Secondly, values inquiry depends upon different points of view. If something is uncontroversial and everyone is of the same opinion, then there is no motivation for inquiry. Inquiry arises in situations where something is uncertain, puzzling, contentious or in some way problematic. The collaborative inquiry is organic, synergistic and evolving, a kind of moral practice based on a principle of democracy. Consider such elementary aspects of philosophical practice as: learning to hear someone out when you disagree with what they are saying; learning to explore the source of your disagreement rather than engaging in personal attacks; developing the habit of giving reasons for what you say and expecting the same of others; being disposed to take other people's interests and concerns into account; and generally becoming more communicative and inclusive. To see values education as continuous with all of our other efforts to educate our young in the ways of inquiry is to place it firmly in the tradition of reflective education rather than traditional religious instruction. Religious instruction cannot take on the burden of a systematic exploration of the ethical issues involved in the various areas of the curriculum as they are presented throughout the rest of the week. If we are to cultivate good moral judgement we need to make it integral to the material that we teach and not something we attempt to establish in such a disconnected fashion. From a pedagogical perspective, while it would be possible for religious instructors to introduce students to values inquiry, they are under no obligation to do so and many of them come from traditions that are likely to use the occasion to moralize and engage in indoctrination instead. This is not to say that religious education is incompatible with values inquiry. It is rather to acknowledge the need for change. Much of traditional religious instruction is antithetical to the educational requirements of an inquiring society; and if we are to develop such a society, such an outdated approach should not retain its foothold in our schools. This still leaves it open as to whether the school takes a philosophical approach to values education, or insists upon indoctrination rather than education. We should not think of philosophy and religion as representing two incompatible options when it comes to values education. They are representative, however, of a deeper choice that must be made in relation to values education, the choice between appeal to reason and dogmatism as central to the way we teach. Footnotes 1 Editor's Note : This article has been substantially edited and modified since it was delivered as a keynote address in December 2010. The context in which it was written reflects an ongoing tension between the didactic teaching of ethics through religious education and a more organic process of teaching ethics by modelling it and discussing it in philosophical discussion. In New South Wales (NSW) religious education was not compulsory, but Education Department policy forbade schools from offering alternative lessons to students who chose not to take part in scripture. The NSW government tasked St James Ethics Centre, under the guidance of Professor Cam, to develop and deliver ethics education classes in urban, regional and rural primary schools as an alternative to religious education. St James Ethics Centre promptly established Primary Ethics Limited, an independent not-for-profit organization, to develop an engaging, age-appropriate, interconnected curriculum that spans the primary years from Kindergarten to Year 6 and to then deliver ethics education free of charge via a network of specially trained and accredited volunteers. Despite protests from Church leaders in NSW that they should have sole responsibility for values education, on 1 December 2010 Parliament amended the NSW Education Act to give students who do not attend special religious education/scripture classes in NSW public schools the legal right to attend philosophical ethics classes as an alternative to supervised 'private study'. Because of the popularity of secular ethics classes, pressure from Church leaders and a change to a conservative state government, it was legislated in 2012 that parents should be told of the availability of ethics classes in their school only after they have opted out of special religious education or scripture. 2 Since the early 1990s Lipman's followers have extended his work and this general approach is now represented in schools in many countries around the world. For a selection of Australasian resources see http://www.fapsa.org.au/resources/catalogue References Bruner, J. S. (1960). The process of education. Cambridge, MA: Harvard University Press. Cahn, S. E. (Ed.). The philosophical foundations of education. New York: Harper & Row. 3 Dewey, J. (1910). How we think. Chicago, IL: D. C. Heath & Co. 4 Dewey, J. (1957). Reconstruction in philosophy (enlarged ed.). Boston, MA: Beacon Press. (Original work published 1920). 5 Dewey, J. (1966). Democracy and education. London: Collier Macmillan. (Original work published 1916). 6 Dewey, J. (1980). The quest for certainty. New York: Perigee Books. (Original work published 1929). 7 Lipman, M. (1988). Philosophy goes to school. Philadelphia, PA: Temple University Press. 8 Lipman, M. (2002). Thinking in education (2nd ed.). New York: Cambridge University Press. 9 Ryle, G. (1970). Teaching and training. In S. M. Cahn (Ed.), The philosophical foundations of education (pp. 413–424). New York: Harper & Row. ~~~~~~~~ By Philip Cam Reported by Author
    1. Professional organizations integrated the importance of professional identity in training, higher education, and professional development (CACREP, 2009). Eventually, some state licensing boards began to require a counselor identity that was based on CACREP standards for individuals to become licensed professional counselors.

      SECTION 1A - Connects PI to credentialing/regulation. Supports "regulation" and "training standards" as PI components.

    2. The American Counseling Association (ACA) and affiliated organizations such as the National Board of Certified Counselors and the Council for Accreditation of Counseling and Related Educational Programs (CACREP) made a commitment to unify counselors in a shared professional identity

      SECTION 1A - Shows professional associations' role in defining unified identity. Supports "professional associations" as PI component.

    1. Transfer-focused associate’s degrees may be called Associate of Arts (AA) or Associate of Science (AS), or other titles, depending on the focus of study.

      What do they mean by "transfer-focused"?

    1. You’re not done. In most dramatic worlds there are hidden, or at least unseen,spaces. Ask questions about them as well. What are their characteristics of space, time,tone, and mood? How do they relate to the represented world, the world you can see?Finally, while you’re looking at this planet, listen to its “music.” Every dramaticworld will have, or suggest, characteristic sounds — of mourning, celebration, chil-dren’s patter, incantation. It will alternate sounds of human and landscape, or soundand silence. Listen for the pattern of the sound.II. The So cial Wor ld of the Pl ay : A Cl oser Lo okYou are still not ready to examine the beings who inhabit this world. Before you inquireinto their individual traits and motives, there are other things you need to know.

      Unseen aspects of the world still impact the visible story

    Annotators

    1. La Coéducation : Synergie entre Milieux Scolaire et Familial

      Synthèse de direction

      La coéducation est définie comme une alliance stratégique entre tous les adultes gravitant autour de l'enfant — enseignants, parents, professionnels et personnel de soutien — visant à optimiser le développement de son plein potentiel.

      Cette approche repose sur la reconnaissance et l'acceptation des rôles complémentaires de chaque acteur.

      L'établissement de cette relation doit idéalement débuter dès la première rencontre parents-enseignants, bien qu'elle puisse se mobiliser à tout moment, notamment lors de situations critiques.

      Le succès de cette démarche repose sur une posture de bienveillance créant un climat de sécurité psychologique, favorisant ainsi une communication transparente et une action concertée.

      L'intégration des technologies numériques, encadrée par le Plan d'action numérique en éducation, vient renforcer cette collaboration en offrant de nouveaux leviers d'apprentissage et en rassurant les parents sur l'usage pédagogique des outils technologiques.

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      1. Fondements et Définition de la Coéducation

      La coéducation n'est pas une simple communication occasionnelle, mais une véritable mentalité de partenariat.

      Elle se structure autour de trois piliers : reconnaître, accepter et mettre en action les rôles respectifs de chacun.

      Une rencontre d'univers : Elle représente la fusion de l'univers familial et de l'univers scolaire pour former un écosystème unique et cohérent dans la vie du jeune.

      Une mission commune : L'objectif central est l'accompagnement de l'élève dans le développement de ses compétences et de son bien-être.

      Une alliance durable : Cette relation doit perdurer tout au long de l'année scolaire, assurant une continuité entre les différents milieux de vie de l'enfant.

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      2. L'Établissement d'une Posture de Bienveillance

      Pour que la coéducation soit effective, les acteurs doivent adopter une posture spécifique favorisant l'ouverture et l'écoute.

      Le climat de sécurité psychologique

      L'état de bienveillance est le moteur de la coéducation. Il permet de :

      • Créer un contexte où chacun se sent à l'aise de nommer ses véritables préoccupations.

      • Établir une écoute mutuelle authentique.

      • Réduire les malentendus et les confrontations.

      Processus d'ancrage de la bienveillance

      Pour cultiver cet état, les intervenants sont invités à :

      1. Se référer à une expérience passée de bienveillance pour en retrouver les codes (ton, attitude).

      2. Pratiquer l'auto-bienveillance avant de l'étendre à l'autre.

      3. S'interroger sur les meilleures conditions pour rester dans l'ouverture lors des échanges.

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      3. Rôles et Responsabilités : Complémentarité des Acteurs

      Bien que les objectifs finaux convergent, les rôles des enseignants et des parents sont distincts et complémentaires.

      | Acteur | Mandat et Objectifs Spécifiques | Domaine d'Influence | | --- | --- | --- | | Enseignant | Instruire, socialiser et qualifier dans un cadre temporel limité (180 jours). Application du programme et progression des apprentissages. | Milieu scolaire (classe) | | Parent | Premier éducateur de l'enfant. Accompagnement dans les transitions, les défis de vie et les étapes de développement. | Milieu familial et social | | Rôles Communs | Se rassurer mutuellement, valider les informations, partager le vécu de l'enfant et s'informer des stratégies efficaces. | Global (Co-responsabilité) |

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      4. Stratégies de Communication et d'Action

      La coéducation se manifeste par un questionnement constant orienté vers l'impact positif pour l'enfant.

      L'intention politique commune : Avant chaque intervention, les adultes devraient se demander : "Quel est l'impact positif que mon intervention va avoir pour le bien de l'enfant ?"

      La résolution de problèmes : Face aux difficultés (comportements nuisibles ou retards d'apprentissage), l'approche préconisée est de se demander : "Comment pourrions-nous travailler ensemble pour répondre aux besoins de l'enfant ?"

      Inclusion de l'enfant : Il est recommandé d'inclure le jeune dans le questionnement pour s'assurer que les stratégies développées répondent réellement à ses besoins.

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      5. Bénéfices et Manifestations de la Réussite

      Une coéducation réussie transforme la dynamique éducative et génère des résultats tangibles :

      Engagement accru : La clarté des rôles et le climat bienveillant stimulent la motivation des adultes à s'investir.

      Sentiment d'efficacité personnelle : Les expériences positives répétées renforcent la croyance des parents et des enseignants en leur capacité de réussir l'éducation du jeune.

      Progrès accélérés : L'action concertée et continue entre la maison et l'école permet une multiplication des progrès de l'enfant.

      Gestion émotionnelle : Les acteurs parviennent mieux à se détacher d'une surcharge émotionnelle lors des communications pour se recentrer sur l'objectif pédagogique.

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      6. La Coéducation à l'Ère du Numérique

      Le numérique agit comme un levier pour soutenir la relation entre l'école et la famille.

      Le Plan d'action numérique

      Ce plan offre un cadre de référence inspiré des meilleures pratiques mondiales. Il vise deux dimensions centrales :

      1. Développer un citoyen éthique à l'ère du numérique.

      2. Mobiliser les compétences technologiques des jeunes.

      Manifestations concrètes en classe

      L'intégration technologique se traduit par de nouvelles méthodes d'apprentissage où l'enfant est placé en mode création :

      • Ateliers de robotique, de programmation et de codage.

      • Utilisation de la réalité virtuelle (ex: pour des exposés oraux).

      • Usage de tablettes pour la lecture et d'autres contributions pédagogiques.

      Cette structure numérique, encadrée par des pédagogues, sert également à rassurer les parents sur l'accompagnement technologique de leurs enfants, renforçant ainsi le lien de confiance nécessaire à la coéducation.

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      Reply to the reviewers

      COMBINED REVIEW REPORTS

      __1.1. The biochemical and biophysical experiments performed in this study were well designed, data were clear and the conclusions were well supported by the results. One potential improvement is to check whether NLS could affect the normal activation targets of ΔNp63α, such as KRT14 and other epithelial genes. This could complement the experiments testing the inhibition effect of ΔNp63α on p53-mediated gene activation. This will be interesting, as ΔNp63α is a master regulator in epithelial cells via regulation of diverse epithelial genes. __

      We thank the Review for such useful comment. In order to further investigate the relationship between p63 nuclear import and function, and the importance of the oligomerization driven tolerance to point mutations in the latter, we have now performed a number of novel experiments. First of all, we have included both DNp63a NLSn and NLSc mutants in DNA binding/p53 -inhibition assays shown in original Figure 7. The new data is shown in Figure 4E and Supplementary Figure__ S5__. As expected, such mutants had a much smaller effect on DNA binding/p53-inhibition as compared to the NLSbip mutant, further establishing a functional link between p63 nuclear levels and transcriptional activity, and proving the functional relevance of the compensatory mechanism evolved by p63 to tolerate the effect of mutations inactivating either NLSn or NLSc.

      In addition, and as specifically suggested by the Reviewer, we have measured the effect of NLS impairing mutations on the ability of DNap63 to transactivate the K14 and the Bax promoters, which. Our results, shown in revised Figure 4F and 4G, as well as in Supplementary Figure S6 clearly show that both DNp63a NLSn and NLSc mutants transactivate the promoters at undistinguishable levels compared to the wild-type, consistent with their minimal effect on DNA binding and nuclear transport, while the NLSbip mutation, which prevents nuclear localization and DNA binding, also prevents transcriptional transactivation.

      __1.2. A minor suggestion: authors could consider use p63 rather than ΔNp63α in the manuscript. The heterogenous sequences of NLS regions are relevant for the delta isoform of p63. In addition, all experiments performed in the study are not necessarily specific for the biology of the ΔNp63α isoform, but they are probably informative for all p63 isoforms. __

      We thank the Reviewer for this suggestion. We have modified the text in the discussion to introduce this concept. Indeed, we expect the bipartite NLS to mediate nuclear transport of most p63 isoforms, whereas the p63 delta isoform, which lacks NLSn, would be transported into the nucleus by NLSc. We have modified the text in the Discussion section to make this point clearer and more explicit "the bipartite NLS identified here is responsible for nuclear localization of most p63 isoforms, while p63 delta is transported into the nucleus by NLSc: SIKKRRSPD)." To further corroborate this statement, we have also included new data obtained with the TAp63a and gNp63a isoforms. Our data clearly show that nuclear import of both isoforms depends on the NLSbip identified here and is mediated by the IMPa/b1 heterodimer, so that the findings obtained for the ΔNp63α isoform can be generalized to others. The new data is shown in Figure 3 and in Supplementary Figure S3.

      __1.3. Another minor suggestion: As p63 forms a tetramer when binding to DNA sequence for gene regulation, it would be good for authors to speculate the role of NLS and its variations in tetramerization. __

      We thank the Reviewer for such comment. Since the NLS is located outside of the tetramerization domain, it is not expected to play a direct role in tetramerization. We have addressed this issue by generating computational models of ΔNp63α and DNp63α;mNLS dimers and tetramers to allow a direct comparison. The new data is shown in Figure 5A-D and Supplementary Figure S11A-D. The data suggests that mutation of the NLS residues, which lies outside of the oligomerizaiton domain, does not affect ΔNp63α oligomerization abilities supporting the experimental evidences from Figure 5E (BRET experiments).

      __

      2.1. In immunofluorescence images it is sometime difficult to see nuclear accumulation. Single channels of the GFP signal may help to make the point. __

      We thank the Reviewer for pointing out this issue. We have provided single channels for every microscopic image in Supplemental Figures.

      __ 2.2. The binding assays in Fig. 3 would profit from using the most efficient imp a variant together with imp beta to show potential cooperative binding.__

      We thank the Reviewer for such comment, which helped enhancing the physiological relevance of our binding data. We have now introduced the requested data in Supplementary Figure S2A. In the revised Figure panel, we compared binding of FITC-labelled p63-NLS peptide to either full length IMPa1 alone, IMPa1DIBB and pre-heterodimerized IMPa1/IMPb1 complex. The data are consistent with a classical binding mode whereby interaction with IMPb1 releases full length IMPa1 binding minor and major binding sites by engaging with the autoinhibitory IBB domain. To corroborate our results even further and demonstrate the bipartite nature of p63 NLS identified here, we have also performed FP experiments between p63-NLS and LTA SV40 NLS (a well characterized monopartite NLS) in the presence of either wt IMPa1DIBB or its minor and major site mutants. As expected from a bipartite NLS, either mutation impaired binding significantly, whereas the mutation of the minor site had a much smaller effect on binding of SV40 LTA NLS. The new data, shown in Supplementary Figure S2BC and Supplementary Table S3 confirm our hypothesis by highlighting a very strong binding affinity reduction of p63 NLS peptide for IMPa1 major site mutant (

      __2.3. please mention that NTR can also recognize 3D structures of structural RNAs, e.g. tRNAs or miRNAs __

      We thank the Reviewer for this very useful suggestion. We have now introduced this concept in the Introduction and added two references to support our statement. The paragraph is as follows: "Additionally, Exportin 5 and Exportin-T evolved to recognize specific RNA structures within pre-miRNAs and t-RNAs, respectively (5, 6)."

      2.4. longer TA isoforms

      We have added corrected the typo and we thank the Reviewer for noticing it.

      __ 2.5. homologues or orthologues? __

      We thank the reviewer for pointing out this issue. We have corrected the text, so now IMPas and members of the p53 family are referred to as paralogs and not as orthologs

      __3.1. The major function of DNp63a seems to be that of a bookmarking factor that ensures the establishment of an epithelial transcriptional program. It is found to bind more to enhancer than to promoter regions. While it might also act for a few genes as a classical transcription factor (K14). this bookmarking and interaction with other transcriptional regulators seems to be its major task. This should be included in the introduction. __

      We thank the Reviewer for this suggestion. The Introduction has been modified as requested to incorporate this important concept "Additionally, p63 has been shown to act as a pioneer factor, shaping the chromatin and enhancer landscape, thus regulating accessibility to activating and repressing transcription factors (18-20)."

      __ 3.2. "DNp63a can be imported into the nucleus as a dimer" What is the evidence that DNp63a is imported as a dimer and not as a tetramer? Although functional not really relevant, because all conclusions drawn for a dimer are true for a tetramer (such as the mutation compensation), this statement (and others in the text) should either be substantiated or modified. __

      The Reviewer is correct in pointing out that, while p63 isoforms bind DNA as tetramers (7), the precise oligomeric state at which nuclear import occurs is not firmly established. Indeed, little is known about the regulation of the p63 oligomerization process during nucleocytoplasmic trafficking. While TA isoforms are generally maintained in an inactive, closed, and dimeric conformation-requiring external stimuli such as phosphorylation to undergo activation and tetramerization-ΔNp63α has been reported to form tetramers even in the absence of such stimuli (4, 8). In light of this, we have modified the text to explicitly acknowledge the possibility that ΔNp63α may be transported into the nucleus either as a dimer or as a tetramer, rather than implying a single obligatory oligomeric state.

      Importantly, to directly address the Reviewer's concern, we have broadened the scope of the manuscript to include additional p63 isoforms, particularly TAp63α, which is predominantly present as a dimer under basal conditions. Our new data (Figure 3) demonstrate that TAp63α is efficiently translocated into the nucleus via the IMPα/β1 heterodimer in an NLSbip-dependent manner. Notably, despite its inability to form tetramers, TAp63α displays a similar tolerance to mutations that inactivate individual basic clusters within the bipartite NLS, analogous to what is observed for ΔNp63α (Supplementary Figure S11).

      Together, these results formally demonstrate that dimerization is sufficient to support efficient nuclear import in the presence of NLS-inactivating mutations, and that higher-order oligomerization (i.e., tetramerization) is not required for this property. We have therefore revised the manuscript accordingly to avoid over-interpretation and to more accurately reflect the experimental evidence.

      __ 3.3. The explanation for the difference in the sensitivity of mutations in the bipartite NLS in the isolated peptide experiments and experiments with the full length DNp63a is intriguing. Unfortunately, it is not based on direct experimental evidence. To proof their model (which is the central claim of this manuscript) they should fuse the bipartite NLS to any dimerization module (e.g. a leucine zipper sequence) and show that by dimerization of the bipartite NLS the same results towards mutations are obtained as for full length DNp63a. This would strongly support their model. __

      We agree that the model for nuclear transport is a central claim of our work, and deserves additional experimental validation. In order to support our hypothesis, in the revised manuscript we have generated a number of additional DNp63a mutants uncapable of self-interaction, based on deletion of residues 301-347(p63-DOD).

      We have now:

      (i) Validated the inability of the DOD mutant to self-interact by means of BRET assays in living cells, whereby a strong decrease in BRET ratio is observed compared to wild-type DNp63a (New Figure 6E and New Supplementary Figure S8).

      (ii) Shown that, in such context, substitution of either the N-terminal or C-terminal basic stretch of amino acids in the NLS is sufficient to impact p63 nuclear import, whereas in the context of the full-length protein, they are not (New Figure 6F-H, and New Supplementary Figure S9).

      (iii) Shown that while FLAG-p63 wt could relocalize to the nucleus YFP-p63mNLSbip but not YFP-p63;DOD;mNLSbip (New Supplementary Figure S10).

      We believe that these new data further demonstrate the impact of p63 self-association on subcellular localization and strongly support our hypothesis. We greatly thank the Reviewer for their inspiring comment, which led to a significant improvement of our manuscript.

      References

      Lotz R, Osterburg C, Chaikuad A, Weber S, Akutsu M, Machel AC, et al. Alternative splicing in the DBD linker region of p63 modulates binding to DNA and iASPP in vitro. Cell Death Dis. 2025;16(1):4. Ciribilli Y, Monti P, Bisio A, Nguyen HT, Ethayathulla AS, Ramos A, et al. Transactivation specificity is conserved among p53 family proteins and depends on a response element sequence code. Nucleic Acids Res. 2013;41(18):8637-53. Monti P, Ciribilli Y, Bisio A, Foggetti G, Raimondi I, Campomenosi P, et al. ∆N-P63alpha and TA-P63alpha exhibit intrinsic differences in transactivation specificities that depend on distinct features of DNA target sites. Oncotarget. 2014;5(8):2116-30. Pitzius S, Osterburg C, Gebel J, Tascher G, Schafer B, Zhou H, et al. TA*p63 and GTAp63 achieve tighter transcriptional regulation in quality control by converting an inhibitory element into an additional transactivation domain. Cell Death Dis. 2019;10(10):686. Okada C, Yamashita E, Lee SJ, Shibata S, Katahira J, Nakagawa A, et al. A high-resolution structure of the pre-microRNA nuclear export machinery. Science. 2009;326(5957):1275-9. Kutay U, Lipowsky G, Izaurralde E, Bischoff FR, Schwarzmaier P, Hartmann E, et al. Identification of a tRNA-specific nuclear export receptor. Mol Cell. 1998;1(3):359-69. Enthart A, Klein C, Dehner A, Coles M, Gemmecker G, Kessler H, et al. Solution structure and binding specificity of the p63 DNA binding domain. Scientific reports. 2016;6:26707. Deutsch GB, Zielonka EM, Coutandin D, Weber TA, Schafer B, Hannewald J, et al. DNA damage in oocytes induces a switch of the quality control factor TAp63alpha from dimer to tetramer. Cell. 2011;144(4):566-76.

    1. si Twitter propose des cœurs, c’est pour mieux sélectionner les messages qui vont être affichés à nos abonnés. Et donc les affecter ?

      exemple venant appuyer son argument que cela affecte ceux qui utilisent la signalisation émotionnelle: en ciblant davantage ce qui sera diffusé

    2. multitude : c’est d’un gros volume de critiques en ligne (par exemple) que l’on va pouvoir tirer des enseignements utiles au déploiement de stratégies spécifiques.

      la récolte de données en masse permet d'établir des stratégies

    3. Ces premières recherches nous ont amené à percevoir le lien entre deux « boites noires », que les professionnels de la communication rêvent de pouvoir illuminer : notre cerveau et les systèmes de computation des dispositifs numériques

      Conclusion de sa recherche: comment l'utilisation du numérique par l'individu permet une récolte de données sur son fonctionnement cognitif, qui en retour est utilisé pour l'influencer

    4. penser l’économie du numérique au travers d’un prisme affectif

      ici ce serait l'intention de l'auteur: observer la place des affects dans l'activité économique du web tout en annonçant l'objectif critique ensuite

    1. Il y avait une anomalie à découvrir via les tests exploratoires : l’affichage du message d’erreur au moment de cliquer sur le bouton "Valider le paiement".

      Il y en a 2 et non 1, le panier est vide, un test exploratoire devrait justement remonter ce cas en plus du fait de l'erreur à la validation, sinon le test exploratoire n'a pas d'utilité dans cette situation vis à vis d'un simple scénario de test en 3 étapes...

    2. Exécutez le cahier de recette. Combien d’anomalies avez-vous trouvées en lien avec les scénarios de test ?

      Si on suit strictement à la lettre les indications du scénario de test il devrait y en avoir au moins 6 voir 9 si on prend en considération que les libellés doivent être identiques entre le cahier de test et l'application.

    3. En utilisant Postman ou directement à partir du swagger, exécuter une requête GET sur le endpoint /products/{id} avec l’id 9. À quoi ressemble la réponse ?

      Réponse incorrecte côté correction. Le résultat dans le swagger est différent.

    1. Author response:

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

      Public Reviews:

      Reviewer #1 ( Public review):

      The strength of the current study lies in their establishing the molecular mechanism through which PRMT1 could alter craniofacial development through regulation of the transcriptome, but the data presented to support the claim that a PRMT1-SFPQ axis directly regulates intron retention of the relevant gene networks should be robust and with multiple forms of clear validation. For example, elevated intron retention findings are based on the intron retention index, and according to the manuscript, are assessed considering the relative expression of exons and introns from a given transcript. However, delineating between intron retention and other forms of alternative splicing (i.e., cryptic splice site recognition) requires a more comprehensive consideration of the intron splicing defects that could be represented in data. A certain threshold of intron read coverage (i.e., the percent of an intron that is covered by mapped reads) is needed to ascertain if those that are proximal to exons could represent alternative introns ends rather than full intron retention events. In other words, intron retention is a type of alternative splicing that can be difficult to analyze in isolation given the confounding influence of cryptic splicing and cryptic exon inclusion. If other forms of alternative splicing were assessed and not detected, more confident retention calls can be made.

      This manuscript is a mechanistic exploration that follows previous work we published on the role of Prmt1 in craniofacial development, in which genetic deletion of Prmt1 in CNCCs leads to cleft palate and mandibular hypoplasia (PMID: 29986157).

      As the reviewer pointed out, a certain threshold of intron read coverage is needed to assess intron retention events. We employed IRTools to assess the collective changes of intron retention between cell-states associated with certain biological function or pathway. IRTools incorporated considerations for intron read coverage by checking the evenness of read distribution in an intron. Specifically, every constitutive intronic regions (CIR) is divided into 10 equally sized bins and the proportion of reads that map to each bin is calculated. CIRs are then ranked according to their imbalance in bin-wise reads distribution, represented by the proportion of reads in its most populated bin. Those among top 1% are considered to contain potentially false IR events and excluded. We further addressed this question by developing another measure of intron retention, intron retention coefficient (IRC), which assesses IR events using the junction reads (Supplemental Figure-S8). Junction reads that straddle two exons are called exon-exon junction reads (spliced reads), and those that straddle an exon and a neighboring intron are called exon-intron junction reads (retained reads). The IRC of an intron is defined as the fraction of junction reads that are exon-intron junction reads: IRC = exon-intron read-count / (exon-exon read-count + exon-intron read-count), where exon-intron read-count = (5’ exon-intron read-count + 3’ exon-intron read-count) / 2. The IRC of a gene is defined as the exon-intron fraction of all junction reads overlapping or over the constitutive introns of this gene. In the calculation of the IRC, only exon-intron junction reads that cover the junction point and overlap both of each side for at least 8 bps were counted, and only exon-exon junction reads that jump over the relevant junction points and overlap each of the respective exons for at least 8 bps were counted. In this process, evenness of the proportion of exon-intron junction reads that are 5’ or 3’ exon-intron junction reads are taken into account. As shown in the Supplemental Figure S7A and S7B, IRC analysis generated consistent results with those obtained from using IRI (Figure 3A and 3I).

      In addition, as the reviewer pointed out, intron retention can be difficult to analyze in isolation. We followed the reviewer’s suggestion that “If other forms of alternative splicing were assessed and not detected, more confident retention calls can be made“ and analyzed other forms of alternative splicing for all ECM and GAG genes with significant IRI increase (genes highlighted in Figure-3A and 3I) using rMATS (Supplemental Figure-S9). Among these genes, only 5 genes (Cthcr1, Mmp23, Adamts10, Ccdc80 and Col25a1) showed statistically significant changes in skipped exon, 1 gene (Bmp7) showed significant changes in mutually exclusive exons, and none showed significant changes in alternative 5’ or 3’ splicing. SE and MXE changes detected were marginal (Supplemental figure S8), while the majority of matrix genes with significant intron retention didn’t exhibit other forms of alternative splicing, further supporting the confidence of intron retention calls.

      While data presented to support the PRMT1-SFPQ activation axis is quite compelling, that this is directly responsible for the elevated intron retention remains enigmatic. First, in characterizing their PRMT1 knockout model, it is unclear whether the elevated intron retention events directly correspond to downregulated genes.

      In the revised manuscript, we demonstrate IR-triggered NMD as a mechanism for transcript decay and downregulation of matrix genes. When IR-triggered NMD was blocked by chemical inhibitor NMDI14, the intron-retaining transcripts showed significant accumulation (new Figure-4). NMD is the RNA surveillance system to degrade aberrant RNAs. Intron retention-triggered NMD in cancer has both promotive and suppressive roles and NMD inhibitors has been tested for cancer therapy including immunotherapy. During embryonic development, the functional significance of NMD machinery is suggested by human genetic findings and mouse genetic models. NMD is driven by a protein complex composed of SMG and UPF proteins. Smg6, Upf1, Upf2 and Upf3a knockout mouse die at early embryonic stages (E5.5-E9.5), and Smg1 gene trap mutant mice die at E12.5 (PMID: 29272451). SMG9 mutation in human patients causes malformation in the face, hand, heart and brain (PMID: 27018474).

      We show that in CNCCs NMD functions both as a physiological mechanism and invoked by molecular insult. Blocking NMD in CNCCs caused significant accumulation of intron-retaining Adamts2, Alpl, Eln, Matn2, Loxl1 and Bgn transcripts, suggesting a basal role for NMD to degrade intron-retaining transcripts (Figure-4Ba-4Bf). We further demonstrated the accumulation of Adamts2 and Fbln5 using semi-quantitative PCR with the detection of a longer product from Adamts2 intron 19 and Fbln5 intron 7 (Figure-4Ca-4Ch). In CNCCs and ST2 cells, NMD is further invoked by Prmt1 and Sfpq deficiency. In Prmt1 deficient CNCCs, NMD blockage led to higher accumulation of intron-retaining Adamts2 and Alpl transcripts, suggesting that Prmt1 deficiency triggers NMD to reduce intron-containing transcripts (Figure-4Aa, 4Ab). In Sfpq-depleted ST2 cells, blocking NMD caused accumulation of intron-retaining transcripts Col4a2, St6galnac3 and Ptk7 (Figure-9B, 9C).

      Moreover, intron splicing is a well-documented node for gene regulation during embryogenesis and in other proliferation models, and craniofacial defects are known to be associated with 'spliceosomopathies'. However, reproduction of this phenotype does not suggest that the targets of interest are inherently splicing factors, and a more robust assessment is needed to determine the exact nature of alternative splicing in this system. Because there are several known splicing factors downstream of PRMT1 and presented in the supplemental data, the specific attribution of retention to SFPQ would be additionally served by separating its splicing footprint from that of other factors that are primed to cause alternative splicing.

      We have previously shown that a group of splicing factors depends on Prmt1 for arginine methylation, including SFPQ (PMID: 31451547). We tested additional splicing factors that are highly expressed in CNCCs and depends on PRMT1 for arginine methylation: SRSF1, EWSR1, TAF15, TRA2B and G3BP1 (Figure-5, 6 and 10). Among these factors, EWSR1 and TRA2B are both methylated in CNCCs and depend on PRMT1 for methylation (Fig. 5 and Supplemental Figure-S3B, S3C). We weren’t able to assess TAF15 methylation because of lack of efficient antibody for the PLA assay. We also demonstrated that their protein expression or subcellular localization was not altered by Prmt1 deletion in CNCCs, unlike SFPQ (Supplemental Figure-S4). To define their splicing footprint, we performed siRNA-mediated knockdown in ST2 cells, followed by RNA-seq and IRI analysis to define differentially regulated genes and introns, which revealed distinct biological pathways regulated by SFPQ, EWSR1, TRA2B and TAF15, but minimal roles of EWSR1, TRA2B and TAF15 on intron retention when compared to SFPQ (Fig. 10F-10S, Supplemental Figure S7A-S7F, Supplemental Tables S4-S6). ECM genes are significantly downregulated by all four splicing factors (Fig. 10F-10I), but EWSR1, TRA2B and TAF15 function through IR-independent mechanisms, such as exon skipping, as exemplified by Postn (Fig. 10J-10S).

      Clarifying the relationship between SFPQ and splicing regulation is important given that the observed splicing defects are incongruous with published data presented by Takeuchi et al., (2018) regarding SFPQ control of neuronal apoptosis in mice. In this system, SFPQ was more specifically attributed to the regulation of transcription elongation over long introns and its knockout did not result in significant splicing changes. Thus, to establish the specificity for the SFPQ in regulating these retention events, authors would need to show that the same phenotype is not achieved by mis-regulation of other splicing factors. That the authors chose SFPQ based on its binding profile is understandable but potentially confounding given its mechanism of action in transcription of long introns (Takeuchi 2018). Because mechanisms and rates of transcription can influence splicing and exon definition interactions, the role of SFPQ as a transcription elongation factor versus a splicing factor is inadequately disentangled by authors.

      To test whether SFPQ acts as a transcription elongation factor, we performed Pol II Cut&Tag in ST2 cells and demonstrated that depletion of SFPQ only caused marginal changes in either the promoter region or gene body of ECM genes, suggesting that the role of SFPQ as a transcriptional activator or elongation factor is minimal (Fig. 7G, 7H). This finding is distinct from SFPQ function in neurons (PMID: 29719248), suggesting that the activation or recruitment of SFPQ in transcriptional regulation may involve tissue-specific factors in neurons.

      Reviewer #2 (Public review):

      Summary:

      The manuscript by Lima et al examines the role of Prmt1 and SFPQ in craniofacial development. Specifically, the authors test the idea that Prmt1 directly methylates specific proteins that results in intron retention in matrix proteins. The protein SFPQ is methylated by Prmt1 and functions downstream to mediate Prmt1 activity. The genes with retained introns activate the NMD pathway to reduce the RNA levels. This paper describes an interesting mechanism for the regulation of RNA levels during development.

      Strengths:

      The phenotypes support what the authors claim that Prmt1 is involved in craniofacial development and splicing. The use of state-of-the-art sequencing to determine the specific genes that have intron retention and changes in gene expression is a strength.

      Weaknesses:

      Some of the data seems to contradict the conclusions. And it is unclear how direct the relationships are between Prmt1 and SFPQ.

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      First, the claims regarding the effect of PRMT1 loss on splicing are unclear by the section title. In other words, does loss PRMT1 change the incidence of baseline alternative splicing events, or does it introduce new retention events that are responsible for underwriting the craniofacial phenotype? Consistent with this idea, the narrative could benefit from more cellular and/or histological validations of the transcriptomic defects discovered in the RNAseq, which could help contextualize the bioinformatics data with the developmental defects. Moreover, the conclusions drawn about intron retention could be clarified in terms of how applicable the mechanism is likely to be outside of this tissue-specific set of responsive introns.

      Loss of Prmt1 did not cause a global shift in intron retention, as shown in Supplemental Figure S2. Instead, Prmt1 deletion caused increase of intron retention specifically in genes enriched in cartilage development, glycosaminoglycan biology, dendrite and axon, and decreased intron retention in mitochondria and metabolism genes (Table. S1). We also tested matrix protein expression by histology to confirm that transcriptomic defects revealed at the RNA level resulted in lower protein production. The new data are in Figure 3E-3H.

      Additionally, invoking NMD to align splicing and differential gene expression data understandable but lacking sufficient controls to be conclusive, such as positive control genes to confirm inhibition of NMD.

      To validate the blockage of NMD, glutathione peroxidase 1 (Gpx1) intron 1, a well-documented substrate for NMD, is tested as positive control (Fig 4Ac, 4Ad, 9B).

      Additionally, it should be clarified whether NMD is a basal mechanism for the regulation of these introns or whether it is an induced mechanism that is invoked by the molecular insult.

      In CNCCs, NMD functions both as a physiological mechanism and invoked by molecular insult. Please refer to responses to Reviewer 1’s public review for detailed explanations.

      Further, authors present data downstream of two siRNAs for the same gene target, but it remains unclear how siRNAs for the same gene target produce different effects. It may be helpful for authors to clarify how many of the transcriptomic defects are shared versus unique between the siRNAs.

      To address this question, we used bioinformatic analysis of the whole genome data to the similarity in changes caused by the two SFPQ-targeting siRNAs. As shown in the new Fig. 7Ba & 7Bb, transcriptomic and intron changes are consistent between the two siRNAs, suggesting that genes targeted by the two siRNA predominantly overlap. This overlap is illustrated by scatter plot analysis of RNAseq DEG and IRI data from each siRNA against SFPQ.

      Finally, we stress the importance of presenting the full conceptual basis for SFPQ's potential role in splicing and gene expression. It is significant to note that SFPQ has been previously studied as a splicing factor and was instead determined to function in support of the transcription elongation rather than in splicing. Thus, if authors are confident that the SFPQ manifests directly in splicing changes they encumber the burden of proof to show that its role in transcription, nor another splicing factor, are driving splicing changes.

      We demonstrated that depletion of SFPQ only caused marginal changes in either the promoter region or gene body of ECM genes, suggesting that the role of SFPQ as a transcriptional activator or elongation factor is minimal (Fig. 7G, 7H). Please refer to responses to Reviewer 1’s public review for detailed explanations.

      Reviewer #2 (Recommendations for the authors):

      (1) It is not clear why the authors focused on intron retention targets vs the other possibilities. Skipped Exon is much higher in terms of the number of changes, please clarify. For the intron retention how is this quantified? The traces are nice, but it is hard to tell which part is retained at this magnification. Also, because the focus is on extracellular matrix (ECM) and NMD it would be nice to show some of those targets here. In the tbx1 trace, some are up and some are down. What does that mean for the gene expression?

      We have investigated SE initially and found that genes with significant changes in Prmt1 CKO CNCCs fall into diverse functional pathways. Among them, a few genes are critical for skeletal formation, including Postn and Fn, and the function of their exon skipping has been documented. For example, the two exons that are skipped in Postn, Exon17 and 21, have been shown to regulate craniofacial skeleton shape and mandibular condyle hypertrophic zone thickness using transgenic mouse models (PMID: 36859617). As illustrated by Figure 10, the skipped exon of Postn is regulated by multiple splicing factors that may perform overlapping functions in vivo.

      Intron retention of each gene is quantified by the ratio of the overall read density of its constitutive intronic regions (CIRs) to the overall read density of its constitutive exonic regions (CERs) and defined as the intron retention index (IRI). In the first section of Response to Reviewer 1’s comments, we explained additional bioinformatic analysis that was performed to address reviewers’ questions, support the confidence of intron event calls and rule out the possibility of other alternative splicing mechanisms, such as by SE, MXE, A5SS or A3SS (Supplemental Figure S5, S6, Table S7).

      (2) RNA-Sequencing of Prmt1 mutants nicely shows gene expression changes, including in ECM and GAG genes. While validation of the sequencing results is not necessarily required, it would be very interesting to show the expression in situ. In addition, the heat map shows both downregulated but also upregulated transcripts. This is expected since this protein regulates many genes. However, the volcano plot shows a significant number of genes upregulated. It would be interesting to show what the upregulated genes are. And what is the proposed mechanism for Prmt1 regulation of upregulated genes?

      Validation for the transcriptomic changes is shown in Fig. 3E-3H using immunostaining.

      As for upregulated genes in Prmt1 mutant, top pathways include cytokine-mediated signaling pathway, signal transduction by p53 signaling pathway and cell morphogenesis (Figure 2E), which are consistent with our previous reports that Prmt1 deletion induces cytokine production in oral epithelium and leads to p53 accumulation in embryonic epicardium (PMID: 32521264, 29420098). Besides these pathways, Prmt1 deletion also caused upregulation of genes involved in adult behavior, postsynaptic organization and apoptotic process, which is consistent with findings from other labs on PRMT1 function in neuronal and cancer cells (PMID: 34619150, 33127433).

      (3) Specific transcripts were shown to have elevated intron retention involved in the ECM and GAG pathway. However in Figure 3D it seems to show the opposite with intronic expression decreased and exonic increases and intronic decrease. This is very important to the final conclusion of the paper. In addition, is there a direct relationship between increased intron and downregulation of this specific gene expression? It seems a bit correlational as it could also be an indirect mechanism. One way to test this is to do in vitro translation with and without the specific intron to test if it results in lower expression.

      We apologize for the mis-labeling in previous version of Figure 3D, which is now corrected. We also tried to test the direct relationship between intron and downregulation of matrix genes such as Adamts2 using in vitro experiments, however, the introns of matrix genes with high retention tends to be long, many 10 to 50kb in length, making it challenging to generate mini-gene constructs for molecular analysis. We used a different approach and demonstrated that inhibition of NMD with a chemical inhibitor NMDI14 caused dramatic accumulation of the Adamts2, Alpl, Eln, Matn2, Loxl1 and Bgn transcripts, suggesting that retained introns triggered NMD to regulate gene expression and this mechanism acts as a physiological level in CNCCs (Fig. 4). We also blocked NMD in control and Prmt1 null CNCCs, where NMD blockage led to higher accumulation of Adamts2 and Alpl transcripts, suggesting that upon Prmt1 deficiency, NMD is further utilized to degrade intron-containing transcripts (Fig. 4). Similarly, in Sfpq-depleted ST2 cells, blocking NMD caused accumulation of intron-retaining transcripts Col4a2, St6galnac3 and Ptk7 (Fig. 9A, 9B).

      (4) While Figure 4 nicely shows the methylation of SFPQ is reduced in Prmt1 CKO cells, it is unclear which reside this methylation occurs. Also the overall expression of SFPQ is also down so it is possible that the methylation is indirect ie Prmt1 regulates some other methyltransferase that regulates SFPQ. Or that because the overall level of SFPQ is down, there is no protein to methylate. How do the authors differentiate between these possibilities?

      Previously, arginine methylation of SFPQ has been characterized using in vitro reaction and cell lines with biochemical assays by Snijders., et al in 2015 (PMID: 25605962). Among all PRMTs that catalyze asymmetric arginine dimethylation (ADMA), SFPQ is methylated by only PRMT1 and PRMT3, with PRMT1 showing higher efficiency while PRMT3 showing a lower efficiency. However, PRMT3 is mainly cytosolic. Its expression in CNCCs is about 100-fold lower than PRMT1 (Fig. 1). Based on these knowledges, PRMT1 is the primary arginine methyltransferase for SFPQ, a nuclear protein in CNCCs. We and others have shown in a previous publication that SFPQ methylation on arginine 7 and 9 depends on PRMT1 (PMID: 31451547).

      To investigate SFPQ protein degradation in CNCCs, we used MG132 to block proteasomal degradation and observed a partial rescue of SFPQ protein degradation in Prmt1 mutant embryos, suggesting that SFPQ is degraded through proteasomal-mediated mechanism. To address the relationship between SFPQ methylation and protein expression, we assessed arginine methylation of SFPQ that accumulated after MG132 treatment. The accumulated SFPQ was not methylated, confirming the absence of methylation even when SFPQ protein expression is restored.

      Snijders., et al, also shown that citrullination induced by PADI4 regulate SFPQ stability (Snijders 2015). We considered this possibility and assessed the expression levels of PADIs. In E13.5 and E15.5 CNCCs, PADI1-4 mRNA expression levels are very low (TPM<5), suggesting that PADIs may not regulate SFPQ stability in CNCCs. A detailed mechanism as to how PRMT1-mediated SFPQ methylation controls stability awaits further investigation.

      (5) For the Sfpq deleted experiment, it seems that the two knockdowns are not similar in the gene targets and GO terms different except Wnt signaling. This makes this data difficult to interpret. The genes identified as intron retention are different than the ones identified in Prmt1 deletion and not reduced as much. How does this fit in with the Prmt1 story? If working through Sfpq, it assumes that the targets will be similar and more the 8% would be in common.

      To address the first concern, we used bioinformatic analysis of the whole genome data to the similarity in changes caused by the two SFPQ-targeting siRNAs. As shown in the new Fig. 7Ba & 7Bb, transcriptomic and intron changes are consistent between the two siRNAs, suggesting that genes targeted by the two siRNA predominantly overlap. This overlap is illustrated by scatter plot analysis of RNAseq DEG and IRI data from each siRNA against SFPQ.

      We have previously identified a group of splicing factors that depends on PRMT1 for arginine methylation, including SFPQ (PMID: 31451547). In the new data in Figures 5, 6 and 10, we tested an additional five PRMT1-dependent splicing factors that are highly expressed in CNCCs: SRSF1, EWSR1, TAF15, TRA2B and G3BP1 (Fig. 5, 6 and 10). Among these factors, SRSF1 and G3BP1 are predominantly expressed in the cytosol of NCCs at E13.5. As splicing activity in the nucleus is needed for pre-mRNA splicing, we excluded these two and focused on the other three proteins. EWSR1 and TRA2B are both methylated in CNCCs and depend on PRMT1 for methylation (Fig. 5). We weren’t able to assess TAF15 methylation because of lack of efficient antibody for the PLA assay. We also demonstrated that their protein expression or subcellular localization was not altered by Prmt1 deletion in CNCCs, unlike SFPQ (Fig. S2). To define their splicing footprint, we performed siRNA-mediated knockdown in ST2 cells, followed by RNA-seq and IRI analysis to define differentially regulated genes and introns, which revealed distinct biological pathways regulated by SFPQ, EWSR1, TRA2B and TAF15, but minimal roles of EWSR1, TRA2B and TAF15 on intron retention when compared to SFPQ (Fig. 10F-10I, Supplemental Figure S7A-S7F). ECM genes are significantly downregulated by all four splicing factors (Fig. 10J-10M), but EWSR1, TRA2B and TAF15 regulate transcription or exon skipping instead of IR, as exemplified by Alpl and Postn (Fig. 10N-10T).

      (6) The addition of an NMD mechanism is interesting but not surprising that when inhibiting the pathway broadly, there is an increase in gene expression in the mesoderm cell line. How specific is this to craniofacial development?

      NMD is driven by a protein complex composed of SMG and UPF proteins. We show in the revised manuscript that NMD is both a physiological mechanism in CNCCs and triggered by genetic disturbance (Fig. 4). These data are in line with human patient reports where SMG9 mutation in human causes malformation in the face, hand, heart and brain (PMID: 27018474). Mouse genetic studies also demonstrated roles of NMD components during embryonic development.Smg6, Upf1, Upf2 and Upf3a knockout mouse die at early embryonic stages (E5.5-E9.5), and Smg1 gene trap mutant mice die at E12.5 (Han 2018). Additionally, intron retention-triggered NMD in cancer has both promotive and suppressive roles and NMD inhibitors has been tested for cancer therapy and recently cancer immunotherapy. Our findings highlight matrix genes as one of the key targets for NMD during craniofacial development.

      Minor:

      (1) The supplemental figures are difficult to understand. In the first upload there are many figures and tables, some excel files that are separate uploads and some not. Please upload as separate files so it is clear. And also put them in order that they are in the manuscript.

      (2) For the heat map in figure 2B, it would be good to show all the genes or none at all. It seems a bit like cherry-picking to highly only a few. And they are not labeled where they are located in the graph. Are these the top lines if so please label.

      (3) Gene names in Figure 3A are difficult to read. I would also not consider BMP7 an ECM gene.

      (4) A summary diagram of the interactions proposed will help to make this more understandable.

      The supplemental figures are reorganized and uploaded as separate word and excel documents. For Heat map in Fig. 2B, we have removed the gene names. For Fig. 3A, only the most significantly changed gene are labeled in red dots with names. We didn’t label all the genes because of the large number of genes. For the new Figure 3B, we have replaced BMP7. A schematic summary is also added to Supplemental Fig. S9 to illustrate the PRMT1-SFPQ pathway.

    1. Author response:

      The following is the authors’ response to the previous reviews

      Public Reviews:

      Reviewer #2 (Public review):

      In this valuable manuscript, Lin et al attempt to examine the role of long non coding RNAs (lncRNAs) in human evolution, through a set of population genetics and functional genomics analyses that leverage existing datasets and tools. Although the methods are incomplete and at times inadequate, the results nonetheless point towards a possible contribution of long non coding RNAs to shaping humans, and suggest clear directions for future, more rigorous study.

      Comments on revisions:

      I thank the authors for their revision and changes in response to previous rounds of comments. As before, I appreciate the changes made in response to my comments, and I think everyone is approaching this in the spirit of arriving at the best possible manuscript, but we still have some deep disagreements on the nature of the relevant statistical approach and defining adequate controls. I highlight a couple of places that I think are particularly relevant, but note that given the authors disagree with my interpretation, they should feel free to not respond!

      (1) On the subject of the 0.034 threshold, I had previously stated: "I do not agree with the rationale for this claim, and do not agree that it supports the cutoff of 0.034 used below."

      In their reply to me, the authors state:

      "What we need is a gene number, which (a) indicates genes that effectively differentiate humans from chimpanzees, (b) can be used to set a DBS sequence distance cutoff. Since this study is the first to systematically examine DBSs in humans and chimpanzees, we must estimate this gene number based on studies that identify differentially expressed genes in humans and chimpanzees. We choose Song et al. 2021 (Song et al. Genetic studies of human-chimpanzee divergence using stem cell fusions. PNAS 2021), which identified 5984 differentially expressed genes, including 4377 genes whose differential expression is due to trans-acting differences between humans and chimpanzees. To the best of our knowledge, this is the only published data on trans-acting differences between humans and chimpanzees, and most HS lncRNAs and their DBSs/targets have trans-acting relationships (see Supplementary Table 2). Based on these numbers, we chose a DBS sequence distance cutoff of 0.034, which corresponds to 4248 genes (the top 20%), slightly fewer than 4377."

      I have some notes here. First, Agoglia et al, Nature, 2021, also examined the nature of cis vs trans regulatory differences between human and chimps using a very similar set up to Song et al; their Supplementary Table 4 enables the discovery of genes with cis vs trans effects although admittedly this is less straightforward than the Song et al data. Second, I can't actually tell how the 4377 number is arrived at. From Song et al, "Of 4,671 genes with regulatory changes between human-only and chimpanzee-only iPSC lines, 44.4% (2,073 genes) were regulated primarily in cis, 31.4% (1,465 genes) were regulated primarily in trans, and the remaining 1,133 genes were regulated both in cis and in trans (Fig. 2C). This final category was further broken down into a cis+trans category (cis- and transregulatory changes acting in the same direction) and a cis-trans category (cis- and trans-regulatory changes acting in opposite directions)." Even when combining trans-only and cis&trans genes that gives 2,598 genes with evidence for some trans regulation. I cannot find 4,377 in the main text of the Song et al paper.

      Elsewhere in their response, the authors respond to my comment that 0.034 is an arbitrary threshold by repeating the analyses using a cutoff of 0.035. I appreciate the sentiment here, but I would not expect this to make any great difference, given how similar those numbers are! A better approach, and what I had in mind when I mentioned this, would be to test multiple thresholds, ranging from, eg,0.05 to 0.01 <DBS dist =0.01 -> 0.034 -> 0.05> at some well-defined step size.

      (1) We sincerely thank the reviewer for this critical point. Our initial purpose, based on DBS distances from the human genome to chimpanzee genome and archaic genomes, was that genes with large DBS distances may have contributed more to human evolution. However, our ORA (overrepresentation analysis) explored only genes with large DBS distances (the legend of old Figure 2 was “1256 target genes whose DBSs have the largest distances from modern humans to chimpanzees and Altai Neanderthals are enriched in different Biological Processes GO terms”), with the use of the cutoff (threshold) of 0.034 for defining large distance. The cutoff is not totally unreasonable (as our new results and the following sensitivity analysis indicate), but this approach was indirect and flawed.

      (2) We have now performed ORA using two methods. The first uses only DBS distances. Instead of using a cutoff, we now sort genes by DBS distance (human-chimpanzee distances and human-Altai Neanderthal distance, respectively, see Supplementary Table 5) and use the top 25% and bottom 25% of genes to perform ORA. This directly examines whether DBS distances along indicate that genes with large DBS distances contribute more to human evolution than genes with small DBS distances. The second also explores the ASE genes (allele-specific expression, genes undergoing human/chimpanzee-specific regulation in the tetraploid human–chimpanzee hybrid iPS) reported by Agoglia et al. 2021. We select the top 50% and bottom 50% of genes with large and small DBS distances, intersect them with ASE genes from Agoglia et al. 2021 (their Supplementary Table 4), and apply ORA to the intersections. Both the results are that: (a) more GO terms are obtained from genes with large DBS distances, (b) more human evolution-related GO terms are obtained from genes with large DBS distances (Supplementary Table 5,6,7; Figure 2; Supplementary Fig. 15). These results directly suggest that genes with large DBS distances contribute more to human evolution than genes with small DBS distances, which is a key theme of the study.

      (3) Regarding Song et al 2021, the statement of “we differentiated…allotetraploid (H1C1a, H1C1b, H2C2a, H2C2b) lines into ectoderm, mesoderm, and endoderm” made us assume that their differentiated hybrid cell lines cover more tissue types than those of Agoglia et al. 2021. Now, upon re-examining Supplementary Table 5 of Song et al. and Supplementary Table 4 of Agoglia et al. 2021, we find that the latter more clearly indicates significant ASE genes (p-adj<0.01 and |LFC>0.5| in GRCh38 and PanTro5).

      (4) We have also performed two additional analyses in response to the suggestion of “test multiple thresholds, ranging from, eg, 0.05 to 0.01 <DBS dist =0.01 -> 0.034 -> 0.05> at some well-defined step size”. First, we performed a multi-threshold sensitivity analysis using a spectrum of cutoffs (0.03, 0.034, 0.04, 0.05), and tracked the number of genes identified and the enrichment significance of key GO terms (e.g., "neuron projection development," "behavior") across these thresholds. The result confirms that while the absolute number of genes varies with the cutoffs, the core biological conclusion (specifically, the significant enrichment of target genes in neurodevelopmental and cognitive functions) remains stable and significant. For instance, "behavior" maintains strong statistical significance (FDR<0.01) in both the human-chimpanzee and human-Altai Neanderthal comparisons across all tested cutoffs, and "Neuron projection development" also remains significant across three (0.03, 0.034, 0.04) of the four cutoffs in the Altai comparison. This pattern suggests that our core findings regarding neurodevelopmental functions are robust across a range of cutoffs. Nevertheless, we did not extend the analysis to smaller cutoffs (e.g., 0.01 or 0.02) because such values would identify an excessively large number of genes (>10000) for ORA, which would render the GOterm enrichment analysis less meaningful due to a loss of specificity.

      Second, we have performed an additional validation to directly evaluate whether the 0.034 cutoff itself represents a stringent and biologically meaningful value. We sought to empirically determine how often a DBS sequence distance of 0.034 or greater might occur by chance in promoter regions, thereby testing its significance as a marker of potential evolutionary divergence. We randomly sampled 10,000 windows from annotated promoter regions across the hg38 genome, each with a size matching the average length of DBSs (147 bp). We then calculated the per-base sequence distances for these random windows between modern humans and chimpanzees, as well as between modern humans and the three archaic humans (Altai, Denisovan, Vindija). The analysis reveals that a distance of ≥0.034 is a rare event in random promoter sequences: for Human-Chimp, Human-Altai, HumanDenisovan, and Human-Vindija, 5.49% (549/10000), 0.31% (31/10000), 4.47% (447/10000), and0.03% (3/10000) of random windows reach this distance. This empirical evidence suggests that 0.034 is a sufficiently strong cutoff for defining large DBS distance, it would occur very unlikely in a random genomic background (P<0.1 for Chimpanzee and P<0.05 for the archaic humans), and DBSs exceeding this cutoff are significantly enriched for sequences that have undergone substantial evolutionary change instead of being random neutral variations.  

      (5) We present new Figure 2, Supplementary Table 5,6,7, and Supplementary Fig. 15. We have substantially revised section 2.3, related sections in Results, Supplementary Note 3, and Supplementary Table 8. We have removed related descriptions and explanations in the main text and Supplementary Notes. The results of the above two analyses are presented here as two Author response images.

      Author response table 1.

      Sensitivity analysis of GO-term enrichment across different DBS sequence distance cutoffs. The table shows the numbers of target genes identified and the false discovery rates (FDR) for the enrichment of three selected GO terms at four different distance cutoffs. Note that, unlike in the old Figure 2, the results for chimpanzees and Altai Neanderthals are not directly comparable here, as the numbers of target genes used for the enrichment analysis differ between them at each cutoff.

      Author response image 1.

      Distribution of per-base sequence distances for DBS size-matched random genomic windows in Ensembl-annotated promoter regions, calculated between modern humans and (A) chimpanzee, (B) Altai Neanderthal, (C) Denisovan, and (D) Vindija Neanderthal genomes.

      (2) The authors have introduced a new TFBS section, as a control for their lncRNAs - this is welcome, though again I would ask for caution when interpreting results. For instance, in their reply to me the authors state: "The number of HS TFs and HS lncRNAs (5 vs 66) <HS TF vs all HS lncRNAs> alone lends strong evidence suggesting that HS lncRNAs have contributed more significantly to human evolution than HS TFs (note that 5 is the union of three intersections between <many2zero + one2zero> and the three <human TF list>)."

      But this assumes the denominator is the same! There are 35899 lncRNAs according to the current GENCOVE build; 66/35899 = 0.0018, so, 0.18% of lncRNAs are HS. The authors compare this to 5 TFs. There are 19433 protein coding genes in the current GENCOVE build, which naively (5/19433) gives a big depletion (0.026%) relative to the lnc number. However, this assumes all protein coding genes are TFs, which is not the case. A quick search suggests that ~2000 protein coding genes are TFs (see, eg, https://pubmed.ncbi.nlm.nih.gov/34755879/); which gives an enrichment (although I doubt it is a statistically significant one!) of HS TFs over HS lncRNAs (5/2000 = 0.0025). Hence my emphasis on needing to be sure the controls are robust and valid throughout!

      We thank the reviewer for this comment. While 5 vs 66 reveals a difference, a direct comparison is too simplified. The real take-home message of the new TFBS section is not the numbers but the distributions of HS TFs’ targets and HS lncRNAs’ targets across GTEx organs and tissues (Figure 3 and Supplementary Figures 24, 25) - correlated HS lncRNA-target transcript pairs are highly enriched in brain regions, but correlated HS TF-target transcript pairs are distributed broadly across GTEx tissues and organs. We have now removed the simple comparison of “5 vs 66” and more carefully explained our comparison in section 2.6.

      (3) In my original review I said: line 187: "Notably, 97.81% of the 105141 strong DBSs have counterparts in chimpanzees, suggesting that these DBSs are similar to HARs in evolution and have undergone human-specific evolution." I do not see any support for the inference here. Identifying HARs and acceleration relies on a far more thorough methodology than what's being presented here. Even generously, pairwise comparison between two taxa only cannot polarise the direction of differences; inferring human-specific change requires outgroups beyond chimpanzee.

      In their reply to me, the authors state:

      Here, we actually made an analogy but not an inference; therefore, we used such words as "suggesting" and "similar" instead of using more confirmatory words. We have revised the latter half sentence, saying "raising the possibility that these sequences have evolved considerably during human evolution".

      Is the aim here to draw attention to the ~2.2% of DBS that do not have a counterpart? In that case, it would be better to rewrite the sentence to emphasise those, not the ones that are shared between the two species? I do appreciate the revised wording, though.

      (1) Our original phrasing may be misleading, and we agree entirely that “pairwise comparison between two taxa only cannot polarise the direction of differences; inferring human-specific change requires outgroups beyond chimpanzee”. As explained in that reply, we know and think that DBSs and HARs are two different classes of sequences, and indeed, identifying HARs and acceleration relies on a far more thorough methodology. Yet, three factors prompted us to compare them. First, both suggest the importance of sequences outside genes. Second, both are quite “old” sequences and have undergone considerable evolution recently (although the references are different). Third, both have contributed greatly to human brain evolution.  

      (2) Here, our stress is 97.81% but not 2.2%, and we have made this analogy more clearly and cautiously. Relevant revisions have been made in the Results, Discussion, and Methods sections.   

      (3) We also have further determined whether the 2.2% DBSs are human-specific gains by analyzing them using the UCSC Multiz Alignments of 100 Vertebrates. The result confirms that all 2248 DBSs are present in the human genome but are absent from the chimpanzee genome and all other aligned vertebrate genomes. We add this result into the manuscript.

      (4) Finally, Line 408: "Ensembl-annotated transcripts (release 79)" Release 79 is dated to March 2015, which is quite a few releases and genome builds ago. Is this a typo? Both the human and the chimpanzee genome have been significantly improved since then!

      (1) We thank the reviewer for this comment, which prompts us to provide further explanation and additional data. First, we began predicting HS lncRNAs’ DBSs when Ensembl release 79 was available, but did not re-predict DBSs when new Ensembl releases were published because (a) these new Ensembl releases are based also on hg38, (b) we did not find any fault in the LongTarget program during our use, nor received any one from users, (c) predicting lncRNAs’ DBSs using the LongTarget program is highly time-consuming.  

      (2) Second, to assess the influence of newer Ensembl releases, we compared the promoters annotated in release 79 and in release 115. We found that the vast majority (87.3%) of promoters newly annotated in release 115 belong to non-coding genes. Thus, using release 115 may predict more DBSs in non-coding genes, but downstream analyses based on protein-coding genes would be essentially the same (meaning that all figures and tables would be the same).

      (3) Third, a key element of this study is GTEx data analysis, and these data were also published years ago.  

      (4) Finally, some lncRNA genes have new gene symbols in new Ensembl releases. To allow researchers to use our data conveniently, we have added a new column titled "Gene symbol (Ensembl release115)" to Supplementary Tables 2A and 2B.  

      Summary:

      Major changes based on Reviewer’s comments:

      (1) The following revisions are made to address the comment on “the 0.034 threshold”: (a) Section 2.3, section 2.4, Supplementary Note 3, and related contents in Discussion and Methods are revised, (b) new Figure 2, Supplementary Figure 15, new Supplementary Table 5,6,7, (c) Table 2 and Supplementary Table 8 are revised.

      (2) To address the comment on “new TFBS section”, section 2.6 and section 4.13 are revised.  

      (3) To address the comment on “97.81% and 2.2% of DBSs”, section 2.3 is revised.

      (4) The following revisions are made to address the comment on “release 79”: (a) the old Supplementary Table 2, 3 are merged to Supplementary Table 2AB, and the new column "Gene symbol (Ensembl release115)" is added to Supplementary Table 2AB, (b) accordingly, Supplementary Table 4,5 are renamed to Supplementary Table 3,4.

      Additional revisions:

      (1) Section 2.5 “Young weak DBSs may have greatly promoted recent human evolution” is moved into Supplementary Note 3 (which now has the subtitle “Target genes with specific DBS features are enriched in specific functions”), because this section is short and lacking sufficient cross-validation.

      (2) Considerable minor revisions of sentences have been made.

      (3) Since there are many supplementary figures, the main text now cites only Supplementary Notes, as the reader can easily access supplementary figures in Supplementary Notes.

    1. Author response:

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

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      Lesser et al provide a comprehensive description of Drosophila wing proprioceptive sensory neurons at the electron microscopy resolution. This “tour-de-force” provides a strong foundation for future structural and functional research aimed at understanding wing motor control in Drosophila with implications for understanding wing control across other insects.

      Strengths:

      (1) The authors leverage previous research that described many of the fly wing proprioceptors, and combine this knowledge with EM connectome data such that they now provide a near-complete morphological description of all wing proprioceptors.

      (2) The authors cleverly leverage genetic tools and EM connectome data to tie the location of proprioceptors on the wings with axonal projections in the connectome. This enables them to both align with previous literature as well as make some novel claims.

      (3) In addition to providing a full description of wing proprioceptors, the authors also identified a novel population of sensors on the wing tegula that make direct connections with the B1 wing motor neurons, implicating the role of the tegula in wing movements that was previously underappreciated.

      (4) Despite being the most comprehensive description so far, it is reassuring that the authors clearly state the missing elements in the discussion.

      Weaknesses:

      (1) The authors do their main analysis on data from the FANC connectome but provide corresponding IDs for sensory neurons in the MANC connectome. I wonder how the connectivity matrix compares across FANC and MANC if the authors perform a similar analysis to the one they have done in Figure 2. This could be a valuable addition and potentially also pick up any sexual dimorphism.

      We agree that systematic comparisons will provide valuable insights as more connectome datasets become available. However, the primary goal of this study was to link central axon morphology with peripheral structures in the wing. We deliberately omitted more detailed and quantitative analyses of the downstream VNC circuitry, apart from providing a global view of the connectivity matrix and using it to cluster the sensory axon types. A more detailed and systematic comparison of wing sensorimotor circuit connectivity across different connectome datasets (FANC, MANC, BANC, IMAC) is the subject of ongoing work in our lab, which we feel is beyond the scope of this study. Here, we chose to match the wing proprioceptors to axons in MANC to demonstrate their stereotypy across individuals and to make them more accessible to other researchers. We found no obvious sexual dimorphism at the level of wing sensory neurons. We now note this in the Discussion.

      (2) The authors speculate about the presence of gap junctions based on the density of mitochondria. I’m not convinced about this, given that mitochondrial densities could reflect other things that correlate with energy demands in sub-compartments.

      We have moved speculation about mitochondria and gap junctions to the Discussion.

      (3) I’m intrigued by how the tegula CO is negative for iav. I wonder if authors tried other CO labeling genes like nompc. And what does this mean for the nature of this CO. Some more discussion on this anomaly would be helpful.

      Based on this suggestion, we have added an image showing that tegula CO neurons are labeled by nompC-Gal4.

      (4) The authors conclude there are no proprioceptive neurons in sclerite pterale C based on Chat-Gal4 expression analysis. It would be much more rigorous if authors also tried a pan-neuronal driver like nsyb/elav or other neurotransmitter drivers (Vglut, GAD, etc) to really rule this out. (I hope I didn’t miss this somewhere.)

      To address this, we imaged OK371-GFP, which labels glutamatergic neurons, in the wing and wing hinge. We saw expression in the wing, as others have reported (Neukomm et. al., 2014), but we saw no expression at the wing hinge. Apart from a handful of glutamatergic gustatory neurons in the leg, we are not aware of any other sensory neurons in the fly that are not labeled by Chat-Gal4.

      Overall, I consider this an exceptional analysis that will be extremely valuable to the community.

      We sincerely appreciate the reviewer’s positive feedback.

      Reviewer #2 (Public review):

      Summary:

      Lesser et al. present an atlas of Drosophila wing sensory neurons. They proofread the axons of all sensory neurons in the wing nerve of an existing electron microscopy dataset, the female adult fly nerve cord (FANC) connectome. These reconstructed sensory axons were linked with light microscopy images of full-scale morphology to identify their origin in the periphery of the wing and encoded sensory modalities. The authors described the morphology and postsynaptic targets of proprioceptive neurons as well as previously unknown sensory neurons.

      Strengths:

      The authors present a valuable catalogue of wing sensory neurons, including previously undescribed sensory axons in the Drosophila wing. By providing both connectivity information with linked genetic drive lines, this research facilitates future work on the wing motor-sensory network and applications relating to Drosophila flight. The findings were linked to previous research as well as their putative role in the proprioceptive and nerve cord circuitry, providing testable hypotheses for future studies.

      Weaknesses:

      (1) With future use as an atlas, it should be noted that the evidence is based on sensory neurons on only one side of the nerve cord. Fruit flies have stereotyped left/right hemispheres in the brain and left/right hemisegments in the nerve cord. The comparison of left and right neurons of the nervous system can give a sense of how robust the morphological and connectivity findings are. Here, the authors have not compared the left and right side sensory axons from the wing nerve, leaving potential for developmental variability across samples and left/right hemisegments.

      The right ADMN nerve in the FANC dataset is partially severed, making left/right comparisons unreliable (see Azevedo 2024, Extended Data Figure 4). We have updated the text to explain this within the Methods section of the paper.

      (2) Not all links between the EM reconstructions and driver lines are convincing. To strengthen these, for all EM-LM matches in Figures 3-7, rotated views of the driver line (matching the rotated EM views) should be shown to provide a clearer comparison of the data. In particular, Figure 3G and Figure 7B are not very convincing based on the images shown. MCFO imaging of the driver lines in Figure 3G and 7B would make this position stronger if a clone that matches the EM reconstruction could be identified.

      Many of the z-stack images in the paper are from the Janelia FlyLight collection, and unfortunately their imaging parameters were not optimized for orthogonal views. Rotated views are blurry and not especially helpful for comparison to EM reconstruction. We now point out in the text that interested readers can access the z-stacks from FlyLight to see the dorsal-ventral projections.

      Regarding Figure 3G and 7B, we have added markers to the image with corresponding descriptions in the legend to guide the reader through the image of the busy driver line. Although these lines label many cells in the VNC as a whole, they sparsely label cells in the ADMN, making them nonetheless useful for identifying peripheral sensory neurons.

      (3) Figure 7B looks like the driver line might have stochastic expression in the sensory neuron, which further reduces confidence in the result shown in Figure 7C. Is this expression pattern in the wing consistently seen? Many split-GAL4s have stochastic expressions. The evidence would be strengthened if the authors presented multiple examples (~4-5) of each driver line’s expression pattern in the supplement.

      Figure 7B shows sparse labeling of the driver line using the MCFO technique, as specified in the legend. Its unilateral expression is therefore not due to stochastic expression of the Gal4 line. We have added the “MFCO” label to the image to clarify.

      (4) Certain claims in this work lack quantitative evidence. On line 128, for instance, “Overall, our comprehensive reconstruction revealed many morphological subgroups with overlapping postsynaptic partners, suggesting a high degree of integration within wing sensorimotor circuits.” If a claim of subgroups having shared postsynaptic partners is being made, there should have been quantitative evidence. For example, cosine similar amongst members of each group compared to the cosine similarity of shuffled/randomised sets of axons from different groups. The heat map of cosine similarity in Figure 2B alone is not sufficient.

      We agree that illustrating the extent of shared postsynaptic partners across subgroups strengthens this point. We added a visualization showing pairwise similarity scores for within- and between-cluster neuron pairs (Figure 2B inset). We also performed a permutation test to determine that within-cluster similarity is significantly higher than between clusters, and we report the test in the results as well as the figure legend. This analysis provides a more quantitative summary of the qualitative trends in connectivity that are summarized in Figure 2B.

      (5) Similarly, claims about putative electrical connections to b1 motor neurons are very speculative. The authors state that “their terminals contain very densely packed mitochondria compared to other cells”, without providing a quantitative comparison to other sensory axons. There is also no quantitative comparison to the one example of another putative electrical connection from the literature. Further, it should be noted that this connection from Trimarchi and Murphey, 1997, is also stated as putative on line 167, which further weakens this evidence. Quantification would strongly strengthen this position. Identification of an example of high mitochondrial density at a confirmed electrical connection would be even better. In the related discussion section “A potential metabolic specialization for flight circuitry”, it should be more clearly noted that the dense mitochondria could be unrelated to a putative electrical connection. If the authors have an alternative hypothesis about the mitochondria density, this should be stated as well.

      We agree with the reviewer that the link between mitochondrial density and metabolic specialization is purely speculative in this context. Based on reviewer feedback, we have moved all mention of the relationship between mitochondrial density and gap junction coupling to the Discussion. We acknowledge that this may seem like a somewhat random and not quantitatively supported observation. However, we found the coincidence striking and worthy of mention, though it is only tangentially relevant to the rest of the paper. From conversations with colleagues, we have also heard that this relationship is consistent with as yet unpublished work in other model organisms (e.g., zebrafish, mouse).

      The electrical coupling to b1 motor neurons is well-established (Fayyazuddin and Dickinson, 1999), and we have updated the text to state this more clearly. However, we agree that whether the specific neurons we have identified based on their anatomy are the same ones functionally identified through whole-nerve recordings remains unknown.

      (6) It would be appropriate to cite previous work using a similar strategy to match sensory axons to their cell bodies/dendrites at the periphery using driver lines and connectomics (see Figure 5 for example in the following paper: https://doi.org/10.7554/eLife.40247 ).

      At this point, there are now dozens of papers that match the axons of sensory neurons to their cell bodies/dendrites in the periphery by comparing light microscopy and connectomics. When we dug in, we found examples in C. elegans, Ciona intestinalis, zebrafish, and mouse, all published prior to the study cited above. For basically every animal for which scientists have acquired EM volumes of neural tissue, they have used other anatomical labeling methods to determine cell types inside and outside the imaged volume. In summary, we found it difficult to establish a single primary citation for this approach. In lieu of this, we have added a citation to an earlier review by a pioneer in EM connectomics that discusses the general approach of matching cells across different labeling/imaging modalities (Meinertzhagen et al., 2009).

      The methods section is very sparse. For the sake of replicability, all sections should be expanded upon.

      We have expanded the methods section, and also a STAR methods table.

      Reviewer #3 (Public review):

      Summary:

      The authors aim to identify the peripheral end-organ origin in the fly’s wing of all sensory neurons in the anterior dorsomedial nerve. They reconstruct the neurons and their downstream partners in an electron microscopy volume of a female ventral nerve cord, analyse the resulting connectome, and identify their origin with a review of the literature and imaging of genetic driver lines. While some of the neurons were already known through previous work, the authors expand on the identification and create a near-complete map of the wing mechanosensory neurons at synapse resolution.

      Strengths:

      The authors elegantly combine electron microscopy, neuron morphology, connectomics, and light microscopy methods to bridge the gap between fly wing sensory neuron anatomy and ventral nerve cord morphology. Further, they use EM ultrastructural observations to make predictions on the signaling modality of some of the sensory neurons and thus their function in flight.

      The work is as comprehensive as state-of-the-art methods allow to create a near-complete mapof the wing mechanosensory neurons. This work will be of importance to the field of fly connectomics and modelling of fly behavior, as well as a useful resource to the Drosophila research community.

      Through this comprehensive mapping of neurons to the connectome, the authors create a lot of hypotheses on neuronal function, partially already confirmed with the literature and partially to be tested in the future. The authors achieved their aim of mapping the periphery of the fly’s wing to axonal projections in the ventral nerve cord, beautifully laying out their results to support their mapping.

      The authors identify the neurons in a previously published connectome of a male fly ventral nerve cord to enable cross-individual analysis of connections. Further, together with their companion paper, Dhawan et al. 2025, describing the haltere sensory neurons in the same EM dataset, they cover the entire mechanosensory space involved in Drosophila flight.

      Weaknesses:

      The connectomic data are only available upon request; the inclusion of a connectivity table of the reconstructed neurons would aid analysis reproducibility and cross-dataset comparisons.

      We have added a connectivity table as well as analysis scripts in the github repository for the paper (https://github.com/EllenLesser/Lesser_eLife_2025).

      Recommendations for the authors:

      Reviewer #2 (Recommendations for the authors):

      The methods section should be expanded in every aspect. Most pressing sections are:

      (1) Data and Code availability: All code should be included as a Zenodo database, the suggestion to ask authors for code upon request is inappropriate.

      We have added all code to a public github repository, which is now linked in the Methods section.

      (2) Samples: Standard cornmeal and molasses medium should have a reference, as many institutes use different recipes.

      The recipe used by the University of Washington fly kitchen is based on the Bloomington standard Cornmeal, Molasses and Yeast Medium recipe, which can be found at https://bdsc.indiana.edu/information/recipes/molassesfood.html. The UW recipe is slightly modified for different antifungal ingredients and includes tegosept, propionic acid, and phosophoric acid.

      (3) Table 3: Driver lines labelling wing sensory neurons: The genetic driver lines should have associated Bloomington stock centre numbers. Additionally, relevant information for effector lines used should be included in the methods.

      We now include the Bloomington stock numbers and more information on effector lines in the STAR methods table.

      Minor corrections:

      (1) Lines 119-120: “Notably, many of the axons do not form crisp cluster boundaries, suggesting that multimodal sensory information is integrated at early stages of sensory processing.” We do not follow the logic of this statement and suspect it is a bit too speculative.

      We removed this sentence from the manuscript.

      (2) Figure 1: The ADMN is missing in the schematics and would be helpful to depict for non-experts. Is this what is highlighted in Figure 1D?

      Yes, and we now label 1D as the ADMN wing nerve.

      (3) Figure 1B: Which driver lines are being depicted here? Looking at Table 3 does not clarify. It should be specified at least in the figure legend.

      As stated in the legend, we include a table of all of the driver lines we screened and which sensory structures they label.

      (4) Figure 1C: There are some minor placement issues with the text in the schematic. There is an arrow very close to the “CO” on the top right, which makes the “O” look like the symbol for male. “ax ii” is a bit too close to the wing hinge

      We updated the figure to address this issue.

      (5) Figure 1D: The outlined grey masks are not clear. The use of colour would be very useful for the reader to help understand what the authors are referring to here

      We now use color for the masks.

      (6) Figure 2A: It is unclear if the descending neuron and non-motor efferent neuron are not shown because they are under the described threshold, or to simplify the plot. They should be included in the plot if over the threshold.

      We have updated the legend to specify that the exclusion of the descending and non-motor efferent neurons are to visually simplify the plot. We include % of sensory output to each of these neurons in the legend, and they are included in the connectivity matrix data in the public  GitHub repository associated with the paper, included in the Methods.

      (7) Figure 2B: What clustering is used specifically? The method says it’s from Scikit-learn, but there are many types of clustering available in this package.

      We now include the specific clustering type used in the Methods section, which is agglomerative clustering.

      (8) Figure 3A: What does the green box behind the plot represent?

      The green box represents the tegula CO axons, which we now specify in the legend.

      (9) Figure 3C: the “C” is clipped at the top.

      We updated the figure to address this issue.

      (10) Figure 4A: the main text says a “group of four axons” (line 203) while the figure says 5 axons.

      We updated the text to address this issue.

      (11) Line 360: “We found that the campaniform sensilla on the tegula provide the most direct feedback onto wing steering motor neurons”. We struggled to find where this was directly shown, because several sensory axon types directly synapse onto motor neurons.

      We now specify in the text that this finding is shown in Figure 3.

      Reviewer #3 (Recommendations for the authors):

      I would like to congratulate the authors on their beautiful, easy-to-read, and easy-to-comprehend manuscript, with clear figures and nice visualizations. This work provides a valuable resource that will contribute to the interpretability of connectomic data and further to connectome-based modeling of fly behavior.

      We sincerely appreciate the reviewer’s positive feedback.

    1. Author response:

      The following is the authors’ response to the original reviews

      Public Reviews:

      Reviewer #1 (Public review):

      This article deals with the chemotactic behavior of E coli bacteria in thin channels (a situation close to 2D). It combines experiments and simulations.

      The authors show experimentally that, in 2D, bacteria swim up a chemotactic gradient much more effectively when they are in the presence of lateral walls. Systematic experiments identify an optimum for chemotaxis for a channel width of ~8µm, close to the average radius of the circle trajectories of the unconfined bacteria in 2D. It is known that these circles are chiral and impose that the bacteria swim preferentially along the right-side wall when there is no chemotactic gradient. In the presence of a chemotactic gradient, this larger proportion of bacteria swimming on the right wall yields chemotaxis. This effect is backed by numerical simulations and a geometrical analysis.

      If the conclusions drawn from the experiments presented in this article seem clear and interesting, I find that the key elements of the mechanism of this wall-directed chemotaxis are not sufficiently emphasized. Moreover, the paper would be clearer with more details on the hypotheses and the essential ingredients of the analyses.

      We thank the reviewer for these constructive suggestions. We agree that emphasizing the underlying mechanism is crucial for the clarity of our findings. In the revised manuscript, we have now explicitly highlighted the critical roles of chiral circular motion and the alignment effect following side-wall collisions in both the Abstract (lines 25-27) and the Discussion (lines 391-393). Furthermore, we have added a new analysis of bacterial trajectories post-collision (Fig. S2), which demonstrates that cells predominantly align with and swim along the sidewalls. We have also clarified the assumptions in our numerical simulations, specifically how the radius of circular trajectories and the alignment effect are incorporated into the equations of motion. Please refer to our detailed responses in the "Recommendations for the authors" section for further specifics.

      Reviewer #2 (Public review):

      Summary:

      In this study, the authors investigated the chemotaxis of E. coli swimming close to the bottom surface in gradients of attractant in channels of increasingly smaller width but fixed height = 30 µm and length ~160 µm. In relatively large channels, they find that on average the cells drift in response to the gradient, despite cells close to the surface away from the walls being known to not be chemotactic because they swim in circles.

      They find that this average drift is due to the cell localization close to the side walls, where they slide along the wall. Whereas the bacteria away from the walls have no chemotaxis (as shown before), the ones on the left side wall go down-gradient on average, but the ones on the right-side wall go up-gradient faster, hence the average drift. They then study the effect of reducing channel width. They find that chemotaxis is higher in channels with a width of about 8 µm, which approximately corresponds to the radius of the circular swimming R. This higher chemotactic drift is concomitant to an increased density of cells on the RSW. They do simulations and modeling to suggest that the disruption of circular swimming upon collision with the wall increases the density of cells on the RSW, with a maximal effect at w = ~ 2/3 R, which is a good match for their experiments.

      Strengths:

      The overall result that confinement at the edge stabilises bacterial motion and allows chemotaxis is very interesting although not entirely unexpected. It is also important for understanding bacterial motility and chemotaxis under ecologically relevant conditions, where bacteria frequently swim under confinement (although its relevance for controlling infections could be questioned). The experimental part of the study is nicely supported by the model.

      Weaknesses:

      Several points of this study, in particular the interpretation of the width effect, need better clarification:

      (1) Context:

      There are a number of highly relevant previous publications that should have been acknowledged and discussed in relation to the current work:

      https://pubs.rsc.org/en/content/articlehtml/2023/sm/d3sm00286a

      https://link.springer.com/article/10.1140/epje/s10189-024-00450-7

      https://doi.org/10.1016/j.bpj.2022.04.008

      https://doi.org/10.1073/pnas.1816315116

      https://www.pnas.org/doi/full/10.1073/pnas.0907542106

      https://doi.org/10.1038/s41467-020-15711-0

      http://doi.org/10.1038/s41467-020-15711-0

      http://doi.org/10.1039/c5sm00939a

      We appreciate the reviewer bringing these important publications to our attention. We have now cited and discussed these works in the Introduction (lines 55-62 and 76-85) to better contextualize our study regarding bacterial motility and chemotaxis in confined geometries.

      (2) Experimental setup:

      a) The channels are built with asymmetric entrances (Figure 1), which could trigger a ratchet effect (because bacteria swim in circle) that could bias the rate at which cells enter into the channel, and which side they follow preferentially, especially for the narrow channel. Since the channel is short (160 µm), that would reflect on the statistics of cell distribution. Controls with straight entrances or with a reversed symmetry of the channel need to be performed to ensure that the reported results are not affected by this asymmetry.

      We appreciate the reviewer's insight regarding the potential ratchet effect caused by asymmetric entrances. To rule this out, we fabricated a control device with straight entrances and repeated the measurements. As shown in Figure S3, the chemotactic drift velocity follows the same trend as observed in the original setup, confirming an optimal width of ~9 mm. These results demonstrate that the entrance geometry does not bias the reported statistics. We have updated the manuscript text at lines 233-235.

      b) The authors say the motile bacteria accumulate mostly at the bottom surface. This is strange, for a small height of 30 µm, the bacteria should be more-or-less evenly spread between the top and bottom surface. How can this be explained?

      We apologize for not explaining this clearly in the text. As shown by Wei et al., Phys. Rev. Lett. 135, 188401 (2025), significant surface accumulation occurs in channels with heights exceeding 20 µm. In our specific experimental setup, we did not use Percoll to counteract gravity. Therefore, the bacteria accumulated mostly at the bottom surface under the combined influence of gravity and hydrodynamic attraction. This bottom-surface localization is supported by our observation that the bacterial trajectories were predominantly clockwise (characteristic of the bottom surface) rather than counter-clockwise (characteristic of the top surface). We have added this explanation to Line 141.

      c) At the edge, some of the bacteria could escape up in the third dimension (http://doi.org/10.1039/c5sm00939a). What is the magnitude of this phenomenon in the current setup? Does it have an effect?

      We thank the reviewer for raising this important point regarding 3D escape. We have quantified this phenomenon and found the escape rate from the edge into the third dimension to be 0.127 s<sup>-1</sup>. This corresponds to a mean residence time that allows a cell moving at 20 mm/s to travel approximately 157.5 mm along the edge. Since this distance is comparable to the full length of our lanes (~160 mm), most cells traverse the entire edge without escaping. Furthermore, our analysis is based on the average drift of the surface trajectories per unit of time; this metric is independent of the absolute number of cells present. Therefore, the escape phenomenon does not significantly impact our conclusions. We have added a statement clarifying this at line 154.

      d) What is the cell density in the device? Should we expect cell-cell interactions to play a role here? If not, I would suggest to de-emphasize the connection to chemotaxis in the swarming paper in the introduction and discussion, which doesn't feel very relevant here, and rather focus on the other papers mentioned in point 1.

      The cell density in our experiments was approximately 1.3×10<sup>-3</sup> μm<sup>-2</sup>. Given this low density, we do not expect cell-cell interactions to play a role in the observed behaviors.

      Regarding the connection to swarming chemotaxis: We agree that our low-density setup differs from a high-density swarm; however, we believe the comparison remains relevant for two reasons. First, it provides a necessary contrast to studies showing surface inhibition of chemotaxis. Second, while we eliminate cell-cell interactions, we isolate the geometric aspect of swarming. In a swarm, cells move within narrow lanes created by their neighbors. Our device mimics this specific physical confinement by replacing neighboring cells with PDMS sidewalls. This allows us to decouple the effects of physical confinement from cell-cell interactions. We have added the text (Line 370) to clarify this rationale and have incorporated the additional references in introduction as suggested in point 1.

      e) We are not entirely convinced by the interpretation of the results in narrow channels. What is the causal relationship between the increased density on the RSW and the higher chemotactic drift? The authors seem to attribute higher drift to this increased RSW density, which emerges due to the geometric reasons. But if there is no initial bias, the same geometric argument would induce the same increased density of down-gradient swimmers on the LSW, and so, no imbalance between RSW and LSW density. Could it be the opposite that the increased RSW density results from chemotaxis (and maybe reinforces it), not the other way around? Confinement could then deplete one wall due to the proximity of the other, and/or modify the swimming pattern - 8 µm is very close to the size of the body + flagellum. To clarify this point, we suggest measuring the bacterial distributions in the absence of a gradient for all channel widths as a control.

      We thank the reviewer for this insightful comment regarding the causal relationship between cell density and chemotactic drift. We apologize if the initial explanation was unclear.

      Regarding the no-gradient control: Without an attractant gradient (and no initial bias), there is no breaking of symmetry and the labels of "LSW" and "RSW" are arbitrary. Therefore, there will be no asymmetry in the bacterial distributions on both sides (within experimental fluctuations) in the absence of a gradient for any channel width.

      Regarding the causality and density imbalance: We agree that the increased RSW density is a result of chemotaxis, which is then reinforced by the lane geometry especially at narrow lane width. The mechanism relies on the coupling of chemotactic bias with surface circularity. The angle ranges that lead to RSW-UG accumulation (Fig. 6A-C) coincide with the up-gradient direction. Because these cells experience suppressed tumbling (longer runs), they can maintain the steady circular trajectories required to reach and align with the RSW. Conversely, while pure geometric analysis suggests a similar potential for LSW-DG accumulation, these trajectories coincide with the down-gradient direction. These cells experience enhanced tumbling, which distorts the circular trajectories. This prevents them from effectively reaching the LSW and also increases the probability of them leaving the wall. Therefore, the causality is indeed a positive feedback loop: the attractant gradient creates an initial bias that allows the RSW-UG fraction to form stable trajectories; the optimal lane width (matching the swimming radius) then maximizes this capture efficiency, further enriching the RSW fraction and enhancing the overall drift.

      We have added clarifications regarding these points in the revised manuscript (the last paragraph of “Results”).

      (3) Simulations:

      The simulations treat the wall interaction very crudely. We would suggest treating it as a mechanical object that exerts elastic or "hard sphere" forces and torques on the bacteria for more realistic modeling.

      We appreciate the reviewer's suggestion to incorporate more detailed mechanical interactions, such as elastic or hard-sphere forces, for the wall collisions. While we agree that a full hydrodynamic or mechanical model would offer higher fidelity, our experimental observations suggest that a simplified kinematic approach is sufficient for the specific phenomena studied here.

      As shown in the new Fig. S2, our analysis of cell trajectories in the 44-µm-wide channels reveals that cells colliding with the sidewalls tend to align with the surface almost instantaneously. The timescale required for this alignment is negligible compared to the typical wall residence time (see also Ref. 6). Consequently, to maintain computational efficiency without sacrificing the essential physics of the accumulation effect, we employed a coarse-grained phenomenological model where a bacterium immediately aligns parallel to the wall upon contact, similar to approaches used previously (Ref. 43). We have added relevant text to the manuscript on lines 168-171.

      Notably, the simulations have a constant (chemotaxis independent) rate of wall escape by tumbling. We would expect that reduced tumbling due to up-gradient motility induces a longer dwell time at the wall.

      We apologize for the confusion. The chemotaxis effect is indeed fully integrated into our simulation. Specifically, the simulated cells sense the chemical gradient and adjust their motor CW bias (B) accordingly. This adjustment directly modulates the tumble rate (k), calculated as k \= B/0.31 s<sup>-1</sup>. Consequently, the wall escape rate is not constant but varies with the chemotactic response. We also imposed a maximum detention time limit which, when combined with the variable tumble rate, results in an average wall residence time of approximately 2 s, consistent with our experimental observations (Fig. S6B). We have clarified these details in the final section of 'Materials and Methods'.

      Reviewer #3 (Public review):

      This paper addresses through experiment and simulation the combined effects of bacterial circular swimming near no-slip surfaces and chemotaxis in simple linear gradients. The authors have constructed a microfluidic device in which a gradient of L-aspartate is established to which bacteria respond while swimming while confined in channels of different widths. There is a clear effect that the chemotactic drift velocity reaches a maximum in channel widths of about 8 microns, similar in size to the circular orbits that would prevail in the absence of side walls. Numerical studies of simplified models confirm this connection.

      The experimental aspects of this study are well executed. The design of the microfluidic system is clever in that it allows a kind of "multiplexing" in which all the different channel widths are available to a given sample of bacteria.

      While the data analysis is reasonably convincing, I think that the authors could make much better use of what must be voluminous data on the trajectories of cells by formulating the mathematical problem in terms of a suitable Fokker-Planck equation for the probability distribution of swimming directions. In particular, I would like to see much more analysis of how incipient circular trajectories are interrupted by collisions with the walls and how this relates to enhanced chemotaxis. In essence, there needs to be a much clearer control analysis of trajectories without sidewalls to understand the mechanism in their presence.

      We thank the reviewer for this insightful suggestion. We agree that understanding how circular trajectories are interrupted by wall collisions is central to explaining the enhanced chemotaxis. While we did not explicitly formulate a Fokker-Planck equation, we have addressed the reviewer's core point by employing two complementary mathematical approaches that model the probability distribution of swimming directions and wall interactions:

      (1) Stochastic simulations (Langevin approach): As detailed in the "Simulation of E. coli chemotaxis within lane confinements" subsection of “Results” and Figure 5, we modeled cells as self-propelled particles performing random walks. This model explicitly accounts for the "interruption" of circular trajectories by incorporating a constant angular velocity (circular swimming) and an alignment effect upon collision with sidewalls. These simulations successfully reproduced the experimental trends, confirming that the interplay between circular radius and lane width determines the optimal drift velocity.

      (2) Geometric probability analysis: To provide the "intuitive understanding", we included a specific Geometrical Analysis section (the last subsection of “Results”) and Figure 6. This analysis mathematically formulates the problem by calculating the exact proportion of swimming angles that allow a cell to transition from a circular trajectory in the bulk to an up-gradient trajectory along the Right Sidewall (RSW). By integrating over the possible swimming directions, we derived the probability of wall interception as a function of lane width (w) and swimming radius (r). This analysis reveals that the interruption of circular paths is most favorable for chemotaxis when w » (0.7-0.8)´r.

      (3) Control analysis: regarding the "control analysis of trajectories without sidewalls," we utilized the cells in the Middle Area (MA) of the wide lanes as an internal control. As shown in Fig. 2B and 4A, these cells exhibit typical surface-associated circular swimming (Fig. 3B) but generate zero net drift. This serves as the baseline "no sidewall" condition, demonstrating that the chemotactic enhancement is strictly driven by the rectification of circular swimming into wall-aligned motion at the boundaries.

      The authors argue that these findings may have relevance to a number of physiological and ecological contexts. Yet, each of these would be characterized by significant heterogeneity in pore sizes and geometries, and thus it is very unclear whether or how the findings in this work would carry over to those situations.

      We thank the reviewer for this important observation regarding environmental heterogeneity. We agree that we should be cautious about directly extrapolating to complex ecological contexts without qualification. We have revised the last sentence of the abstract to adopt a more measured tone: "Our results may offer insights into bacterial navigation in complex biological environments such as host tissues and biofilms, providing a preliminary step toward exploring microbial ecology in confined habitats and potential strategies for controlling bacterial infections."

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      Key elements of the mechanism of wall-directed chemotaxis are not sufficiently emphasized:

      For instance, the chirality of the trajectories is an essential part of the analysis but is mentioned only briefly in the introduction. In the geometrical analysis, I understand that one of the critical parameters is the angle at which bacteria "collide" with the walls. But, again, this remains largely implicit in the discussion. This comes to the point that these ideas are not even mentioned in the abstract which doesn't provide any hint of a mechanism. An analysis of the actual trajectories of the cells after they hit the walls, as a function of their initial angle would be helpful in comparison with the simulations and the geometrical analysis.

      We appreciate the reviewer's insightful comment regarding the need to better emphasize the mechanism of wall-directed chemotaxis. We agree that the chirality of trajectories and the geometry of wall collisions are central to our analysis and were previously under-emphasized.

      To address this, we have made the following revisions:

      (1) We have revised the Abstract (lines 25-27) and the Discussion (lines 391-393) to explicitly highlight the crucial role of chiral circular motion and the alignment effect following sidewall collisions.

      (2) We further analyzed bacterial trajectories at different collision angles. Typical examples are shown in Supplementary Fig. S2. We observed that cells tend to align with and swim along the sidewalls regardless of their initial collision angles. This finding is now described in the main text at lines 168-171.

      The motion of the bacteria is modelled as run-and-tumble at several places in the manuscript, and in particular in the simulations. Yet, the trajectories of the bacteria seem to be smooth in this almost 2D geometry, except of course when they directly interact with the walls (I hardly see tumbles in the MA region in Figure 1B). Can the authors elaborate on the assumptions made in the numerical simulations? In particular, how is the radius of the trajectories included in these equations of motion (line 514)?

      We apologize for the lack of clarity regarding the bacterial motion model. It has been established that while bacteria do tumble near solid surfaces, they exhibit a smaller reorientation angle compared to bulk fluids; in fact, the most probable reorientation angle on a surface is zero (Ref. 41). Consequently, tumbles are often difficult to distinguish from runs with the naked eye. Additionally, the trajectories in Figure 1B are plotted on a 44 mm ´ 150 mm canvas with unequal coordinate scales, which may further obscure the visual distinctness of tumbling events.

      Regarding the equations of motion: We modeled the bacteria as self-propelled particles governed by the internal chemotaxis pathway, alternating between run and tumble states. As noted in the equations on lines 286 & 578, we incorporated the circular motion by introducing a constant angular velocity, −ν<sub>0</sub>/r, during the run state. Here, ν<sub>0</sub> represents the swimming speed, r denotes the radius of circular swimming, and the negative sign indicates clockwise chirality. Furthermore, to model the hydrodynamic interaction with the boundaries, we assumed that when a cell collides with a sidewall, its velocity vector instantly aligns parallel to that wall.

      The comparison of Figure 5B (simulations) with Figure 4B (experiments) does not strike me as so "similar". Why are the points at small widths so noisy (Figure 5AB)? Figure 5C is cut at these widths, it should be plotted over the entire scale.

      We acknowledge that the agreement between simulation and experiment is less robust in the narrowest channels. The discrepancy and "noise" at small widths in Figure 5 arise from the limitations of the self-propelled particle model in highly confined geometries. Specifically, our simulation treats bacteria as point particles and does not explicitly calculate the physical exclusion (steric effects) caused by the finite size of the flagella and cell body.

      In the experimental setup, steric constraints within narrow channels (comparable to the cell size) restrict the cells' ability to turn freely, effectively stabilizing their motion. However, because our model allows particles to reorient more freely than actual cells would in such confined spaces, it produces fluctuations and an overestimation of the drift velocity at small widths. If these confinement effects were fully incorporated, the cell density mismatch between the left and right sidewalls would be reduced, leading to lower drift velocities that match the experimental data more closely.

      Regarding Figure 5C: Since the "active particle" assumption loses physical validity in channels narrower than the scale of the bacterium, the simulation results in this regime are not representative of biological reality. Plotting these non-physical points would distort the analysis. Therefore, we have maintained the truncation of Figure 5C at 4 mm to ensure the data presented is physically meaningful. We have added a clear discussion of these model limitations to the manuscript at lines 310-314.

      These important precisions should be added to the text or in a supplementary section. A validated mechanism describing in detail the impact of the walls on the cell trajectories would greatly improve the conclusions.

      We thank the reviewer for the suggestions. As noted in the responses above, we have incorporated the details concerning the simulation assumptions and the model limitations at narrow widths into the revised manuscript. We have performed further analysis of the collision trajectories between bacteria and the sidewalls. As illustrated in the new Fig. S2, the data confirms that cells tend to align with and swim along the sidewalls following a collision, regardless of the initial impact angle.

      Reviewer #2 (Recommendations for the authors):

      Minor points

      (1) Related to swimming in 3D: The authors should specify the depth of field of the objective in their setup.

      We thank the reviewer for pointing this out. We have calculated the depth of field (DOF) of our objective to be approximately 3.7 µm. This estimate is based on the standard formula:

      where l = 610 nm (emission wavelength), n = 1.0 (refractive index), NA = 0.45 (numeric aperture), M = 20 (magnification), and e = 6.5 µm (camera resolution). We have added this specification to the "Microscopy and Data Acquisition" section of “Materials and Methods”.

      (2) Related to the interpretation of the width effect: We think plotting the cell enrichment, ie the probabilities P in Figure 4B normalized to the expected value if cells were homogeneously distributed ((3µm)/w for the side walls, (w - 6µm)/w for the middle) would help understand the strength of the wall 'siphoning' effect.

      We thank the reviewer for the suggestion. We have calculated the cell enrichment by normalizing the observed probabilities against the expected values for a homogeneous distribution, as suggested. The resulting relationship between cell enrichment and lane width is presented in Figure S4.

      Related to simulations:

      (1) Showing vd for the 3 regions in Figure S5 would be helpful also to understand the underlying mechanism.

      We thank the reviewer for the suggestion. The V<sub>d</sub> values for the three regions are shown in Fig. S5.

      (2) Figure 5B vs 4B: There is a mismatch in the right vs left side density at w=6µm in the simulations that is not here in the experiments. What could explain this difference?

      We appreciate the reviewer pointing this out. The mismatch in the simulations is due to the simplified treatment of cells as self-propelled particles, which overlooks the physical volume of the cell body and flagella. In narrow channels (w\=6 mm), these physical constraints would restrict the cells' ability to change direction freely - a factor not fully captured in the simulation. Accounting for these steric effects would trap cells more effectively against the walls, reducing the density asymmetry between the LSW and RSW and lowering the drift velocity. This would bring the simulation results closer to the experimental observations. We have added a discussion of these limitations and effects to the revised manuscript (lines 310-314).

      (3) The simulations essentially assume that the density of motile cells is homogeneous and equal at both x=0 and x=L open ends of the channel. Is it the case in the experiments, even with the gradient, and the walls creating some cell transport?

      We thank the reviewer for pointing this out. The simulation assumption is consistent with our experimental observations. Our data were recorded within 160-μm-long lanes located in the center of the wider (400 μm) cell channel. In this central region, the cells maintain a continuous flux. Furthermore, experiments were performed within 8 min of flow, limiting the time for significant cell density gradients to establish. As illustrated in Author response image 11, the inhomogeneity in the measured cell density distribution is insignificant across the length of the observation window, indicating that the walls and gradient do not create significant heterogeneity at the boundaries of the region of interest.

      Author response image 1.

      The cell density distribution along the gradient field from the data of 44-μm-wide lane.

      (4) Line 506: There is something strange with the definition of the bias. B cannot be the tumbling bias if k=B/0.31 s<sup>-1</sup> and the tumble-to-run rate is 5/s, because then the tumbling bias is B/0.31 / (B/0.31 + 5). Please clarify.

      We apologize for the confusion caused by the notation. In our model, B represents the CW bias of the individual flagellar motor, not the macroscopic tumbling bias of the cell. We assume the run-to-tumble rate is equivalent to the motor CCW-to-CW switching rate (k). Previous studies have shown that this rate increases linearly with the motor CW bias according to k=B/t, where t is a characteristic time (Ref. 50).

      Based on experimental data for wildtype cells, the average run time in the near-surface region is ~2.0 s (corresponding to a run-to-tumble rate of ~0.5 s<sup>-1</sup>) (Ref. 11), and the steady-state wildtype CW bias is ~0.15. Using these values, we determined t ~ 0.31 s. Consequently, the switching rate is defined as k=B/0.31 s<sup>-1</sup>. Since the tumble duration is constant (0.2 s) (Ref. 51), the tumble-to-run rate is fixed at 5 s<sup>-1</sup>. We have clarified these definitions and parameter values in lines 569-573.

      Other minor comments:

      (1) Line 20 and lines 34-35: We think that the connection to infection is questionable here and should be toned down.

      Thank you for the suggestion. We have revised Line 20 to read: “Understanding bacterial behavior in confined environments is helpful to elucidating microbial ecology and developing strategies to manage bacterial infections.” Additionally, we modified lines 34-35 to state: “Our results may offer insights into bacterial navigation in complex biological environments such as host tissues and biofilms, providing a preliminary step toward exploring microbial ecology in confined habitats and potential strategies for controlling bacterial infections.”

      (2) Line 49: Consider highlighting the change in the sense of rotation at the air-liquid interface.

      Thank you for the suggestion. We have now highlighted the difference in chirality between trajectories at the air-liquid interface and those at the liquid-solid interface. The text has been updated to read: “For example, E. coli swim clockwise when observed from above a solid surface, whereas Caulobacter crescentus move in tight, counter-clockwise circles when viewed from the liquid side.”

      (3) Lines 58-59: The sentence should be better formulated, explaining what is CheY-P and that its concentration changes because of a change in phosphorylation (P).

      Thank you for the suggestion. We have reformulated this section to explicitly define CheY-P and explain how its concentration is regulated through phosphorylation. The revised text reads: “The transmembrane chemoreceptors detect attractants or repellents and transmit signals into the cell by modulating the autophosphorylation of the histidine kinase CheA. Attractant binding suppresses CheA autophosphorylation, while repellent binding promotes it. This modulation alters the concentration of the phosphorylated response regulator protein, CheY-P.”

      (4) Lines 63-64: CheR CheB do a bit more than "facilitating" adaptation, they mediate it. The notation CheB(p) may be confusing, since "-P" was used above for CheY.

      Thank you for pointing this out. We have corrected the notation and strengthened the description of the enzymes' roles. The revised text is: “The adaptation enzymes CheR and CheB methylate and demethylate the receptors, respectively, mediating sensory adaptation.”

      (5) Line 130: there must be a typo in the formula.

      We have replaced the ambiguous lag time variable in Fig. 1C with _n_Δt to ensure mathematical consistency.

      (6) Additionally, \Delta t is both the time between the frame here and the lag time in Figure 1.

      Thank you for highlighting this ambiguity. We have updated the notation to distinguish these two values. The lag time in Figure 1 is now explicitly denoted as _n_Δt, while Δt remains the time interval between individual frames.

      (7) Line 162: "Consistent with previous reports," a reference to said reports is missing.

      Thank you for pointing this out. We have now added the reference (Ref. 41) to support this statement.

      (8) Figure 1B: Are these tracks in the presence of a gradient? Same as used in panel C? This needs to be explained.

      Response: Thank you for this question. We confirm that the tracks shown in Figure 1B were indeed recorded in the presence of a gradient and represent a subset of the data used in Figure 1C. We have clarified this in the figure legend as follows: "Thirty bacterial trajectories selected from the data of the 44-mm-wide lane in gradient assays. These represent a subset of the trajectories analyzed in panel C."

      (9) Simulations: the equation for x(t) should also be given for completeness.

      Thank you for the suggestion. For completeness, we have added the position updating equations for the run state to the Materials and Methods section (lines 579-580). The equations are defined as:

      (10) Figure S2: For the swimming directions that are more unstable due to the surface friction torque, RSW-DG, and LSW-UG, one would have expected that the Up-gradient motion is more persistent than the down gradient one. It seems to be the opposite. Is it significant, and what could be the reason for this?

      We apologize for the lack of clarity in our original explanation. While we would generally expect up-gradient motion to be more persistent than down-gradient motion in bulk fluid, our measurements near the surface show a different trend due to the specific contributions of run and tumble states to the escape rate. Cells swimming up-gradient (UG) in the LSW experience higher probability of running. Consequently, they are subjected to the destabilizing surface friction torque for a greater proportion of time compared to cells swimming down-gradient (DG) in the RSW. This can be explained mathematically. The escape rates for RSW-DG and LSW-UG can be expressed as:

      Where B<sup>+</sup> and B<sup>−</sup> represent the tumble bias (probability of tumbling) when swimming up-gradient and down-gradient, respectively, and k<sub>T</sub> and k<sub>R</sub> denote the escape rates during a tumble and a run, respectively. Due to the chemotactic response, 0≤ B<sup>+</sup>< B<sup>−</sup> ≤1. Crucially, our system is characterized by k<sub>R</sub>>k<sub>T</sub> (the escape rate is higher during a run than a tumble). Therefore, the lower tumble bias during up-gradient swimming (B<sup>+</sup>< B<sup>−</sup>) increases the weight of the run-state escape term((1−B<sup>+</sup>)k<sub>R</sub>), leading to a higher overall escape rate for LSW-UG compared to RSW-DG. We have added an intuitive understanding of k<sub>R</sub>>k<sub>T</sub> in the Supplemental text.

    1. Author response:

      The following is the authors’ response to the original reviews

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      This is a careful and comprehensive study demonstrating that effector-dependent conformational switching of the MT lattice from compacted to expanded deploys the alpha tubulin C-terminal tails so as to enhance their ability to bind interactors.

      Strengths:

      The authors use 3 different sensors for the exposure of the alpha CTTs. They show that all 3 sensors report exposure of the alpha CTTs when the lattice is expanded by GMPCPP, or KIF1C, or a hydrolysis-deficient tubulin. They demonstrate that expansion-dependent exposure of the alpha CTTs works in tissue culture cells as well as in vitro.

      Weaknesses:

      There is no information on the status of the beta tubulin CTTs. The study is done with mixed isotype microtubules, both in cells and in vitro. It remains unclear whether all the alpha tubulins in a mixed isotype microtubule lattice behave equivalently, or whether the effect is tubulin isotype-dependent. It remains unclear whether local binding of effectors can locally expand the lattice and locally expose the alpha CTTs.

      Appraisal:

      The authors have gone to considerable lengths to test their hypothesis that microtubule expansion favours deployment of the alpha tubulin C-terminal tail, allowing its interactors, including detyrosinase enzymes, to bind. There is a real prospect that this will change thinking in the field. One very interesting possibility, touched on by the authors, is that the requirement for MAP7 to engage kinesin with the MT might include a direct effect of MAP7 on lattice expansion.

      Impact:

      The possibility that the interactions of MAPS and motors with a particular MT or region feed forward to determine its future interaction patterns is made much more real. Genuinely exciting.

      We thank the reviewer for their positive response to our work. We agree that it will be important to determine if the bCTT is subject to regulation similar to the aCTT. However, this will first require the development of sensors that report on the accessibility of the bCTT, which is a significant undertaking for future work.

      We also agree that it will be important to examine whether all tubulin isotypes behave equivalently in terms of exposure of the aCTT in response to conformational switching of the microtubule lattice.

      We thank the reviewer for the comment about local expansion of the microtubule lattice. We believe that Figure 3 does show that local binding of effectors can locally expand the lattice and locally expose the alpha-CTTs. We have added text to clarify this.

      Reviewer #2 (Public review):

      The unstructured α- and β-tubulin C-terminal tails (CTTs), which differ between tubulin isoforms, extend from the surface of the microtubule, are post-translationally modified, and help regulate the function of MAPs and motors. Their dynamics and extent of interactions with the microtubule lattice are not well understood. Hotta et al. explore this using a set of three distinct probes that bind to the CTTs of tyrosinated (native) α-tubulin. Under normal cellular conditions, these probes associate with microtubules only to a limited extent, but this binding can be enhanced by various manipulations thought to alter the tubulin lattice conformation (expanded or compact). These include small-molecule treatment (Taxol), changes in nucleotide state, and the binding of microtubule-associated proteins and motors. Overall, the authors conclude that microtubule lattice "expanders" promote probe binding, suggesting that the CTT is generally more accessible under these conditions. Consistent with this, detyrosination is enhanced. Mechanistically, molecular dynamics simulations indicate that the CTT may interact with the microtubule lattice at several sites, and that these interactions are affected by the tubulin nucleotide state.

      Strengths:

      Key strengths of the work include the use of three distinct probes that yield broadly consistent findings, and a wide variety of experimental manipulations (drugs, motors, MAPs) that collectively support the authors' conclusions, alongside a careful quantitative approach.

      Weaknesses:

      The challenges of studying the dynamics of a short, intrinsically disordered protein region within the complex environment of the cellular microtubule lattice, amid numerous other binders and regulators, should not be understated. While it is very plausible that the probes report on CTT accessibility as proposed, the possibility of confounding factors (e.g., effects on MAP or motor binding) cannot be ruled out. Sensitivity to the expression level clearly introduces additional complications. Likewise, for each individual "expander" or "compactor" manipulation, one must consider indirect consequences (e.g., masking of binding sites) in addition to direct effects on the lattice; however, this risk is mitigated by the collective observations all pointing in the same direction.

      The discussion does a good job of placing the findings in context and acknowledging relevant caveats and limitations. Overall, this study introduces an interesting and provocative concept, well supported by experimental data, and provides a strong foundation for future work. This will be a valuable contribution to the field.

      We thank the reviewer for their positive response to our work. We are encouraged that the reviewer feels that the Discussion section does a good job of putting the findings, challenges, and possibility of confounding factors and indirect effects in context. 

      Reviewer #3 (Public review):

      Summary:

      In this study, the authors investigate how the structural state of the microtubule lattice influences the accessibility of the α-tubulin C-terminal tail (CTT). By developing and applying new biosensors, they reveal that the tyrosinated CTT is largely inaccessible under normal conditions but becomes more accessible upon changes to the tubulin conformational state induced by taxol treatment, MAP expression, or GTP-hydrolysis-deficient tubulin. The combination of live imaging, biochemical assays, and simulations suggests that the lattice conformation regulates the exposure of the CTT, providing a potential mechanism for modulating interactions with microtubule-associated proteins. The work addresses a highly topical question in the microtubule field and proposes a new conceptual link between lattice spacing and tail accessibility for tubulin post-translational modification.

      Strengths:

      (1) The study targets a highly relevant and emerging topic-the structural plasticity of the microtubule lattice and its regulatory implications.

      (2) The biosensor design represents a methodological advance, enabling direct visualization of CTT accessibility in living cells.

      (3) Integration of imaging, biochemical assays, and simulations provides a multi-scale perspective on lattice regulation.

      (4) The conceptual framework proposed lattice conformation as a determinant of post-translational modification accessibility is novel and potentially impactful for understanding microtubule regulation.

      Weaknesses:

      There are a number of weaknesses in the paper, many of which can be addressed textually. Some of the supporting evidence is preliminary and would benefit from additional experimental validation and clearer presentation before the conclusions can be considered fully supported. In particular, the authors should directly test in vitro whether Taxol addition can induce lattice exchange (see comments below).

      We thank the reviewer for their positive response to our work. We have altered the text and provided additional experimental validation as requested (see below).

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      (1) The resolution of the figures is insufficient.

      (2) The provision of scale bars is inconsistent and insufficient.

      (3) Figure 1E, the scale bar looks like an MT.

      (4) Figure 2C, what does the grey bar indicate?

      (5) Figure 2E, missing scale bar.

      (6) Figure 3 C, D, significance brackets misaligned.

      (7) Figure 3E, consider using the same alpha-beta tubulin / MT graphic as in Figure 1B.

      (8) Figure 5E, show cell boundaries for consistency?

      (9) Figure 6D, stray box above the y-axis.

      (11) Figure S3A, scale bar wrong unit again.

      (12) S3B "fixed" and mount missing scale bar in the inset.

      (13) S4 scale bars without scale, inconsistency in scale bars throughout all the figures.

      We apologize for issues with the figures. We have corrected all of the issues indicated by the reviewer.

      (10) Figure 6F, surprising that 300 mM KCL washes out rigor binding kinesin

      We thank the reviewer for this important point. To address the reviewer’s concern, we have added a new supplementary figure (new Figure 6 – Figure Supplement 1) which shows that the washing step removes strongly-bound (apo) KIF5C(1-560)-Halo<sup>554</sup> protein from the microtubules. In addition, we have made a correction to the Materials and Methods section noting that ATP was added in addition to the KCl in the wash buffer. We apologize for omitting this detail in the original submission. We also added text noting that the wash out step was based on Shima et al., 2018 where the observation chamber was washed with either 1 mM ATP and 300 mM K-Pipes or with 10 mM ATP and 500 mM K-Pipes buffer. In our case, the chamber was washed with 3 mM ATP and 300 mM KCl. It is likely that the addition of ATP facilitates the detachment of strongly-bound KIF5C.

      (14) Supplementary movie, please identify alpha and beta tubules for clarity. Please identify residues lighting up in interaction sites 1,2 & 3.

      Thank you for the suggestions. We have made the requested changes to the movie.

      Reviewer #2 (Recommendations for the authors):

      There appear to have been some minor issues (perhaps with .pdf conversion) that leave some text and images pixelated in the .pdf provided, alongside some slightly jarring text and image positioning (e.g., Figure 5E panels). The authors should carefully look at the figures to ensure that they are presented in the clearest way possible.

      We apologize for these issues with the figures. We have reviewed the figures carefully to ensure that they are presented in the clearest way possible.

      The authors might consider providing a more definitive structural description of compact vs expanded lattice, highlighting what specific parameters are generally thought to change and by what magnitude. Do these differ between taxol-mediated expansion or the effects of MAPs?

      Thank you for the suggestion. We have added additional information to the Introduction section.

      Reviewer #3 (Recommendations for the authors):

      (1) Figure 1 should include a schematic overview of all constructs used in the study. A clear illustration showing the probe design, including the origin and function of each component (e.g., tags, domains), would improve clarity.

      Thank you for the suggestion. We have added new illustrations to Figure 1 showing the origin and design (including domains and tags) of each probe.

      (2) Add Western blot data for the 4×CAP-Gly construct to Figure 1C for completeness.

      We thank the reviewer for this suggestion. We carried out a far-western blot using the purified 4xCAPGly-mEGFP protein to probe GST-Y, GST-DY, and GST-DC2 proteins (new Figure 1 – Figure Supplement 1C). We note that some bleed-through signal can be seen in the lanes containing GST-ΔY and GST-ΔC2 protein due to the imaging requirements and exposure needed to visualize the 4xCAPGly-mEGFP protein. Nevertheless, the blot shows that the purified CAPGly sensor specifically recognizes the native (tyrosinated) CTT sequence of TUBA1A.

      (3) Essential background information on the CAP-Gly domain, SXIP motif, and EB proteins is missing from the Introduction. These concepts appear abruptly in the Results and should be properly introduced.

      Thank you for the suggestion. We have added additional information to the Introduction section about the CAP-Gly domain. However, we feel that introducing the SXIP motif and EB proteins at this point would detract from the flow of the Introduction and we have elected to retain this information in the Results section when we detail development of the 4xCAPGly probe.

      (4) In Figure 2E, it remains possible that the CAP-Gly domain displacement simply follows the displacement of EB proteins. An experiment comparing EB protein localization upon Taxol treatment would clarify this relationship.

      We thank the reviewer for raising this important point. To address the reviewer’s concern, we utilized HeLa cells stably expressing EB3-GFP. We performed live-cell imaging before and after Taxol addition (new Figure 2 – Figure Supplement 1C). EB3-EGFP was lost from the microtubule plus ends within minutes and did not localize to the now-expanded lattice.

      (5) Statements such as "significantly increased" (e.g., line 195) should be replaced with quantitative information (e.g., "1.5-fold increase").

      We have made the suggested changes to the text.

      (6) Phrases like "became accessible" should be revised to "became more accessible," as the observed changes are relative, not absolute. The current wording implies a binary shift, whereas the data show a modest (~1.5-fold) increase.

      We have made the suggested changes to the text.

      (7) Similarly, at line 209, the terms "minimally accessible" versus "accessible" should be rephrased to reflect the small relative change observed; saturation of accessibility is not demonstrated.

      We have made the suggested changes to the text.

      (8) Statements that MAP7 "expands the lattice" (line 222) should be made cautiously; to my knowledge, that has not been clearly established in the literature.

      We thank the reviewer for this important comment. We have added text indicating that MAP7’s ability to induce or presence an expanded lattice has not been clearly established.

      (9) In Figures 3 and 4, the overexpression of MAP7 results in a strikingly peripheral microtubule network. Why is there this unusual morphology?

      The reviewer raises an interesting question. We are not sure why the overexpression of MAP7 results in a strikingly peripheral microtubule network but we suspect this is unique to the HeLa cells we are using. We have observed a more uniform MAP7 localization in other cell types [e.g. COS-7 cells (Tymanskyj et al. 2018), consistent with the literature [e.g. BEAS-2B cells (Shen and Ori-McKenney 2024), HeLa cells (Hooikaas et al. 2019)].

      (10) In Supplementary Figure 5C, the Western blot of detyrosination levels is inconsistent with the text. Untreated cells appear to have higher detyrosination than both wild-type and E254A-overexpressing cells. Do you have any explanation?

      We thank the reviewer for this important comment. We do not have an explanation at this point but plan to revisit this experiment. Unfortunately, the authors who carried out this work recently moved to a new institution and it will be several months before they are able to get the cell lines going and repeat the experiment. We thus elected to remove what was Supp Fig 5C until we can revisit the results. We believe that the important results are in what is now Figure 5 - Figure Supplement 1A,B which shows that the expression levels of the WT and E254E proteins are similar to each other.

      (11) The image analysis method in Figures 5B and 5D requires clarification. It appears that "density" was calculated from skeletonized probe length over total area, potentially using a strict intensity threshold. It looks like low-intensity binding has been excluded; otherwise, the density would be the same from the images. If so, this should be stated explicitly. A more appropriate analysis might skeletonize and integrate total fluorescence intensity relative to the overall microtubule network.

      We have added additional information to the Materials and Methods section to clarify the image analysis. We appreciate the reviewer’s valuable feedback and the suggestion to use the integrated total fluorescence intensity, which is a theoretically sound approach. While we agree that integrated intensity is a valid metric for specific applications, its appropriate use depends on two main preconditions:

      (1) Consistent microscopy image acquisition conditions.

      (2) Consistent probe expression levels across all cells and experiments.

      We successfully maintained consistent image acquisition conditions (e.g., exposure time) throughout the experiment. However, despite generating a stably-expressing sensor cell lines to minimize variation, there remains an inherent, biological variability in probe expression levels between individual cells. Integrated intensity is highly susceptible to this cell-to-cell variability. Relying on it would lead to a systematic error where differences in the total amount of expressed probe would be mistaken for differences in Y-aCTT accessibility.

      The density metric (skeletonized probe length / total cell area) was deliberately chosen as it serves as a geometric measure rather than an intensity-based normalization. The density metric quantifies the proportion of the microtubule network that is occupied by Y-aCTT-labeled structures, independent of fluorescence intensity. Thus, the density metric provides a more robust and interpretable measure of Y-aCTT accessibility under the variable expression conditions inherent to our experimental system. Therefore, we believe that this geometric approach represents the most appropriate analysis for our image dataset.

      (12) In Figure 5D, the fold-change data are difficult to interpret due to the compressed scale. Replotting is recommended. The text should also discuss the relative fold changes between E254A and Taxol conditions, Figure 2H.

      We appreciate the reviewer's insightful comment. We agree that the presence of significant outliers led to a compressed Y-axis scale in Figure 5D, obscuring the clear difference between the WT-tubulin and E254A-tubulin groups. As suggested, we have replotted Figure 5D using a broken Y-axis to effectively expand the relevant lower range of the data while still accurately representing all data points, including the outliers. We believe that the revised graph significantly enhances the clarity and interpretability of these results. For Figure 2, we have added the relative fold changes to the text as requested.

      (13) Figure 6. The authors should directly test in vitro whether Taxol addition can induce lattice exchange, for example, by adding Taxol to GDP-microtubules and monitoring probe binding. Including such an assay would provide critical mechanistic evidence and substantially strengthen the conclusions. I was waiting for this experiment since Figure 2.

      We thank the reviewer for this suggestion. As suggested, we generated GDP-MTs from HeLa tubulin and added it to two flow chambers. We then flowed in the YL1/2<sup>Fab</sup>-EGFP probe into the chambers in the presence of DMSO (vehicle control) or Taxol. Static images were taken and the fluorescence intensity of the probe on microtubules in each chamber was quantified. There was a slight but not statistically significant difference in probe binding between control and Taxol-treated GDP-MTs (Author response image 1). While disappointing, these results underscore our conclusion (Discussion section) that microtubule assembly in vitro may not produce a lattice state resembling that in cells, either due to differences in protofilament number and/or buffer conditions and/or the lack of MAPs during polymerization.

      Author response image 1.

      References

      Hooikaas, P. J., Martin, M., Muhlethaler, T., Kuijntjes, G. J., Peeters, C. A. E., Katrukha, E. A., Ferrari, L., Stucchi, R., Verhagen, D. G. F., van Riel, W. E., Grigoriev, I., Altelaar, A. F. M., Hoogenraad, C. C., Rudiger, S. G. D., Steinmetz, M. O., Kapitein, L. C. and Akhmanova, A. (2019). MAP7 family proteins regulate kinesin-1 recruitment and activation. J Cell Biol, 218, 1298-1318.

      Shen, Y. and Ori-McKenney, K. M. (2024). Microtubule-associated protein MAP7 promotes tubulin posttranslational modifications and cargo transport to enable osmotic adaptation. Dev Cell, 59, 1553-1570.

      Tymanskyj, S. R., Yang, B. H., Verhey, K. J. and Ma, L. (2018). MAP7 regulates axon morphogenesis by recruiting kinesin-1 to microtubules and modulating organelle transport. Elife, 7.

    1. Author response:

      The following is the authors’ response to the original reviews

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      This manuscript uses primarily simulation tools to probe the pathway of cholesterol transport with the smoothened (SMO) protein. The pathway to the protein and within SMO is clearly discovered, and interactions deemed important are tested experimentally to validate the model predictions.

      Strengths:

      The authors have clearly demonstrated how cholesterol might go from the membrane through SMO for the inner and outer leaflets of a symmetrical membrane model. The free energy profiles, structural conformations, and cholesterol-residue interactions are clearly described.

      We thank the reviewer for their kind words.

      (1) Membrane Model: The authors decided to use a rather simple symmetric membrane with just cholesterol, POPC, and PSM at the same concentration for the inner and outer leaflets. This is not representative of asymmetry known to exist in plasma membranes (SM only in the outer leaflet and more cholesterol in this leaflet). This may also be important to the free energy pathway into SMO. Moreover, PE and anionic lipids are present in the inner leaflet and are ignored. While I am not requesting new simulations, I would suggest that the authors should clearly state that their model does not consider lipid concentration leaflet asymmetry, which might play an important role.

      We thank the reviewer for their comment. Membrane asymmetry is inherent in endogenous systems; we acknowledge that as a limitation of our current model. We have addressed the comment by adding this limitation to our discussion in the manuscript.

      Added lines: (End of paragraph 6, Results subsection 2):

      “One possibility that might alter the thermodynamic barriers is native membrane asymmetry, particularly the anionic lipid-rich inner leaflet. This presents as a limitation of our current model.”

      (2) Statistical comparison of barriers: The barriers for pathways 1 and 2 are compared in the text, suggesting that pathway 2 has a slightly higher barrier than pathway 1. However, are these statistically different? If so, the authors should state the p-value. If not, then the text in the manuscript should not state that one pathway is preferred over the other.

      We thank the reviewer for their comment. We have added statistical t-tests for the barriers.

      Changes made: (Paragraph 6, Results subsection 2)

      “However, we also observe that pathway 1 shows a lower thermodynamic barrier (5.8 ± 0.7 kcal/mol v/s 6.5 ± 0.8 kcal/mol, p = 0.0013)”

      (3) Barrier of cholesterol (reasoning): The authors on page 7 argue that there is an enthalpy barrier between the membrane and SMO due to the change in environment. However, cholesterol lies in the membrane with its hydroxyl interacting with the hydrophilic part of the membrane and the other parts in the hydrophobic part. How is the SMO surface any different? It has both characteristics and is likely balanced similarly to uptake cholesterol. Unless this can be better quantified, I would suggest that this logic be removed.

      We thank the reviewer for this suggestion. We have removed the line to avoid confusion.

      Reviewer #2 (Public review):

      Summary:

      In this work, the authors applied a range of computational methods to probe the translocation of cholesterol through the Smoothened receptor. They test whether cholesterol is more likely to enter the receptor straight from the outer leaflet of the membrane or via a binding pathway in the inner leaflet first. Their data reveal that both pathways are plausible but that the free energy barriers of pathway 1 are lower, suggesting this route is preferable. They also probe the pathway of cholesterol transport from the transmembrane region to the cysteine-rich domain (CRD).

      Strengths:

      (1) A wide range of computational techniques is used, including potential of mean force calculations, adaptive sampling, dimensionality reduction using tICA, and MSM modelling. These are all applied rigorously, and the data are very convincing. The computational work is an exemplar of a well-carried out study.

      (2) The computational predictions are experimentally supported using mutagenesis, with an excellent agreement between their PMF and mRNA fold change data.

      (3) The data are described clearly and coherently, with excellent use of figures. They combine their findings into a mechanism for cholesterol transport, which on the whole seems sound.

      (4) The methods are described well, and many of their analysis methods have been made available via GitHub, which is an additional strength.

      Weaknesses:

      (1) Some of the data could be presented a little more clearly. In particular, Figure 7 needs additional annotation to be interpretable. Can the position of the cholesterol be shown on the graph so that we can see the diameter change more clearly?

      We thank the reviewer for this suggestion. We have added the cholesterol positions as requested.

      Changes made: (Caption, Figure 7)

      “The tunnel profile during cholesterol translocation in SMO. (a) Free energy plot of the zcoordinate v/s the tunnel diameter when cholesterol is present in the core TMD. The tunnel shows a spike in the radius in the TMD domain, indicating the presence of a cholesterol-accommodating cavity. (b) Representative figure for the tunnel when a cholesterol molecule is in the TMD. (c) Same as (a), when cholesterol is at the TMD-CRD interface. (e) same as (b), when cholesterol is at the TMD-CRD interface. (e) same as (a), when cholesterol is at the CRD binding site. (f) same as (b), when cholesterol is at the CRD binding site. Tunnel diameters shown as spheres. Cholesterol positions marked on plots using dotted lines. All snapshots presented are frames taken from MD simulations.”

      (2) In Figure 3C, it doesn’t look like the Met is constricting the tunnel at all. What residue is constricting the tunnel here? Can we see the Ala and Met panels from the same angle to compare the landscapes? Or does the mutation significantly change the tunnel? Why not A283 to a bulkier residue? Finally, the legend says that the figure shows that cholesterol can still pass this residue, but it doesn’t really show this. Perhaps if the HOLE graph was plotted, we could see the narrowest point of the tunnel and compare it to the size of cholesterol.

      We thank the reviewer for this suggestion. A283 was mutated to methionine as it presents with a longer heavy tail containing sulfur. We have plotted the tunnel radii for both WT and A283M mutants and added them as a supplemental figure. As shown in the figure, the presence of methionine doesn’t completely block the tunnel, but occludes it, thereby increasing the barrier for cholesterol transport slightly.

      Changes made: (End of Results subsection 1)

      “When we calculated the PMF for cholesterol entry, A<sup>2.60f</sup>M mutant showed restricted tunnel but it did not fully block the tunnel (Figure 3—figure Supplement 3).”

      (3) The PMF axis in 3b and d confused me for a bit. Looking at the Supplementary data, it’s clear that, e.g., the F455I change increases the energy barrier for chol entering the receptor. But in 3d this is shown as a -ve change, i.e., favourable. This seems the wrong way around for me. Either switch the sign or make this clearer in the legend, please.

      We thank the reviewer for this suggestion. We measured ∆PMF as PMF<sub>WT</sub> PMF<sub>mutant</sub>, hence the negative values. We have added additional text to the legend to clarify this.

      Changes made: (Caption, Figure 3)

      “(b) ∆Gli1 mRNA fold change (high SHH vs untreated) and ∆ PMF (difference of peak PMF , calculated as PMF<sub>WT</sub> - PMF<sub>mutant</sub>) plotted for the mutants in Pathway 1. (c) Example mutant A<sup>2_._60f</sup>M shows that cholesterol can enter SMO through Pathway 1 even on a bulky mutation. (d) Same as (b) but for Pathway 2 (e) Example mutant L<sup>5.62f</sup>A shows that cholesterol can enter SMO through Pathway 2 due to lesser steric hindrance. All snapshots presented are frames taken from MD simulations.”

      Changes made: (Caption, Figure 6)

      “(b) ∆Gli1 mRNA fold change (high SHH vs untreated) and ∆ PMF (difference of peak PMF, calculated as PMF<sub>WT</sub> - PMF<sub>mutant</sub>) plotted for mutants along the TMD-CRD pathway. (c, d) Example mutants Y<sup>LD</sup>A and F<sup>5.65f</sup>A show that cholesterol is unable to translocate through this pathway because of the loss of crucial hydrophobic contacts provided by Y207 and F484 and along the solvent-exposed pathway.”

      (4) The impact of G280V is put down to a decrease in flexibility, but it could also be a steric hindrance. This should be discussed.

      We thank the reviewer for this suggestion. We have added it as a possible mechanism of the decrease in activity of SMO.

      Changes made: (Paragraph 5, Results subsection 1)

      “We mutated G280<sup>2.57f</sup>  to valine - G<sup>2.57f</sup>V to test whether reducing the flexibility of TM2 prevents cholesterol entry into the TMD. Consequently, the activity of mSMO showed a decrease. However, this decrease could also be attributed to steric hindrance added by the presence of a bulky propyl group in valine.”

      (5) Are the reported energy barriers of the two pathways (5.8plus minus0.7 and 6.5plus minus0.8 kcal/mol) significantly and/or substantially different enough to favour one over the other? This could be discussed in the manuscript.

      We thank the reviewer for this suggestion. We have added statistical t-tests for the barriers.

      Changes made: (Paragraph 6, Results subsection 2)

      “However, we also observe that pathway 1 shows a lower thermodynamic barrier (5.8 ± 0.7 kcal/mol v/s 6.5 ± 0.8 kcal/mol, p = 0.001)”

      (6) Are the energy barriers consistent with a passive diffusion-driven process? It feels like, without a source of free energy input (e.g., ion or ATP), these barriers would be difficult to overcome. This could be discussed.

      We thank the reviewer for this suggestion. We have added a discussion to further clarify this point.

      Discussion: (Paragraph 6, Results subsection 2)

      “These values are comparable to ATP-Binding Cassette (ABC) transporters of membrane lipids, which use ATP hydrolysis (-7.54 ± 0.3 kcal/mol) (Meurer et al., 2017) to drive lipid transport from the membrane to an extracellular acceptor. Some of these transporters share the same mechanism as SMO, where the lipid from the inner leaflet is flipped and transported to the extracellular acceptor protein (Tarling et al., 2013). Additionally, for secondary active transporters that do not use ATP for the transport of substrates, a thermodynamic barrier of 5-6 kcal/mol has been reported in literature. (Chan et al., 2022; Selvam et al., 2019; McComas et al., 2023; Thangapandian et al., 2025).”

      (7) Regarding the kinetics from MSM, it is stated that the values seen here are similar to MFS transporters, but this then references another MSM study. A comparison to experimental values would support this section a lot.

      We thank the reviewer for this suggestion. We have added a discussion discussing millisecond-scale timescales measured for MFS transporters.

      Changes made: (Paragraph 2, Results subsection 5)

      “These timescales are comparable to the substrate transport timescales of Major Facilitator Superfamily (MFS) transporters (Chan et al., 2022). Furthermore, several experimental studies have also resolved the millisecond-scale kinetics of MFS transporters (Blodgett and Carruthers, 2005; Körner et al., 2024; Bazzone et al., 2022; Smirnova et al., 2014; Zhu et al., 2019), further corroborating the results from our study.”

      Reviewer #2 (Recommendations for the authors):

      (1) The heatmaps in Figures 2a and 4a are great. On these, an arrow denotes what looks like a minimum energy path. Is it possible to see this plotted, as this might show the height of the energy barriers more clearly?

      We thank the reviewer for this suggestion. We have computed the minimum energy paths for both pathways and presented them in a supplementary figure.

      Added lines: (Paragraph 4, Results subsection 1):

      For further clarity, we have plotted the minimum energy path taken by cholesterol as it translocates along this pathway (Figure 2—figure Supplement 3)a,b)

      Added lines: (Paragraph 4, Results subsection 2):

      For further clarity, we have plotted the minimum energy path taken by cholesterol as it translocates along this pathway (Figure 2—figure Supplement 3)c,d)

      (2) The tiCA data in S15 is first referred to on line 137, but the technique isn’t introduced until line 222. This makes understanding the data a little confusing. Reordering this might improve readability.

      We thank the reviewer for this suggestion. We have reordered the text to make it clearer.

      Changes made: (Paragraph 2, Results subsection 1) This provides evidence for multiple stable poses along the pathway as observed in the multiple stable poses of cholesterol in Cryo-EM structures of SMO bound to sterols (Deshpande et al., 2019; Qi et al., 2019b, 2020). A reliable estimate of the barriers comes from using the time-lagged Independent Components (tICs), which project the entire dataset along the slowest kinetic degrees of freedom. Overall, the highest barrier along Pathway 1 is 5.8 ± 0.7 kcal/mol, and it is associated with the entry of cholesterol into the TMD (Figure 2—Figure Supplement 2).

      Changes made: (Paragraph 3, Results subsection 2)

      “On plotting the first two components of tICs, (Figure 2—Figure Supplement 2), we observe that the energetic barrier between η and θ is ∼6.5 ± 0.8 kcal/mol.”

      (3) Missing bracket on line 577.

      We thank the reviewer for this suggestion. The typo has been fixed.

      (4) Line 577: Fig. S2nd?

      We thank the reviewer for this suggestion. This typo has been fixed.

      Reviewer #3 (Public review):

      Summary:

      This manuscript presents a study combining molecular dynamics simulations and Hedgehog (Hh) pathway assays to investigate cholesterol translocation pathways to Smoothened (SMO), a G protein-coupled receptor central to Hedgehog signal transduction. The authors identify and characterize two putative cholesterol access routes to the transmembrane domain (TMD) of SMO and propose a model whereby cholesterol traverses through the TMD to the cysteine-rich domain (CRD), which is presented as the primary site of SMO activation. The MD simulations and biochemical experiments are carefully executed and provide useful data.

      Weaknesses:

      However, the manuscript is significantly weakened by a narrow and selective interpretation of the literature, overstatement of certain conclusions, and a lack of appropriate engagement with alternative models that are well-supported by published data-including data from prior work by several of the coauthors of this manuscript. In its current form, the manuscript gives a biased impression of the field and overemphasizes the role of the CRD in cholesterol-mediated SMO activation. Below, I provide specific points where revisions are needed to ensure a more accurate and comprehensive treatment of the biology.

      (1) Overstatement of the CRD as the Orthosteric Site of SMO Activation

      The manuscript repeatedly implies or states that the CRD is the orthosteric site of SMO activation, without adequate acknowledgment of alternative models. To give just a few examples (of many in this manuscript):

      (a) “PTCH is proposed to modulate the Hh signal by decreasing the ability of membrane cholesterol to access SMO’s extracellular cysteine-rich domain (CRD)” (p. 3).

      (b) “In recent years, there has been a vigorous debate on the orthosteric site of SMO” (p. 3).

      (c) “cholesterol must travel through the SMO TMD to reach the orthosteric site in the CRD” (p. 4).

      (d) “we observe cholesterol moving along TM6 to the TMD-CRD interface (common pathway, Fig. 1d) to access the orthosteric binding site in the CRD” (p. 6).

      While the second quote in this list at least acknowledges a debate, the surrounding text suggests that this debate has been entirely resolved in favor of the CRD model. This is misleading and not reflective of the views of other investigators in the field (see, for example, a recent comprehensive review from Zhang and Beachy, Nature Reviews Molecular and Cell Biology 2023, which makes the point that both the CRD and 7TM sites are critical for cholesterol activation of SMO as well as PTCH-mediated regulation of SMO-cholesterol interactions).

      In contrast, a large body of literature supports a dual-site model in which both the CRD and the TMD are bona fide cholesterol-binding sites essential for SMO activation. Examples include:

      (a) Byrne et al., Nature 2016: point mutation of the CRD cholesterol binding site impairs-but does not abolish-SMO activation by cholesterol (SMO D99A, Y134F, and combination mutants - Fig 3 of the 2016 study).

      (b) Myers et al., Dev Cell 2013 and PNAS 2017: CRD deletion mutants retain responsiveness to PTCH regulation and cholesterol mimetics (similar Hh responsiveness of a CRD deletion mutant is also observed in Fig. 4 Byrne et al, Nature 2016).

      (c) Deshpande et al., Nature 2019: mutation of residues in the TMD cholesterol binding site blocks SMO activation entirely, strongly implicating the TMD as a required site, in contrast to the partial effects of mutating or deleting the CRD site.

      Qi et al., Nature 2019, and Deshpande et al., Nature 2019, both reported cholesterol binding at the TMD site based on high-resolution structural data. Oddly, Deshpande et al., Nature 2019, is not cited in the discussion of TMD binding on p. 3, despite being one of the first papers to describe cholesterol in the TMD site and its necessity for activation (the authors only cite it regarding activation of SMO by synthetic small molecules).

      Kinnebrew et al., Sci Adv 2022 report that CRD deletion abolished PTCH regulation, which is seemingly at odds with several studies above (e.g., Byrne et al, Nature 2016; Myers et al, Dev Cell 2013); but this difference may reflect the use of an N-terminal GFP fusion to SMO in the Kinnebrew et al 2022, which could alter SMO activation properties by sterically hindering activation at the TMD site by cholesterol (but not synthetic SMO agonists like SAG); in contrast, the earlier work by Byrne et al is not subject to this caveat because it used an untagged, unmodified form of SMO.

      Although overexpression of PTCH1 and SMO (wild-type or mutant) has been noted as a caveat in studies of CRD-independent SMO activation by cholesterol, this reviewer points out that several of the studies listed above include experiments with endogenous PTCH1 and low-level SMO expression, demonstrating that SMO can clearly undergo activation by cholesterol (as well as regulation by PTCH1) in a manner that does not require the CRD.

      Recommendation: The authors should revise the manuscript to provide a more balanced overview of the field and explicitly acknowledge that the CRD is not the sole activation site. Instead, a dual-site model is more consistent with available structural, mutational, and functional data. In addition, the authors should reframe their interpretation of their MD studies to reflect this broader and more accurate view of how cholesterol binds and activates SMO.

      We thank the reviewer for this comprehensive overview of the existing literature. We agree that cholesterol binding to both the TMD and CRD sites is required for full activation of SMO. As described below in responses to comments, we have made changes to the manuscript to make this point clear. For instance, in the revised manuscript, we refrain from calling the CRD cholesterol binding site the “orthosteric site”. Instead, we highlight that the goal of the manuscript is not to resolve the debate over whether the TMD or CRD site is more important for PTCH1 regulation by SMO but rather to use molecular dynamics to understand the fascinating question of how cholesterol in the membrane can reach the CRD, located at a significant distance above the outer leaflet of the membrane. We believe that this is an important goal since there is an abundance of evidence that supports the view that PTCH1 inhibits SMO by reducing cholesterol access to the CRD. This evidence is now summarized succinctly in the introduction:

      Changes made: (Paragraph 4, Introduction)

      “While cholesterol binding to both the TMD and CRD sites is required for full SMO activation, our work focuses on how cholesterol gains access to the CRD site, perched above the outer leaflet of the membrane (Luchetti et al., 2016; Kinnebrew et al., 2022). Multiple lines of evidence suggest that PTCH1-regulated cholesterol binding to the CRD plays an instructive role in SMO regulation both in cells and animals. Mutations in residues predicted to make hydrogen bonds with the hydroxyl group of cholesterol bound to the CRD reduced both the potency and efficacy of SHH in cellular signaling assays (Kinnebrew et al., 2022; Byrne et al., 2016) and, more importantly, eliminated HH signaling in mouse embryos (Xiao et al., 2017). Experiments using both covalent and photocrosslinkable sterol probes in live cells directly show that PTCH1 activity reduces sterol access to the CRD (Kinnebrew et al., 2022; Xiao et al., 2017). Notably, our simulations evaluate a path of cholesterol translocation that includes both the TMD and CRD sites: cholesterol first enters the 7-transmembrane domain bundle from the membrane; it then engages the TMD site before continuing along a conduit to the CRD site. Thus, we analyze translocation energetics and residue-level contacts along a path that includes both the TMD and the CRD.”

      However, Reviewer 3 makes several comments below that are biased, inaccurate, or selective. We feel it is important to address these so readers can approach the literature from a balanced perspective. Indeed, the eLife review forum provides an ideal venue to present contrasting views on a scientific model. We encourage the editors to publish both Reviewer 3’s comments and our response in full so readers can read the original papers and reach their own conclusions. It is important to note these issues are not relevant to the quality of the computational and experimental data presented in this paper.

      We have now removed the term “orthosteric” to describe the CRD site throughout the paper and clearly state in the introduction that “both the CRD and TMD sites are required for SMO activation” but that our focus is on how cholesterol moves from the membrane to the CRD site. There is no doubt that cholesterol binding to the CRD plays a key role in SMO activation– our focus on this path is justified and does not devalue the importance of the TMD site. Our prior models (see Figure 7 of Kinnebrew 2022 explicitly include contributions of both sites).

      Now we respond to some of the concerns outlined, individually:

      (1) Byrne et al., Nature 2016: point mutation of the CRD cholesterol binding site impairs-but does not abolish-SMO activation by cholesterol (SMO D99A, Y134F, and combination mutants - Fig 3 of the 2016 study)

      The fact that a point mutation dramatically diminishes (but does not abolish signaling) does not mean that the CRD cholesterol binding site is not important for SMO regulation. Indeed, the reviewer fails to mention that Song et. al. (Molecular Cell, 2017) found that a SMO protein carrying a subtle mutation at D99 (D95/99N, a residue that makes a hydrogen bond with the cholesterol hydroxyl) completely abolishes SMO signaling in mouse embryos. Thus, the CRD site is critical for SMO activation in an intact animal, justifying our focus on evaluating the path of cholesterol translocation to the CRD site.

      (2) Myers et al., Dev Cell 2013 and PNAS 2017: CRD deletion mutants retain responsiveness to PTCH regulation and cholesterol mimetics (similar Hh responsiveness of a CRD deletion mutant is also observed in Fig 4 Byrne et al, Nature 2016).

      The Reviewer fails to note that CRD-deleted versions of SMO have markedly (>10-fold) higher basal (i.e. ligand-independent) activity compared to full-length SMO. The response to SHH is minimal (∼2-fold), compared to >50-100-fold with full-length SMO. Thus, CRD-deleted SMO is likely in a non-native conformation. Local changes in cholesterol accessibility caused by PTCH1 inactivation or cholesterol loading can cause small fluctuations in delta-CRD activity, but this cannot be used to infer meaningful insights about how native, full-length SMO (with >10-fold lower basal activity) is regulated. We encourage the reviewer to read our previous paper (Kinnebrew et. al. 2022), which presents a unified view of how the TMD and CRD sites together regulate SMO activation.

      A more physiological experiment, reported in Kinnebrew et. al. 2022, tested mutations in residues that make hydrogen bonds with cholesterol at the CRD and TMD sites in the context of full-length SMO. These mutants were stably expressed at moderate levels in Smo<sup>−/−</sup> cells. Mutations at the CRD site reduced the fold-increase in signaling output in response to SHH, as would be expected for a PTCH1-regulated site. In contrast, analogous mutations in the TMD site reduced the magnitude of both basal and maximal signaling, without affecting the fold-change in response to SHH. In signaling assays, the key parameter in evaluating the impact of a mutation is whether it impacts the change in output in response to a signal (in this case PTCH1 inactivation by SHH). A mutation in SMO that affects PTCH1 regulation is expected to decrease the fold-change in signaling in response to SHH, a criterion that is fulfilled by mutations in the CRD site. Accordingly, mutations in the CRD site abolish SMO signaling in mouse embryos (Xiao et al., 2017).

      (3) Deshpande et al., Nature 2019: mutation of residues in the TMD cholesterol binding site blocks SMO activation entirely, strongly implicating the TMD as a required site, in contrast to the partial effects of mutating or deleting the CRD site.

      Introduction of bulky mutations at the TMD site (V333F) that abolish SMO activity were first reported by Byrne et. al. 2016 and were used to markedly increase the stability of SMO for protein expression. These mutations indeed stabilize the inactive state of SMO, increasing protein abundance and completely preventing its localization at primary cilia. SMO variants carrying such bulky mutations cannot be used to infer the importance of the TMD site since they do not distinguish between the following possibilities: (1) SMO is inactive because the sterol cannot bind, or (2) SMO is inactive because it is locked in an inactive conformation, or (3) SMO is inactive because it cannot localize to primary cilia (where it must be localized to activate downstream signaling).

      As described in Response 3.3, a better evaluation of the importance of the TMD site is the use of mutations in residues that make hydrogen bonds with the hydroxyl group of TMD cholesterol. These mutations do not markedly increase protein stability or prevent ciliary localization (Kinnebrew 2022, Fig.S2). While a TMD site mutation decreases the magnitude of maximal (and basal) SMO signaling, it does not impact the fold-increase in signal output in response to Hh ligands (the key parameter that should be used to evaluate PTCH1 activity).

      (4) Qi et al., Nature 2019, and Deshpande et al., Nature 2019, both reported cholesterol binding at the TMD site based on high-resolution structural data. Oddly, Deshpande et al., Nature 2019 not cited in the discussion of TMD binding on p. 3, despite being one of the first papers to describe cholesterol in the TMD site and its necessity for activation (the authors only cite it regarding activation of SMO by synthetic small molecules)

      The reference has now been added at this location in the manuscript.

      (5) Kinnebrew et al., Sci Adv 2022 report that CRD deletion abolished PTCH regulation, which is seemingly at odds with several studies above (e.g., Byrne et al, Nature 2016; Myers et al, Dev Cell 2013); but this difference may reflect the use of an N-terminal GFP fusion to SMO in the Kinnebrew et al 2022, which could alter SMO activation properties by sterically hindering activation at the TMD site by cholesterol (but not synthetic SMO agonists like SAG); in contrast, the earlier work by Byrne et al is not subject to this caveat because it used an untagged, unmodified form of SMO.

      The reviewer fails to note that CRD deleted versions of SMO have markedly (>10-fold) higher basal activity than full-length SMO. The response to SHH is minimal (∼2fold), compared to >50-fold with full-length SMO. Thus, CRD-deleted SMO is likely in a non-native conformation. Local changes in cholesterol accessibility caused by PTCH1 inactivation or cholesterol loading can cause small fluctuations in delta-CRD activity, but this cannot be used to infer meaningful insights about how native, full-length SMO (with >10-fold lower basal activity) is regulated. Please see Response 3.3 for further details.

      Reviewer 3 presents an incomplete picture of the extensive experiments reported in Kinnebrew et. al. to establish the functionality of YFP-tagged delta-CRD SMO. Most importantly, a TMDselective sterol analog (KK174) can fully activate YFP-tagged delta-CRD, showing conclusively that the YFP fusion does not block sterol access to the TMD site. The fact that this protein is nearly unresponsive to SHH highlights the critical role of the CRD-bound cholesterol in SMO regulation by PTCH1. Indeed, the YFP-tagged, CRD-deleted SMO was made purposefully to test the requirement of the CRD in a construct that had normal basal activity. Again, this data justifies the value of investigating the path of cholesterol movement from the membrane via the TMD site to the CRD.

      (6) Although overexpression of PTCH1 and SMO (wild-type or mutant) has been noted as a caveat in studies of CRD-independent SMO activation by cholesterol, this reviewer points out that several of the studies listed above include experiments with endogenous PTCH1 and low-level SMO expression, demonstrating that SMO can clearly undergo activation by cholesterol (as well as regulation by PTCH1) in a manner that does not require the CRD.

      This comment is inaccurate. The data presented in Deshpande et. al. (and prior work in Myers et. al.) used transient transfection to overexpress SMO in Smo<sup>−/−</sup> cells. At the individual cell level transient transfection produces expression levels that are markedly higher (10-1000-fold) than stable expression (in addition to being more variable). Most scientists would agree that stable expression (as used in Kinnebrew 2022) at a moderate expression level is a better system to compare mutant phenotypes, assess basal and activated signaling, and provide an accurate measure of the fold-change in signal output in response to SHH. Notably, introduction of a mutation in the CRD cholesterol binding site at the endogenous mouse Smo locus (an even better experiment than stable expression) leads to complete loss of SMO activity (PMID 28344083). This result again justifies our investigation of the pathway of cholesterol movement from the membrane to the CRD site.

      We have changed the initial discussion and reflect a more general outlook.

      Changes made: (Paragraph 1, Introduction)

      “PTCH modulates the availability of accessible cholesterol at the primary cilium and thereby regulates SMO, with models invoking effects on both the CRD and 7TM pockets.”

      Changes made: (Results subsection 3, paragraph 1)

      “According to the dual-site model, to reach the binding site in the CRD (ζ), cholesterol translocate along the TMD-CRD interface from the TM binding site (α∗) is required.”

      Added lines: (Paragraph 5, Results subsection 3):

      “The computational investigation showed here covers the dual-site model, where cholesterol reaches the CRD site via binding to the TM binding site first. In comparison to the CRD site, the TM site is more stable by ∼ 2 kcal/mol (Figure 2—Figure Supplement 3b, d).”

      Added lines: (Paragraph 2, Conclusions):

      “Here we have explored the role the CRD-site plays in SMO activation. In addition, through simulating the CRD site-dependent SMO activation hypothesis, we have also simulated the TMD site-dependent activation. We show that the overall stability of cholesterol is higher than the CRD site by ∼ 2 kcal/mol.”

      (2) Bias in Presentation of Translocation Pathways

      The manuscript presents the model of cholesterol translocation through SMO to the CRD as the predominant (if not sole) mechanism of activation. Statements such as: "Cholesterol traverses SMO to ultimately reach the CRD binding site" (p. 6) suggest an exclusivity that is not supported by prior literature in the field. Indeed, the authors’ own MD data presented here demonstrate more stable cholesterol binding at the TMD than at the CRD (p 17), and binding of cholesterol to the TMD site is essential for SMO activation. As such, it is appropriate to acknowledge that cholesterol may activate SMO by translocating through the TM5/6 tunnel, then binding to the TMD site, as this is a likely route of SMO activation in addition to the CRD translocation route they highlight in their discussion.

      The authors describe two possible translocation pathways (Pathway 1: TM2/3 entry to TMD; Pathway 2: TM5/6 entry and direct CRD transfer), but do not sufficiently acknowledge that their own empirical data support Pathway 2 as more relevant. Indeed, because their experimental data suggest Pathway 2 is more strongly linked to SMO activation, this pathway should be weighted more heavily in the authors’ discussion. In addition, Pathway 2 is linked to cholesterol binding to both the TMD and CRD sites (the former because the TMD binding site is at the terminus of the hydrophobic tunnel, the latter via the translocation pathway described in the present manuscript), so it is appropriate that Pathway 2 figures more prominently than Pathway 1 in the authors’ discussion.

      The authors also claim that "there is no experimental structure with cholesterol in the inner leaflet region of SMO TMD" (p 16). However, a structural study of apo-SMO from the Manglik and Cheng labs (Zhang et al., Nat Comm, 2022) identified a cholesterol molecule docked at the TM5/6 interface and also proposed a "squeezing" mechanism by which cholesterol could enter the TM5/6 pocket from the membrane. The authors do not consider this SMO conformation in their models, nor do they discuss the possibility that conformational dynamics at the TM5/6 interface could facilitate cholesterol flipping and translocation into the hydrophobic conduit, despite both possibilities having precedent in the 2022 empirical cryoEM structural analysis.

      Recommendation: The authors should avoid oversimplifying the SMO cholesterol activation process, either by tempering these claims or broadening their discussion to better reflect the complexity and multiplicity of cholesterol access and activation routes for SMO. They should also consider the 2022 apo-SMO cryoEM structure in their analysis of the TM5/6 translocation pathway.

      We thank the reviewer for this comprehensive overview of the existing literature and parts we have missed to include in the discussion. We agree with the reviewer, since our data shows that both pathways are probable. Through our manuscript, we have avoided using a competitive approach (that one pathway dominates over the other). Instead, we have evaluated both pathways independently and presented a comparative rather than competitive overview of both pathways from our observations. While we agree that experimental evidence suggests the inner leaflet pathway is possible, we cannot discount the observations made in previous studies that support the outer leaflet pathway, particularly Hedger et al. (2019), Bansal et al. (2023), and Kinnebrew et al. (2021). Therefore, considering the reviewer’s comments have made the following changes:

      (1) Added lines: (Paragraph 3, Conclusions):

      “We show that the barriers associated with the pathway starting from the outer leaflet are lower by ∼0.7 kcal, (p=0.0013). We also provide evidence that cholesterol can enter SMO via both leaflets, considering that multiple computational and experimental studies have found cholesterol entry sites and activation modulation via the outer leaflet, between TM2TM3. This is countered by evidence from multiple experimental and computational studies corroborating entry via the inner leaflet, between TM5-TM6, including this study. Overall, we posit that cholesterol translocation from either pathway is feasible.”

      (2)nChanges made: (Paragraph 6, Results subsection 2)

      “Based on our experimental and computational data, we conclude that cholesterol translocation can happen via either pathway. This is supported on the basis of the following observations: mutations along pathway 2 affect SMO activity more significantly, and the presence of a direct conduit that connects the inner leaflet to the TMD binding site. In addition, a resolved structure of SMO in the presence of cholesterol shows a cholesterol situated at the entry point from the membrane into the protein between TM5 and TM6, in the inner leaflet. However, we also observe that pathway 1 shows a lower thermodynamic barrier (5.8 ± 0.7 kcal/mol vs. 6.5 ± 0.8 kcal/mol, p \= 0.0013). Additionally, PTCH1 controls cholesterol accessibility in the outer leaflet. This shows that there is a possibility for transport from both leaflets. One possibility that might alter the thermodynamic barriers is native membrane asymmetry, particularly the anionic lipid-rich inner leaflet. This presents as a limitation of our current model.”

      (3)nChanges made: (Paragraph 1, Results subsection 2)

      “In a structure resolved in 2022, cholesterol was observed at the interface between the protein and the membrane, in the inner leaflet, between TMs 5 and 6. However, cholesterol in the inner leaflet has a downward orientation, with the polar hydroxyl group pointing intracellularly (η). A striking observation is that this cholesterol binding site pose was never used as a starting point for simulations and was discovered independent of the pose described in Zhang et al. (2022) (Figure 4—Figure Supplement 1).”

      (3) Alternative Possibility: Direct Membrane Access to CRD

      The possibility that the CRD extracts cholesterol directly from the membrane outer leaflet is not considered. While the crystal structures place the CRD in a stable pose above the membrane, multiple cryo-EM studies suggest that the CRD is dynamic and adopts a variety of conformations, raising the possibility that the stability of the CRD in the crystal structures is a result of crystal packing and that the CRD may be far more dynamic under more physiological conditions.

      Recommendation: The authors should explicitly acknowledge and evaluate this potential mechanism and, if feasible, assess its plausibility through MD simulations.

      We thank the reviewer for the suggestion. We have addressed this comment by calculating the distance from the lipid headgroups for each lipid in the membrane to the cholesterol binding site. We show that in our study, we do not observe any bending of the CRD over the membrane, precluding any cholesterol from being extracted from the membrane directly.

      Added lines: (Paragraph 3, Conclusions):

      “An alternative possibility states that the flexibility associated with the CRD would allow it to directly access the membrane, and consequently, cholesterol. In the extensive simulations reported in this study, the binding site of cholesterol in the CRD remains at least 20 Å away from the nearest lipid head group in the membrane, suggesting that such direct extraction and the bending of the CRD do not occur within the timescales sampled (Appendix 2 – Figure 6).

      The mechanistic details of this process are still unexplored and form the basis of future work.”

      (4) Inconsistent Framing of Study Scope and Limitations

      The discussion contains some contradictory and misleading language. For example, the authors state that "In this study we only focused on the cholesterol movement from the membrane to the CRD binding site," and then several sentences later state that "We outline the entire translocation mechanism from a kinetic and thermodynamic perspective." These statements are at odds. The former appropriately (albeit briefly) notes the limited scope of the modeling, while the latter overstates the generality of the findings.

      In addition, the authors’ narrow focus on the CRD site constitutes a major caveat to the entire work. It should be acknowledged much earlier in the manuscript, preferably in the introduction, rather than mentioned as an aside in the penultimate paragraph of the conclusion.

      Recommendation: The authors should clarify the scope of the study and expand the discussion of its limitations. They should explicitly acknowledge that the study models one of several cholesterol access routes and that the findings do not rule out alternative pathways.

      We thank the reviewer for the suggestion. We have addressed this comment by explicitly mentioning the scope of the study.

      Changes made: (Paragraph 3, Conclusions)

      “We outline the entire translocation mechanism from a kinetic and thermodynamic perspective for one of the leading hypotheses for the activation mechanism of SMO.”

      (5) Summary:

      This study has the potential to make a useful contribution to our understanding of cholesterol translocation and SMO activation. However, in its current form, the manuscript presents an overly narrow and, at times, misleading view of the literature and biological models; as such, it is not nearly as impactful as it could be. I strongly encourage the authors to revise the manuscript to include:

      (1) A more balanced discussion of the CRD vs. TMD binding sites.

      (2) Acknowledgment of alternative cholesterol access pathways.

      (3) More comprehensive citation of prior structural and functional studies.

      (4) Clarification of assumptions and scope.

      Of note, the above suggestions require little to no additional MD simulations or experimental studies, but would significantly enhance the rigor and impact of the work.

      We thank the reviewer for the suggestions. We have taken into account the literature and diverse viewpoints. We have changed the initial discussion and reflected a more general outlook. In the revised version of the manuscript, we have refrained from referring to the CRD site as the orthosteric site. Instead, we refer to it as the CRD sterol-binding site. To better represent the dual-site model, we add further discussion in the Introduction. Through our manuscript, we have avoided using a competitive approach (that one pathway dominates over the other). Instead, we have evaluated both pathways independently and presented a comparative rather than competitive overview of both pathways from our observations. We explicitly mention the scope of the study.

    1. Storia della vaccinazione, Edward Jenner

      🥸 Ecco perché Edward Jenner era un ignorante e cialtrone. ☣.. 💥💫 Sir Charles Creighton, MD, nella voce “Vaccinazioni” pubblicata nell’Enciclopedia Britannica del 1888, demolisce il “Jennerismo” con tre passaggi che, nel suo impianto argomentativo, ne fanno crollare la pretesa di scientificità: 👉 1. Oggetto instabile: il cow-pox viene descritto come fenomeno condizionato, non come entità naturale semplice, univoca e costante. 👉 2. Mezzo non unico: la “linfa vaccinale” appare come una filiera di stock e pratiche (passaggi, selezioni, sostituzioni), non come una sostanza unica con effetti invariabili. 👉 3. Prova fallace: l’impianto dimostrativo è accusato di post hoc ergo propter hoc, mentre l’uso delle statistiche dell’epoca viene messo duramente alla prova. ⚡️🔥 Un caso di invalidazione di una teoria pseudoscientifica attraverso testimonianze storiche. 😎 Articolo completo (con rimandi alle pagine originali): 👇https://autoimmunityreactions.org/wp/2026/01/07/creighton-britannica-1888-la-storia-naturale-del-cow-pox-come-leva-per-smontare-la-vaccinazione-jenneriana/

    1. Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

      Learn more at Review Commons


      Reply to the reviewers

      Reviewer #1

      Evidence, reproducibility and clarity

      Authors should be commended for the availability of data/code and detailed methods. Clarity is good. Authors have clearly spent a lot of time thinking about the challenges of metabolomics data analysis.

      Significance

      Schmidt et al. present MetaProViz, a comprehensive and modular platform for metabolomics data analysis. The tool provides a full suite of processing capabilities spanning metabolite annotation, quality control, normalization, differential analysis, integration of prior knowledge, functional enrichment, and visualization. The authors also include example datasets, primarily from renal cancer studies, to demonstrate the functionality of the pipeline. The MetaProViz framework addresses several long-standing challenges in metabolomics data analysis, particularly issues of reproducibility, ambiguous metabolite annotation, and the integration of metabolite features with pathway knowledge. The platform is likely to be a valuable addition for the community, but the reviewer has some comments that need to be addressed prior to publication.

      We thank the reviewer for this positive feedback.

      Comments:

      (1) (Planned)

      The section "Improving the connection between prior knowledge and metabolomics features" could benefit from additional clarification. It is not entirely clear to the reader what specific steps were taken beyond using RaMP-DB to translate metabolite identifiers. For example, how exactly were ambiguous mappings ("different scenarios") handled in practice, and to what extent does this process "fix" or merely flag inconsistencies? A more explicit description or example of how MetaProViz resolves these cases would help readers better understand the improvements claimed.

      We thank the reviewer for pointing this out and we agree that this section requires extension to ensure clarity. Beyond using RaMP-DB, we are characterising the mapping ambiguity (one-to-none, one-to-many, many-to-one, many-to-many) within and across metabolite-sets (i.e. pathways) and return this information to the user together with the translated identifiers. This is important to understand potential inflation/deflation of metabolite-sets that occur due to the translation. Moreover, we also offer the manually curated amino-acid collection to ensure L-, D- and zwitterion without chirality IDs are assigned for aminoacids (Fig. 2b). Ambiguous mappings are handled based on the measured data (Fig. 2e). Indeed, many translation cases that deflate (many-to-one mapping) or inflate (one-to-many mapping) the metabolite-sets are resolved when merging the prior knowledge with actual measured data (i.e. Fig. 2e, one-to-many in scenario 1, which becomes obsolete as only one/none of the many potential metabolite IDs is detected). By sorting each mapping into one of those scenarios, we only flag those cases. The reason for this decision has been that in many cases multiple decisions are valid (i.e. Fig. 2e, Scenario 5: Here the values of the two detected metabolites could be summed or the metabolite value with the larger Log2FC could be kept) and it should really be up to the user to make those dependent on their knowledge of the biological system and the analytical LC-MS method used.

      Since these points have not been clear enough, we will add a more explicit description to the results section by showcasing more details on how we exactly tackled this problem in the ccRCC example data. This has also been suggested by Reviewer 3 (Minor Comment 7 and 8), so feel free to also see the responses below.

      (2) (Planned)

      The introduction of MetSigDB is intriguing, but its construction and added value are not sufficiently described. It would be helpful to clarify what specific advantages MetSigDB provides over directly using existing pathway resources such as KEGG, Reactome, or WikiPathways. For example, how many features, interactions, or metabolite-set relationships are included, and in what way are these pathways improved or extended compared to those already available in public databases?

      We thank the reviewer for this valuable comment and we apologise that this was not described sufficiently. One of the major advantages is that all the resources are available in one place following the same table format without the need to visit the different original resources and perform data wrangling prior to enrichment analysis. In addition, where applicable, we have removed metabolites that are not detectable by LC-MS (i.e. ions, H2O, CO2) to circumvent pathway inflation with features that are never within the data and hence impacting the statistical testing in enrichment analysis workflows.

      During the revision, we will compile an Extended Data Table listing all the resources present in MetSigDB, their number of features and interactions. We will also extend the methods section "Prior Knowledge access" about MetSigDB and how we removed metabolites.

      (3)

      Figure 1D/1E: The reviewer appreciates the inclusion of the visualizations illustrating the different mapping scenarios, as these effectively convey the complexity of metabolite ID translation. However, it took some time to interpret what each scenario represented. It would be helpful to include brief annotations or explanatory text directly on the figures to clarify what each scenario depicts and how it relates to the underlying issue being addressed.

      *We think the reviewer refers to Fig. 2D/E and we acknowledge that this is a complex problem we try to convey. We received a similar comment from Reviewer 2 (Minor Comment 1), who asked to extend the figure legend description of what the different scenarios display. *

      We have extended the figure legend and specifically explained each displayed case and its meaning (Line 222-242):

      "d-e) Schematics of possible mapping cases between metabolite IDs (= each circle corresponds to one ID) of a pathway-metabolite set (e.g. KEGG) to metabolites IDs of a different database (e.g. HMDB) with (d) showing many-to-many mappings that can occur within and across pathway-metabolite sets and (e) additionally showing the mapping to metabolite IDs that were assigned to the detected peaks within and across pathway-metabolite sets. (d) __Translating the metabolite IDs of a pathway-metabolite set can lead to special cases such as many-to-one mappings (Pathway 1), where for example the original resource used the ID for L-Alanine (Pathway 1, green) and D-Alanine (Pathway 1, yellow) in the amino-acid pathway, whilst the translated resources only has an entry for Alanine zwitterion (Pathway 1, blue). Additionally, many-to-one mappings can also occur across pathways (Pathway 2-4), where this mapping is only detected when mappings are analysed taking all pathways into account. Both of these cases deflate the pathways, which can also happen for one-to-none mappings (Pathway 1, white). There are also cases that inflate the pathway such as one-to-many mappings (e.g. Pathway 2-4, orange mapping to pink and violet). (e)__ Showcasing the different scenarios when merging measured data (detected) based on the translated metabolites within pathways (scenario 1-5) and across pathways (scenario 6-8) highlighting problematic scenarios (4-7) that require further actions. Unproblematic scenarios (1-3 and 8) can include special cases between original and translated (i.e. one-to-many in scenario 1), which become obsolete as only one/none of the many potential metabolite IDs is detected. Yet, if multiple metabolites are detected action is required (scenario 5), which can include building the sum of the multiple detected features or only keeping the one with the highest Log2FC between two conditions. Other special cases between original and translated (i.e. many-to-one in scenario 4 and 6) also depend on what has been mapped to the measured features. If features have been measured in those scenarios, pathway deflation (i.e. only one original entry remains) or measured feature duplication (the same measurement is mapped to many features in the prior knowledge) are the possible results within and across pathways. Those scenarios should be addressed on a case-by-case basis as they also require biological information to be taken into account."

      We have also rearranged the Scenarios in Fig. 2e. We hope that together with the extended figure legend this is now clear.

      (4) (Planned)

      "By assigning other potential metabolite IDs and by translating between the present ID types, we not only increase the number of features within all ID types but also increase the feature space with HMDB and KEGG IDs (Fig. 2a, right, SFig. 2 and Supplementary Table 1)". The reviewer would appreciate additional clarification on how this was done. It is not clear what specific steps or criteria were used to assign additional metabolite IDs or to translate between identifier types. The reviewer also appreciates the inclusion of the UpSet plots. However, simply having the plots side-by-side makes it difficult to determine the specific differences. An alternative visualization, such as stacked bar plots, scatter plots summarizing the changes in feature counts, or other representation that more clearly highlights the deltas, might make these results easier to interpret.

      The main Fig. 2a shows the original (left) metabolite ID availability per detected metabolite feature in the ccRCC data and the adapted (right) metabolite IDs. The individual steps taken to extend the metabolite ID coverage of the measured features and obtain Fig 2a (right), are shown in SFig. 2 for HMDB (SFig. 2a) and KEGG (SFig. 2b). We did not include the plots for the pubchem IDs as they follow the same principle. The individual steps we are showcasing with SFig. 2 are (I) How many of the detected features (577) have a HMDB ID (341, red bar + grey bar), (II) How this distribution changed after equivalent amino-acid IDs are added, which does not change the number of features with an HMDB ID, but the number of features with a single HMDB ID, and (III) How this distribution changed after translating from the other available ID types (KEGG and PubChem) to HMDB IDs using RaMP-DBs knowledge, which leads to 430 detected features with one or multiple HMDB IDs. The exact numbers can be extracted from Supplementary Table 1, Sheet "Feature metadata", where for example N-methylglutamate had no HMDB ID assigned in the original publication (see column HMDB_Original), yet by translating HMDB from KEGG (hmdb_from_kegg) and PubChem (see column hmdb_from_pubchem) we obtain in both cases the same HMDB ID "HMDB0062660". In order to clarify this in the manuscript, we have extended the figure legend of SFig. 2: "a-b) Bargraphs showing the frequency at which a certain number of metabolite IDs per integrated peak are available as per ccRCC patients feature metadata provided in the original publication (left), after potential equivalent IDs for amino-acid and amnio-acid-related features were assigned (middle), which increases the number of features with multiple (middle: grey bars) and after IDs were translated from the other available ID types (right). for a) Of 577 detected features, 341 had at least one HMDB IDs assigned (left graph, red + grey bar) according to the original publication (left). Translating from KEGG-to-HMDB and from PubChem-to-HMDB increased the number of features with an HMDB ID from 341 to 430 (left). and __b) __Of 577 detected features, 306 had at least one KEGG IDs assigned (left graph, red + grey bar) according to the original publication (left). Translating from HMDB-to-KEGG and from PubChem-to-KEGG did not increase the total number of features with an KEGG ID (left)."

      We like the suggestion of the reviewer to provide representations of the deltas and will add additional plots to SFig. 2 as part of our planned revision.

      (5) (Planned)

      MetaboAnalyst is mentioned several times in the manuscript. The reviewer is familiar with some of the limitations and practical challenges associated with using MetaboAnalyst and its R package. Given that MetaboAnalyst already offers some overlapping functionality with MetaProViz (and offers it in the form of an interactive website and a sometimes functional R package), a more explicit comparison between the two tools would help readers fully understand the unique advantages and improvements provided by MetaProViz.

      This is a good point the reviewer raises. As part of the revisions, we plan to create a supplementary data table that includes both tools and their respective features. We will refer to this table within the manuscript text.

      (6)

      Page 11: The authors state that they used limma for statistical testing, including for the analysis of exometabolomics data, where the values appear to represent log2-transformed distances or ratios rather than normally distributed intensities. Since limma assumes approximately normal residuals, please provide evidence or justification that this assumption holds for these data types. If the distributions deviate substantially from normality, a non-parametric alternative might be more appropriate.

      For exometabolomics data we use data normalised to media blank and growth factor (formula (1)). Limma is performed on those data, not on the log2-transformed distances. The Log2(Distance) is calculated separately to the statistical results using the normalised exometabolomics data. In addition, we always perform the Shapiro-Wilk test as part of MetaProViz differential analysis function on each metabolite to understand the distribution. In this particular case we have the following distributions:

      Cell line

      Metabolites normal distribution [%]

      Metabolites not-normal distribution [%]

      HK2

      82.35

      17.65

      786-O

      95.71

      4.29

      786-M1A

      97.14

      2.86

      786-M2A

      88.57

      11.43

      OSRC2

      92.86

      7.14

      OSLM1B

      85.71

      14.29

      RFX631

      97.14

      2.86

      If a user would have distributions that deviate substantially from normality, non-parametric alternatives are also available in MetaProViz (see methods section for all options).

      7)

      Page 13: why were young and old defined this way? Authors should provide their reasoning and/or citations for this grouping.

      We thank the reviewer for pointing this out. The explanation of our choices of the age groups is purely based on the literature:

      First, ccRCC can be sporadic (>96%) or familial (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308682/pdf/nihms362390.pdf). This was also observed in other cohorts, where of 1233 patients only 93 were under 40 years of age (%, whilst 1140 (%) were older than 40 years (https://www.europeanurology.com/article/S0302-2838(06)01316-9/fulltext). Second, given the high frequency of sporadic cases it is unsurprising that ccRCC incidences were found to peak in patients aged 60 to 79 years with more male than female incidences (https://journals.lww.com/md-journal/Fulltext/2019/08020/Frequency,_incidence_and_survival_outcomes_of.49.aspx). Third, it was shown that sex impacts on the renal cancer-specific mortality and is modified by age, which is a proxy for hormonal status with premenopausal period below 42 years and postmenopausal period above 58 years (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361860/pdf/srep09160.pdf). Putting all of this information together, we decided on our age groups of young (58years) following the hormonal period in order to account for sex impact. Additionally, our young age group is representative of the age of familial ccRCC, whilst our old age group summarises the age group where incidences were found to peak.

      To make this clear in the manuscript we have extended the method section of the manuscript (Line 547-548):

      "For the patient's ccRCC data, we compared tumour versus normal of two patient subset, "young" (58years)."

      (8)

      Figure 4e: It may help with interpretation to have these Sankey-like graph edges be proportional to the number of metabolites.

      We thank the reviewer for this suggestion, which we also pondered. When we tested this visualisation, the plot became convoluted, hard to interpret and not all potential flows exist in the data. This is why we have opted to create an overview graph of each potential flow, with each edge representing a potentially existing flow. The number of times a flow exists is shown in Fig. 4f.

      (9)

      Figure 4h: The values appear to be on an intensity scale (e.g., on the order of 3e10), yet some of them are negative, which would not be expected for raw or log-transformed mass spectrometry intensities. It is unclear whether these represent normalized abundance values, distances, or some other transformation. In addition, for the comparison of tumour versus normal tissue, it is not specified what statistical test was applied. Since mass spectrometry data are typically log2-transformed to approximate a log-normal distribution before performing t-tests or similar parametric methods, clarification is needed on how these data were processed.

      Thanks for pointing this out, it made us realize that we need to extend our figure legend for clarity for Fig. 4h (Line 343-345). In both cases we show normalized intensities following the workflow described in Fig. 3a. In case of the left graph labelled "CoRe", we are plotting an exometabolomics experiment, were additionally normalised using both media blanks (samples where no cells were cultured in) and growth factor (accounts for cell growth during experiment) as growth rate (accounts for variations in cell proliferation) has not been available (see also formula (1) in methods section). A result has a negative value if the metabolite has been consumed from the media, or a positive value if the metabolite has been released from the cell into the culture media.

      In addition, the reviewer refers to the comparison of tumour versus normal (Fig. 4a __and 4d__) and the missing description of the chosen statistical test. We have added the details to the figure legend (Lines 334 and 345).

      Adapted legend Fig. 4: "a) Differential metabolite analysis results for exometabolomics data comparing 786-O versus HK2 cells using Annova and false discovery rate (FDR) for p-value adjustment. b) __Heatmap of mean consumption-release of the measured metabolites across cell lines. c) Heatmap of normalised ccRCC cell line exometabolomics data for the selected metabolites of amino acid metabolism for a sample subset. __d) __Differential metabolite analysis results for intracellular data comparing 786-O versus HK2 cells using Annova and false discovery rate (FDR) for p-value adjustment. __e) __Schematics of bioRCM process to integrate exometabolomics with intracellular metabolomics and __f) __number of metabolites by their combined change patterns in intracellular- and exometabolomics in 786-M1A versus HK2. g)__ Heatmap of the metabolite abundances in the "Both_DOWN (Released/Comsumed)" cluster. __h) __Bar graphs of normalised methionine intensity for exometabolomics (CoRe: negative value, if the metabolite has been consumed from the media, or a positive value, if the metabolite has been released from the cell into the culture media) and intracellular metabolomics (Intra)."


      (10)

      Figure 5: "Tukey's p.adj We thank the reviewer for pointing this out. We have used the TukeyHSD (Tukey's Honestly Significant Difference) test in R on the Anova results. We have added more details into the figure legend (Line 384): "(Tukey's post-doc test after anova p.adj<br /> (11)

      The potential for multi-omics is mentioned. Please clarify how generalizable this framework is. Can it readily accommodate transcriptomics, proteomics, or fluxomics data, or does it require custom logic or formatting for each new data type?

      Thanks for raising this question. MetaProViz can readily accommodate transcriptomics and proteomics data for combined enrichment analysis using for example MetalinksDB metabolite-receptor pairs. Yet, MetaProViz does not support modelling fluxomics data into metabolic networks. We state in the discussion that this could be future development ("Beyond current capabilities, future developments could also incorporate mechanistic modeling to capture metabolic fluxes, subcellular compartmentalization, enzyme kinetics, regulatory feedback loops, and thermodynamic constraints to dissect metabolic response under perturbations."). To clarify on the availability of multi-omics integration for combined enrichment analysis, we have added some more details into the discussion section.

      Line 467-469: "In addition, providing knowledge of receptor-, transporter- and enzyme-metabolite pairs, MetaProViz can readily accommodate transcriptomics and proteomics data for combined enrichment analysis."

      (12)

      Please clarify if/how enrichment analyses account for varying set sizes and redundant metabolite memberships across pathways, which can bias over-representation analysis results.

      This is a very relevant point, which we have already been working on. Indeed, we agree that enrichment results from enrichment analyses can be biased due to varying set sizes and redundant metabolite memberships across pathways. MetaProViz explicitly accounts for varying set sizes when running over representation analysis (functions standard_ora()and cluster_ora()), which uses a model that computes the p-value under a hypergeometric distribution. Thereby, larger pathways are penalized unless the overlap is proportionally large, while smaller pathways can be significant with fewer overlaps. Hence, the test quantifies whether the observed overlap between the query set and a pathway is larger than would be expected under random sampling. In addition, we explicitly filter by gene‑set size using min_gssize/max_gssize, which further controls for extreme small or large sets. So both the statistical test itself and the size filters incorporate gene‑set size variation.

      Regarding the redundant metabolite-set (i.e. pathways) memberships, we have now implemented a new function (cluster_pk()) to cluster metabolite-sets like pathways based on overlapping metabolites. Thereby we allow investigation of enrichment results in regard to redundancy and similarity. For given metabolite-sets, the function calculates pathway similarities via either overlap- or correlation-based metrics. After optional thresholding to remove weak similarities, we implemented three clustering algorithms (connected-components clustering, Louvain community detection and hierarchical clustering) to group similar pathways. We then visualize the clustering results as a network graph using the new function viz_graph based on igraph. We have added all information into our methods section "Metabolite-set clustering" (Lines 656-671). In addition, we have also added the results of the clustering into Fig. 5f.

      New Fig. 5f:"f) *Network graph of top enriched pathways (p.adjusted

      Reviewer #2

      Evidence, reproducibility and clarity

      Schmidt et al report the development of MetaProViz, an integrated R package to process, analyze and visualize metabolomics data, including integration with prior knowledge. The authors then go on to demonstrate utility by analyzing several metabolomes of cell lines, media and patient samples from kidney cancer. The manuscript provides a concise description of key challenges in metabolomics that the authors identify and address in their software. The examples are helpful and illustrative, although I should point out that I lack the expertise to evaluate the R package itself. I only have a few very minor comments.

      Significance

      This is a very significant advance from one of the leading groups in the field that is likely to enhance metabolomics data analysis in the wider community.

      We thank the reviewer for this positive feedback on our package. We appreciate that there are no major comments from the reviewer.

      Minor comments:

      (1)

      Figure 2D, E: While the schematics are fairly intuitive, a brief figure legend description of what the different scenarios etc. represent would make this easier to grasp.

      We thank the reviewer for pointing this out and we acknowledge that this is a complex problem we try to convey. We received a similar comment from Reviewer 1 (Comment 3), so please see the extensive response there. In brief, we have extended the figure legend and specifically explained each displayed case and its meaning (Line 222-242) and extended the Figure itself by adding additional categories to Fig. 2e.

      Extended legend Fig.2 d-e: "d-e) Schematics of possible mapping cases between metabolite IDs (= each circle corresponds to one ID) of a pathway-metabolite set (e.g. KEGG) to metabolites IDs of a different database (e.g. HMDB) with (d) showing many-to-many mappings that can occur within and across pathway-metabolite sets and (e) additionally showing the mapping to metabolite IDs that were assigned to the detected peaks within and across pathway-metabolite sets. (d) __Translating the metabolite IDs of a pathway-metabolite set can lead to special cases such as many-to-one mappings (Pathway 1), where for example the original resource used the ID for L-Alanine (Pathway 1, green) and D-Alanine (Pathway 1, yellow) in the amino-acid pathway, whilst the translated resources only has an entry for Alanine zwitterion (Pathway 1, blue). Additionally, many-to-one mappings can also occur across pathways (Pathway 2-4), where this mapping is only detected when mappings are analysed taking all pathways into account. Both of these cases deflate the pathways, which can also happen for one-to-none mappings (Pathway 1, white). There are also cases that inflate the pathway such as one-to-many mappings (e.g. Pathway 2-4, orange mapping to pink and violet). (e)__ Showcasing the different scenarios when merging measured data (detected) based on the translated metabolites within pathways (scenario 1-5) and across pathways (scenario 6-8) highlighting problematic scenarios (4-7) that require further actions. Unproblematic scenarios (1-3 and 8) can include special cases between original and translated (i.e. one-to-many in scenario 1), which become obsolete as only one/none of the many potential metabolite IDs is detected. Yet, if multiple metabolites are detected action is required (scenario 5), which can include building the sum of the multiple detected features or only keeping the one with the highest Log2FC between two conditions. Other special cases between original and translated (i.e. many-to-one in scenario 4 and 6) also depend on what has been mapped to the measured features. If features have been measured in those scenarios, pathway deflation (i.e. only one original entry remains) or measured feature duplication (the same measurement is mapped to many features in the prior knowledge) are the possible results within and across pathways. Those scenarios should be addressed on a case-by-case basis as they also require biological information to be taken into account."

      (2) Fig. 4: The authors briefly state that they integrate prior knowledge to identify the changes in methionine metabolism in kidney cancer, but it is not clear how exactly they contribute to this conclusion. It could be helpful to expand a bit on this to better illustrate how MetaProViz can be used to integrate prior knowledge into the analysis workflow.

      We think the reviewer refers to this section in the text (Line 363-370):

      "Next, we focused on the cluster "Both_DOWN (Released-Consumed)" and found that several amino acids are consumed by the ccRCC cell line 786-M1A but released by healthy HK2 cells. At the same time, intracellular levels are significantly lower than in HK2 (Log2FC = -0.9, p.adj = 4.4e-5) (Fig. 4g). To explore the role of these metabolites in signaling, we queried the prior knowledge resource MetalinksDB, which includes metabolite-receptor, metabolite-transporter and metabolite-enzyme relationships, for their known upstream and downstream protein interactors for the measured metabolites (Supplementary Table 5). This approach is especially valuable for exometabolomics, as it allows us to generate hypotheses about cell-cell communication. Notably, we identified links involving methionine (Fig. 4h), enzymes such as BHMT, and transporters such as SLC43A2 that were previously shown to be important in ccRCC25,42 (Supplementary Table 5)."

      We have now extended this part to clearly state that here MetalinkDB is the prior knowledge resource we used to identify the links for methionine (Line 363-364). In addition we have extended our summary statement to ensure clarity for the reader that we combine the biological clustering, which revealed the amino acid changes, with prior knowledge for the mechanistic insight (Line 380-381):

      "In summary, calculating consumption-release and combining it with intracellular metabolomics via biological regulated clustering reveals metabolites of interest. Further combining these results with prior knowledge using the MetaproViz toolkit facilitates biological interpretation of the data."

      (3)

      Given the functional diversity among metabolites -central to diverse pathways, are key signaling molecules, restricted functions, co-variation within a pathway - I wonder how informative approaches such as PCA or enrichment analyses are for identifying metabolic drivers of a (patho)physiological state. To some extent, this can be addressed by integrating prior knowledge, and it would be helpful if the authors could comment on (and if applicable explain) whether/how this is integrated into MetaProViz.

      The reviewer is correct in stating the functional diversity of metabolites, which is also why prior knowledge is needed to add mechanistic interpretation to the finding from the metadata analysis (as we showcased by focusing on the separation of age (Fig. 5c-d)). We think that approaches such as PCA or enrichment can be helpful, even if admittedly limited. For example, in the metadata analysis presented in Fig. 5b and the subsequent enrichment analysis presented in Fig. 5, we used PCA to extract the eigenvector and the loading, which act as weights indicating the contribution of each original metabolite to that specific principal components separation. Hence, the eigenvector of PCA shows the metabolite drivers of the separation. This does not necessarily mean that those metabolites are drivers of a (patho)physiological state - the (patho)physiological state can equally be the reason for those metabolites driving the separation on the Eigenvectors. Thus, the metadata analysis presented in Fig. 5b enables us to extract the metadata variables (patho)physiological states separated on a PC with the explained variance. This can also lead to co-variation, when multiple (patho)physiological states are separated on the same PC, as the reviewer correctly points out. Regarding the enrichment analysis, we provide different types of prior knowledge for classical mapping, but also the prior knowledge we used to create the biological regulated clustering, which together help to identify key metabolic groups as we can first cluster the metabolites and afterwards perform functional enrichment. Yet, this does not account for the technical issues of enrichment analysis. In this context multi-omics integration building metabolic-centric networks could further elucidate the diversity of metabolic pathways and connection to signalling and co-variation, yet this is not the scope of MetaProViz. To sum up, we are aware of the limitations of this analysis and the constraints on the downstream interpretation.

      To capture the functional diversity amongst metabolites, which leads to metabolites being present in multiple pathways of metabolite-pathways sets, we have implemented a new function to cluster metabolite-sets like pathways based on overlapping metabolites and visualize redundant metabolite-set (i.e. pathways) memberships (Fig.5f). For more details also see our response to Reviewer 1, Comment 12. We hope this will circumvent miss- and over-interpretation of the enrichment results.

      In addition, we have extended the text to include the analysis pitfalls explicitly (Line 416-419): "Another variable explaining the same amount of variance in PC1 is the tumour stage, which could point to adjacent normal tissue metabolic rewiring that happens in relation to stage and showcases that biological data harbour co-variations, which can not be disentangled by this method."

      Reviewer #3

      Evidence, reproducibility and clarity

      This manuscript introduces an R package MetaProViz for metabolomics data analysis (post anotation), aiming to solve a poor-analysis-choices problem and enable more people to do the analysis. MetaProViz not only guides people to select the best statistical method, but also enables to solve previously unsolved problems: e.g. multiple and variable metabolite names in different databases and their connections to prior knowledge. They also created exometabolomics analysis and the needed steps to visualise intra-cell / media processes. The authors demonstrated their new package via kidney cancer (clear-cell renal cell carcinoma dataset, steping one step closer to improve biological interpretability of omics data analysis.

      Significance

      This is a great tool and I can't wait to use it on many upcoming metabolomics projects! Authors tackle multiple ongoing issues within the field: from poor selection of statistical methods (they provide guidance or have default safer options) to the messiness of data annotation between databases and improving data interpretability. The field is still evolving quickly, and it's impossible to solve all problems with one package; thus some limitations within the package could be seen as a bit rigid. Nonetheless, this fully steps toward filling an existing methodological gap. All bioinformaticians doing metabolomic analysis, or those learning how to do it, will greatly benefit from this knowledge.

      I myself lead a team of 6 bioinformaticians, and we do analysis for researchers, clinicians, drug discovery, and various companies. We run internal metabolomics pipelines every day and fully sympathise with the problems addressed by the authors.

      Major comments affecting conclusions

      none.

      We thank the reviewer for this positive feedback on evidence, reproducibility and clarity as well as significance of our work given the reviewers experience with metabolomics data analysis mentioned. We appreciate that there are no major comments from the reviewer.

      Minor comments

      Minor comments, important issues that could be addressed and possibly improve the clarity or generally presentation of the tool. Please see all below.

      (1)

      1- You start with separating and talking about metabolomics and lipidomics, but lipidomics quickly dissapears (especially beyond abstract/intro) - no real need to discuss lipidomics.

      Thanks, that's a good note and we have removed it from the abstract and introduction.

      (2)

      2- You refer to the MetImp4 imputation web tool, but I cannot find an active website, manuscript, or R package for it, and the cited link does not load. This raises doubts about whether the tool is currently usable. Additionally, imputation choice should be guided by biological context and study design, not just by testing a few methods and selecting the one that performs best.

      We fully agree with the reviewer on imputation handling. The manuscript we cite from Wei et. al. (https://doi.org/10.1038/s41598-017-19120-0) compared a multitude of missing value imputation methods and made this comparison strategy available as a web-based tool not as any code-based package such as an R-package. Yet, the reviewer is right, the web-tool is no longer reachable. Hence, we have adapted the statement in our introduction (Line 61-62): "Moreover, there are tools that focus on specific steps of the pre-processing of feature intensities, which encompasses feature selection, missing value imputation (MVI)9 and data normalisation. For example, MetImp4 is a web-tool that includes and compares multiple MVI methods9. "

      (3)

      3- The authors address key metabolomics issues such as ambiguous metabolite names and isoforms, and their focus on resolving mapping ambiguities and translating between database identifiers is highly valuable. However, the larger challenge of de novo identification and the "dark matter" of unannotated metabolites remains unresolved (initiatives as MassIVE might help in the future https://massive.ucsd.edu/ProteoSAFe/ ), and readers may benefit from clearer acknowledgement that MetaProViz does not operate on raw spectral data. The introduction currently emphasizes annotation, but since MetaProViz requires already annotated metabolite tables (and then deals with all the messiness), this space might be better used to frame the interpretability and pathway-analysis challenges that the tool directly addresses.

      We appreciate the comment and have highlighted this in the abstract and introduction: "MetaProViz operates on annotated intensity values..." (Line 29 and 88).

      Given the newest advancements in metabolite identification using AI-based methods, MetaProViz toolkit with a focus on connecting metabolite IDs to prior knowledge becomes increasingly valuable. We added this to our discussion (Line 484-488): "Given the imminent shift in metabolite identification through AI-based approaches, including language model-guided48 methods and self-supervised learning49, the growing number of identified metabolites will make the MetaProViz toolkit increasingly valuable for the community to gain functional insights."

      In regards to the introduction, where we mention some tools for peak annotation: The reason why we have this paragraph where peak annotation are named is that we wanted to set the basis by (I) listing the different steps of metabolomics data analysis and (II) pointing to well-known tools of those steps. We also have a dedicated paragraph for pathway-analysis challenges.

      (4)

      4- I also really enjoyed you touching on the point of user-friendly but then inflexible and problem of reproducibility. We truly need well working packages for other bioinformaticians, rather than expecting wet-lab scientists to do all the analysis within the user interface.

      We thank the reviewer for this positive feedback.

      (5)

      5- It would be helpful to explain why the authors chose cancer/RCC samples for the demonstration. Was it because the dataset included both media and cell measurements? Does the tool perform best when multiple layers of information are available from the same experiment?

      We specifically chose the ccRCC cell line data as example since, for a multitude of cell lines, both media (exometabolomics) and intracellular metabolomics had been performed. The combination of both data types is only used in the biological regulated clustering (Fig. 5e-g), all other analyses do not require additional data modalities. We have not specifically tested how performance differs for this particular case as it would require multiple paired data (exometabolomics and intracellular metabolomics) taken at the same time and at different times.

      (6)

      6- Figure 2B: The upset plots effectively show increased overlap after adaptation, but it would be easier to compare changes if the order of the intersection bars in the "adapted" plot matched the original. For example, while total intersections increased (251→285), the PubChem+KEGG overlap decreased (24→5), likely due to reallocation to the full intersection.

      Thanks for raising this point. We initially had ordered the bars based on their intersection size, but we agree with the reviewers that for our point it makes sense to fix the order in the adapted plot to match the order of the original plot. We have done this (Fig 2a) and also extended the figure legend text of SFig. 2, which shows the individually performed adaptations summarized in Fig 2a.

      (7) (Planned)

      7- In your example of D-alanine and L-alanine - you mention how chirality is important biological feature, but up to this point it's not clear how do you do translation exactly and in which situations this would be treated just as "alanine" and when the more precise information would be retained? You mention RaMP-DB knowledge and one to X mappings as well as your general guidance in the "methods" part, but it would be useful to describe in this publication how you exactly tackled this problem in the ccRCC case.

      We thank the reviewer for this suggestion. Since this is a complex problem, we will add a more explicit description to the results section by showcasing more details on how we exactly tackled this problem in the ccRCC example data.

      In regards to D- and L-alanine, even though chirality is an important biological feature, in a standard experiment we can not distinguish if we detect the L- or D-aminoacid. This is why we try to assign all possible IDs to increase the overlap with the prior knowledge. In Fig. 2b we showcase that this can potentially lead to multiple mappings of the same measured feature to multiple pathways. For example, if we measure alanine and assign the pubchem ID for L-Alanine, D-Alanine and Alanine and try to map to metabolite-sets that include both L-Alanine and D-Alanine. In turn this could fall into Scenario 6 (Fig. 2e), where across pathways there is a D-Alanine specific one (Pathway 1) and a L-Alanine specific one (Pathway 2). Now we can decide, if we want to allow both mapping (many-to-one) or if we decide to exclude D-Alanine because we know our biological system is human and should primarily have L-Alanine.

      (8) (Planned)

      8- In one to many mappings, it would be interesting to see quantification how frequently it was happening within a pathway or across pathways. I.e. Would going into pathway analysis "solve" the issue of "lost in translation" or not really?

      We have quantified the frequency for the example of translating the KEGG metabolite-set into HMDB IDs (Fig. 2c, left panel). Yet, we are not showcasing the quantification across the KEGG metabolite-sets with this plot. During the revision we will add the full results available to the Extended Data Table 2, which currently only includes the results displayed in Fig.2c.

      (9)

      9- QC: the coefficient of variation (CV) helps identify features with high variability and thus low detection accuracy. Here it's important to acknowledge that if the feature is very variable between groups it can be extremely important, but if the feature is very variable within the group - only then one would have low trust in the accuracy.

      Yes, we totally agree with the reviewer on this. For this reason, we have applied CV only in instances where this is not leading to any condition-driven CV differences, but is truly feature-focused: (1) Function pool_estimation performs CV on the pool samples only, which are a homogeneous mixture of all samples, and hence can be used to assess feature variability. (2) Function processing performs CV on exometabolomics media samples (=blanks), which are also not impacted by different conditions.

      (10)

      10- Missing value imputation - while missing not at random is a great way to deal with missingness, it would be great to have options for others (not just MNAR), as missingness is of a complex nature. If a pretty strong decision has been made, it would be good to support this by some supplementary data (i.e. how results change while applying various combinations of missingness and why choosing MNAR seems to be the most robust).

      We have decided to only offer support for MNAR, since we would recommend MVI only if there is a biological basis for it.

      As mentioned in the response to your minor comment 2, Wei et. al. (https://doi.org/10.1038/s41598-017-19120-0) compared a multitude of missing value imputation methods. They compared six imputation methods (i.e., QRILC, Half-minimum, Zero, RF, kNN, SVD) for MNAR and systematically measured the performance of those imputation methods. They showed that QRILC and Half-Minimum produced much smaller SOR values, showing consistent good performances on data with different numbers of missing variables. This was the reason for us to only provide Half-minimum.

      (11) (Planned)

      11- In the pre-processing and imputation stages - it would be interesting to see a summary table of how many features are left after each stage.

      This is a good suggestion and refers to the steps described in Fig. 3a. We will create an overview table for this, add it into the Extended Data Table and refer to it in the results section.

      (12)

      12- Is there a reason not to do UMAP or PSL-DA graphs for outlier detection? Doing more than PCA would help to have more confidence in removing or retaining outliers in the cases where biological relevance is borderline.

      The reason we decided to use PCA was the standardly used combination with the Hotelling T2 outlier testing. Since PCA is a linear dimensionality reduction technique that preserves the overall variance in the data and has a clear mathematical foundation linked to the covariance structure, it specifically fits the required assumptions of the Hotelling T2 outlier testing. Indeed, Hotelling T2 relies on the properties of the covariance matrix and the assumption of a multivariate Gaussian distribution. UMAP is a non-linear dimensionality reduction technique, which prioritizes preserving local and global structures in a way that often results in good clustering visualization, but it distorts distances between clusters and does not have the same rigorous statistical underpinnings as PCA. In terms of PLS-DA, which focuses on maximizing the covariance between variables and the class labels, even though not commonly done, one could use the optimal latent variables for discrimination and apply Hotelling's T² to those latent variables. Yet, PLS-DA is supervised and actively tries to separate data points in the latent space, which can be misleading for outlier detection where methods like PCA that are unbiased, unsupervised and preserve global variance are advantageous.

      (13)

      13- Metadata vs metabolite features - can this be used beyond metabolomics (i.e. proteomics, transcriptomics, etc)? It can be always very useful when there are many metadata features and it's hard to pre-select beforehand which ones are the most biologically relevant.

      Yes, definitely. In fact, we have used the metadata analysis strategy also with proteomics data and it will work equally with any omics data type.

      (14)

      14- While authors discussed what KEGG pathways were significantly deregulated, it would be interesting to see all the pathways that were affected (e.g. aPEAR "bubble" graphs can show this (https://github.com/kerseviciute/aPEAR) , or something similar to NES scores). I appreciate the trickiness of it, but it would be quite interesting to see how authors e.g. Figure5e narrowed it down to the two pathways and how all the others looked like.

      We thank the reviewer for the suggestion of the aPEAR graphs. Following this suggestion, we have implemented a new function to enable clustering of the pathways based on overlapping metabolites (cluster_pk()). For more details regarding the method see also our response to Reviewer 1 (Comment 12) and our extended method section "Metabolite-set clustering" (Lines 656-671). We visualize the clustering results as a network graph, which we also included into Fig. 5f.

      The complete result of the KEGG enrichment can be found in Extended Data Table 1, Sheet 13 (Pathway enrichment analysis using KEGG on Young patient subset). The pathways are ranked by p.adjusted value and also include a score (FoldEnrichment) from the fishers exact test (similar to NES scores in GSEA). Here one can find a total of seven pathways with a p.adjusted value For Fig. 5e we narrowed down to these two pathways based on the previous findings of dysregulated dipeptides (Fig. 5d), as we searched for a potential explanation of this observation.

      (15)

      15- Could you comment on the runtime of the pipeline? In particular, do the additional translation steps and use of multiple databases substantially affect computational speed?

      Downloading and parsing databases takes significant time, especially large ones like RaMP or HMDB might take minutes on a standard laptop. Our local cache speeds up the process by eliminating the need for repeated downloads. In the future, database access will be even faster: according to our plans, all prior knowledge will be accessible in an already parsed format by our own API (omnipathdb.org). The ambiguity analysis, which is a complex data transformation pipeline, and plotting by ggplot2, another key component of MetaProViz, are the slowest parts, especially when performing analysis for the first time when no cache can be used. This means there are a few slow operations which complete in maximum a few dozens of seconds. However, the implementation and speed of these solutions doesn't fall behind what we commonly find in bioinformatics packages, and most importantly, the speed of MetaProViz doesn't pose an obstacle or difficulty regarding an efficient use of it in analysis pipelines.

      (16)

      16- I clap to the authors for automated checks if selected methods are appropriate!

      Thank you, this is something we think is important to ensure correct analysis and circumvent misinterpretation.

      (17)

      17- My suggestion would be to also look into power calculation or p-value histogram. In your example you saw some clear signal, but very frequently research studies are under-sampled and while effect can be clearly seen, there are just not enough samples to have statistically significant hits.

      We fully agree that power calculations are very important. Yet, this should ideally happen prior to the user's experiment. MetaProViz analysis starts at a later time-point and power calculations should have been done before. In regards to p-value histogram, we have implemented a similar measure, namely a density plot, which is plotted as a quality control measure within MetaProViz differential analysis function. The density plot is a smoothed version of a histogram that represents the distribution as a continuous probability density function and can be used to assess whether the p-values follow a uniform distribution.

      (18)

      18- Overall functional parts are novel and next step in helping with data interpretability, but I still found it hard to read into functionally clear insights (re to pathways / functional groupings of metabolites) - especially as you have e.g. enzyme-metabolite databases etc. I think clarity there could be improved and would help to get your message more widely across.

      Regarding the clarity to the pathway enrichment and their functional insights, we have extended the Figure legends of Fig. 4 and 5, clearly state that for the functional interpretation MetalinkDB is the prior knowledge resource we used to identify the links for methionine (Line 367-368), and we have extended our summary statement to highlight that we combine the biological clustering with prior knowledge for the mechanistic insight (Line 380-381).

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

      Learn more at Review Commons


      Referee #3

      Evidence, reproducibility and clarity

      This manuscript introduces an R package MetaProViz for metabolomics data analysis (post anotation), aiming to solve a poor-analysis-choices problem and enable more people to do the analysis. MetaProViz not only guides people to select the best statistical method, but also enables to solve previously unsolved problems: e.g. multiple and variable metabolite names in different databases and their connections to prior knowledge. They also created exometabolomics analysis and the needed steps to visualise intra-cell / media processes. The authors demonstrated their new package via kidney cancer (clear-cell renal cell carcinoma dataset, steping one step closer to improve biological interpretability of omics data analysis.

      Major comments affecting conclusions: none.

      Minor comments, important issues that could be addressed and possibly improve the clarity or generally presentation of the tool. Please see all below.

      1. You start with separating and talking about metabolomics and lipidomics, but lipidomics quickly dissapears (especially beyond abstract/intro) - no real need to discuss lipidomics.
      2. You refer to the MetImp4 imputation web tool, but I cannot find an active website, manuscript, or R package for it, and the cited link does not load. This raises doubts about whether the tool is currently usable. Additionally, imputation choice should be guided by biological context and study design, not just by testing a few methods and selecting the one that performs best.
      3. The authors address key metabolomics issues such as ambiguous metabolite names and isoforms, and their focus on resolving mapping ambiguities and translating between database identifiers is highly valuable. However, the larger challenge of de novo identification and the "dark matter" of unannotated metabolites remains unresolved (initiatives as MassIVE might help in the future https://massive.ucsd.edu/ProteoSAFe/ ), and readers may benefit from clearer acknowledgement that MetaProViz does not operate on raw spectral data. The introduction currently emphasizes annotation, but since MetaProViz requires already annotated metabolite tables (and then deals with all the messiness), this space might be better used to frame the interpretability and pathway-analysis challenges that the tool directly addresses.
      4. I also really enjoyed you touching on the point of user-friendly but then inflexible and problem of reproducibility. We truly need well working packages for other bioinformaticians, rather than expecting wet-lab scientists to do all the analysis within the user interface.
      5. It would be helpful to explain why the authors chose cancer/RCC samples for the demonstration. Was it because the dataset included both media and cell measurements? Does the tool perform best when multiple layers of information are available from the same experiment?
      6. Figure 2B: The upset plots effectively show increased overlap after adaptation, but it would be easier to compare changes if the order of the intersection bars in the "adapted" plot matched the original. For example, while total intersections increased (251→285), the PubChem+KEGG overlap decreased (24→5), likely due to reallocation to the full intersection.
      7. In your example of D-alanine and L-alanine - you mention how chirality is important biological feature, but up to this point it's not clear how do you do translation exactly and in which situations this would be treated just as "alanine" and when the more precise information would be retained? You mention RaMP-DB knowledge and one to X mappings as well as your general guidance in the "methods" part, but it would be useful to describe in this publication how you exactly tackled this problem in the ccRCC case.
      8. In one to many mappings, it would be interesting to see quantification how frequently it was happening within a pathway or across pathways. I.e. Would going into pathway analysis "solve" the issue of "lost in translation" or not really?
      9. QC: the coefficient of variation (CV) helps identify features with high variability and thus low detection accuracy. Here it's important to acknowledge that if the feature is very variable between groups it can be extremely important, but if the feature is very variable within the group - only then one would have low trust in the accuracy.
      10. Missing value imputation - while missing not at random is a great way to deal with missingness, it would be great to have options for others (not just MNAR), as missingness is of a complex nature. If a pretty strong decision has been made, it would be good to support this by some supplementary data (i.e. how results change while applying various combinations of missingness and why choosing MNAR seems to be the most robust).
      11. In the pre-processing and imputation stages - it would be interesting to see a summary table of how many features are left after each stage.
      12. Is there a reason not to do UMAP or PSL-DA graphs for outlier detection? Doing more than PCA would help to have more confidence in removing or retaining outliers in the cases where biological relevance is borderline.
      13. Metadata vs metabolite features - can this be used beyond metabolomics (i.e. proteomics, transcriptomics, etc)? It can be always very useful when there are many metadata features and it's hard to pre-select beforehand which ones are the most biologically relevant.
      14. While authors discussed what KEGG pathways were significantly deregulated, it would be interesting to see all the pathways that were affected (e.g. aPEAR "bubble" graphs can show this (https://github.com/kerseviciute/aPEAR) , or something similar to NES scores). I appreciate the trickiness of it, but it would be quite interesting to see how authors e.g. Figure5e narrowed it down to the two pathways and how all the others looked like.
      15. Could you comment on the runtime of the pipeline? In particular, do the additional translation steps and use of multiple databases substantially affect computational speed?
      16. I clap to the authors for automated checks if selected methods are appropriate!
      17. My suggestion would be to also look into power calculation or p-value histogram. In your example you saw some clear signal, but very frequently research studies are under-sampled and while effect can be clearly seen, there are just not enough samples to have statistically significant hits.
      18. Overall functional parts are novel and next step in helping with data interpretability, but I still found it hard to read into functionally clear insights (re to pathways / functional groupings of metabolites) - especially as you have e.g. enzyme-metabolite databases etc. I think clarity there could be improved and would help to get your message more widely across.

      Significance

      This is a great tool and I can't wait to use it on many upcoming metabolomics projects! Authors tackle multiple ongoing issues within the field: from poor selection of statistical methods (they provide guidance or have default safer options) to the messiness of data annotation between databases and improving data interpretability. The field is still evolving quickly, and it's impossible to solve all problems with one package; thus some limitations within the package could be seen as a bit rigid. Nonetheless, this fully steps toward filling an existing methodological gap. All bioinformaticians doing metabolomic analysis, or those learning how to do it, will greatly benefit from this knowledge.

      I myself lead a team of 6 bioinformaticians, and we do analysis for researchers, clinicians, drug discovery, and various companies. We run internal metabolomics pipelines every day and fully sympathise with the problems addressed by the authors.

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

      Learn more at Review Commons


      Referee #1

      Evidence, reproducibility and clarity

      Schmidt et al. present MetaProViz, a comprehensive and modular platform for metabolomics data analysis. The tool provides a full suite of processing capabilities spanning metabolite annotation, quality control, normalization, differential analysis, integration of prior knowledge, functional enrichment, and visualization. The authors also include example datasets, primarily from renal cancer studies, to demonstrate the functionality of the pipeline. The MetaProViz framework addresses several long-standing challenges in metabolomics data analysis, particularly issues of reproducibility, ambiguous metabolite annotation, and the integration of metabolite features with pathway knowledge. The platform is likely to be a valuable addition for the community, but the reviewer has some comments that need to be addressed prior to publication.

      The section "Improving the connection between prior knowledge and metabolomics features" could benefit from additional clarification. It is not entirely clear to the reader what specific steps were taken beyond using RaMP-DB to translate metabolite identifiers. For example, how exactly were ambiguous mappings ("different scenarios") handled in practice, and to what extent does this process "fix" or merely flag inconsistencies? A more explicit description or example of how MetaProViz resolves these cases would help readers better understand the improvements claimed.

      The introduction of MetSigDB is intriguing, but its construction and added value are not sufficiently described. It would be helpful to clarify what specific advantages MetSigDB provides over directly using existing pathway resources such as KEGG, Reactome, or WikiPathways. For example, how many features, interactions, or metabolite-set relationships are included, and in what way are these pathways improved or extended compared to those already available in public databases?

      Figure 1D/1E: The reviewer appreciates the inclusion of the visualizations illustrating the different mapping scenarios, as these effectively convey the complexity of metabolite ID translation. However, it took some time to interpret what each scenario represented. It would be helpful to include brief annotations or explanatory text directly on the figures to clarify what each scenario depicts and how it relates to the underlying issue being addressed.

      "By assigning other potential metabolite IDs and by translating between the present ID types, we not only increase the number of features within all ID types but also increase the feature space with HMDB and KEGG IDs (Fig. 2a, right, SFig. 2 and Supplementary Table 1)". The reviewer would appreciate additional clarification on how this was done. It is not clear what specific steps or criteria were used to assign additional metabolite IDs or to translate between identifier types. The reviewer also appreciates the inclusion of the UpSet plots. However, simply having the plots side-by-side makes it difficult to determine the specific differences. An alternative visualization, such as stacked bar plots, scatter plots summarizing the changes in feature counts, or other representation that more clearly highlights the deltas, might make these results easier to interpret.

      MetaboAnalyst is mentioned several times in the manuscript. The reviewer is familiar with some of the limitations and practical challenges associated with using MetaboAnalyst and its R package. Given that MetaboAnalyst already offers some overlapping functionality with MetaProViz (and offers it in the form of an interactive website and a sometimes functional R package), a more explicit comparison between the two tools would help readers fully understand the unique advantages and improvements provided by MetaProViz.

      Page 11: The authors state that they used limma for statistical testing, including for the analysis of exometabolomics data, where the values appear to represent log2-transformed distances or ratios rather than normally distributed intensities. Since limma assumes approximately normal residuals, please provide evidence or justification that this assumption holds for these data types. If the distributions deviate substantially from normality, a non-parametric alternative might be more appropriate.

      Page 13: why were young and old defined this way? Authors should provide their reasoning and/or citations for this grouping.

      Figure 4e: It may help with interpretation to have these Sankey-like graph edges be proportional to the number of metabolites.

      Figure 4h: The values appear to be on an intensity scale (e.g., on the order of 3e10), yet some of them are negative, which would not be expected for raw or log-transformed mass spectrometry intensities. It is unclear whether these represent normalized abundance values, distances, or some other transformation. In addition, for the comparison of tumour versus normal tissue, it is not specified what statistical test was applied. Since mass spectrometry data are typically log2-transformed to approximate a log-normal distribution before performing t-tests or similar parametric methods, clarification is needed on how these data were processed.

      Figure 5: "Tukey's p.adj < 0.05" . Was this a Tukey's post-hoc test? This should be explicitly stated.

      The potential for multi-omics is mentioned. Please clarify how generalizable this framework is. Can it readily accommodate transcriptomics, proteomics, or fluxomics data, or does it require custom logic or formatting for each new data type?

      Please clarify if/how enrichment analyses account for varying set sizes and redundant metabolite memberships across pathways, which can bias over-representation analysis results.

      Significance

      The MetaProViz framework addresses several long-standing challenges in metabolomics data analysis, particularly issues of reproducibility, ambiguous metabolite annotation, and the integration of metabolite features with pathway knowledge. The platform is likely to be a valuable addition for the community, but the reviewer has some comments that need to be addressed prior to publication.

      Authors should be commended for the availability of data/code and detailed methods. Clarity is good. Authors have clearly spent a lot of time thinking about the challenges of metabolomics data analysis.

    1. Reviewer #1 (Public review):

      Summary:

      In this study, the authors investigated the detailed structural mechanism of activation of ABHD5 upon interaction with lipid structures (bilayer and LD). The authors used an elaborate multiscale computational workflow, incorporating coarse-grained, all-atom, and enhanced-sampling molecular dynamics simulations, to propose a structural mechanism for the interaction and activation of ABHD5, as well as its specific interaction with TAG in LD. The authors then corroborated these observations with experimental studies involving hydrogen-deuterium exchange coupled with mass spectrometry of wild-type ABHD5 to assess the structural and conformational changes in ABHD5 upon binding, as well as mutagenesis with cell-based and in vitro assays monitoring membrane association, defining specific interactions that infer ABHD5 to localize LD.

      Strengths:

      The manuscript is well-written, and the data are reported in high-quality figures. The experimental design and data analysis are rigorous and support the conclusion. One major strength is the multiscale computational work that reveals a mechanism for the insertion of ABHD5 into lipid bilayers and LD involving the insertion of the N-term portion and the lid helix motif. The design of the computational workflow was very elaborate, and the undertaking was quite extensive, with multiple strategies to (GC, all-atom MD and GaMD). The authors then elegantly generate a hypothesis from these observations to experimentally corroborate the proposed mechanism. Particularly, the HDX-MS data support the engagement of the two regions upon binding, and the fluorescence microscopy data show the role of specific residues in localization/specificity to LD.

      Weaknesses:

      The following limitation is noted. Central to this manuscript is the model, as observed computationally, that initial lipid interaction by the N-term insertion is followed by the insertion of lid-helix in the membrane, which undergoes a conformational switch in the process. However, HDX-MS reveals that, in the unbound form, the lid helix region displays a bimodal isotopic envelope, revealing two species, one with low uptake, suggesting a structured species and one with high uptake, suggesting a less structured species. It is unclear from the manuscript whether the authors think the bimodality fits EX1 regime kinetics or not. Regardless, the model of unbound ABHD5 shows a lid-helix region devoid of secondary structure (Figure 5A), which is more consistent with the unprotected species. The authors also mention that previous modeling had pointed to the high flexibility of the insertion domain. Upon binding, the lid-helix region seems to be ordered from computational observations and loses bimodality by HDX-MS with a deuterium uptake consistent with the protected species of the bimodal envelop in the unbound form. The authors fall short of interpreting or even discussing what the bimodality of the lid-helix represents in the unbound form. What does the protected species in the bimodal envelope represent? Is it a transition representing lid-helix formation and unfolding? Does it imply that interaction and insertion into the lipid structures are governed by conformational selection? This issue should be at the very least acknowledged and discussed, or optimally investigated by performing more integrative studies of the HDX-MS data with the extensive computational data at hand, using existing protection factor calculations or HDX-guided ensemble refinement methods.

    1. Analyse Transversale du « Moi » : Identité, Altérité et Nature du Sujet

      Résumé Exécutif

      La présente note de synthèse explore la déconstruction et la reconstruction de la notion de « Moi » à travers le prisme de la philosophie des sciences et de l’évolution.

      La thèse centrale postule que le sujet pensant n'est pas une entité ponctuelle et isolée, mais un « volume pronominal » complexe à plusieurs variables.

      Cette analyse démontre que la frontière entre le sujet (ce qui pense) et l’objet (ce qui est pensé) est poreuse : la matière elle-même (cristaux, fleuves, gènes) possède des fonctions pré-cognitives telles que la mémoire, le choix et l’écriture.

      En définitive, l'identité humaine se définit par une double tension entre le « Nemo » (personne, la carte blanche de l'identité pure) et le « Panonyme » (le mélange de toutes les appartenances, cultures et objets du monde).

      --------------------------------------------------------------------------------

      I. Les Trois Dimensions du Sujet : Logique, Sociale et Biologique

      L'étude du « Moi » nécessite de distinguer l'identité de l'appartenance pour éviter des confusions éthiques et sociopolitiques majeures.

      Le Sujet Logique (« Je ») : Défini par le principe d'identité (A = A). C'est l'individu en première personne.

      Le Sujet Social (« Nous ») : Il représente les appartenances (culturelles, géographiques, professionnelles). Confondre l'identité avec l'appartenance est le moteur du racisme, où l'on réduit l'individu à son groupe.

      Le Sujet Biologique (« On ») : Il désigne l'espèce humaine en général. Ce sujet est le produit d'une « épaisseur temporelle » de millions d'années d'évolution, inscrite dans nos gènes et nos organes.

      --------------------------------------------------------------------------------

      II. Déconstruction du Cogito : La Pensée comme Gestion du Chaos

      L'analyse étymologique du verbe latin cogitare (penser) révèle une réalité ignorée par la philosophie classique.

      L'origine : Co-agitare : Le terme provient de l'action du berger conduisant plusieurs troupeaux ensemble (ago : conduire ; agitare : s'agiter ; co-agitare : mener ensemble des éléments divergents).

      La pensée comme multiplicité : Penser ne signifie pas viser une unité ponctuelle, mais gérer un paysage différencié et chaotique.

      C'est l'effort de maintenir ensemble des éléments d'âges, de sexes et de tempéraments différents (le « troupeau » des pensées).

      Le paysage intérieur : Une fois la « boîte noire » du cogito ouverte, il n'en sort pas un point unique, mais un tableau somptueux, multiplié en couleurs, sons et rumeurs.

      --------------------------------------------------------------------------------

      III. Le Sujet comme Volume Pronominal

      Le « Moi » qui s'exprime est en réalité un mélange de multiples instances qui influencent et infléchissent la pensée.

      1. Les dépendances du sujet

      Le sujet n'est jamais totalement original ; il est traversé par :

      Le Langage : La langue maternelle impose une « obliquité » singulière sur le monde, dictant des angles d'attaque spécifiques pour réfléchir les choses.

      L'Ethnologie et la Culture : La provenance sociale, la religion et l'histoire personnelle formatent la pensée.

      La Communauté : Les normes scientifiques et universitaires imposent des contrôles stricts et des formats de raisonnement.

      2. La fonction à variables multiples

      Le sujet est un volume où se déplacent des positions floues entre :

      Je / Tu / Nous / Vous : Les interactions sociales et collectives.

      Cela / Il : Le corps biologique (organes, neurones, synapses) qui fonctionne indépendamment de la volonté consciente.

      --------------------------------------------------------------------------------

      IV. L'Intelligence de l'Objet : Vers un Réalisme Dur

      L'une des thèses les plus radicales avancées est que les objets possèdent des fonctions que nous croyions exclusives à l'entendement humain.

      | Fonction Cognitive | Manifestation dans l'Objet (Exemples du texte) | | --- | --- | | Écriture | Le vent trace des lignes sur la mer ; le fleuve creuse son lit ; le diamant raye la vitre. | | Mémoire | Les strates géologiques ; les isotopes du plomb (mémoire du temps) ; l'ADN. | | Choix / Décision | Les réactions chimiques sélectives ; les cristaux qui redressent des flux ; l'huile qui refuse de se mélanger à l'eau. | | Savoir | Le gnomon (axe du cadran solaire) qui « connaît » de lui-même la latitude du lieu. |

      Critique de l'Idéalisme

      L'analyse rejette l'idéalisme (qui réduit les choses à nos représentations) au profit d'un réalisme dur.

      Ce réalisme postule que les fonctions cognitives (mémoire, codage) précèdent l'humanité de millions d'années. Les objets sont des « codes » et non de simples « causes » mécaniques.

      --------------------------------------------------------------------------------

      V. L'Interdépendance Globale et le Nouveau Sujet

      L'ère contemporaine voit l'émergence d'un alliage inédit entre l'homme et la nature, notamment à travers les questions climatiques.

      Inversion de la dépendance : Autrefois, le climat était un destin (le hasard). Aujourd'hui, nos techniques globalisantes influent sur le climat, et nous commençons à dépendre de choses qui dépendent de nous.

      Émergence du « Nous pleuvons » : Par nos actions responsables, le « Il pleut » (impersonnel) tend à devenir un « Nous pleuvons », marquant l'intégration de la nature dans la fonction sujet.

      --------------------------------------------------------------------------------

      VI. Conclusion : Les Deux États du Moi

      En fin d'analyse, l'identité humaine se stabilise autour de deux états fondamentaux, comparables à l'état de la mer :

      1. L'état chaotique (Le Panonyme) : C'est le volume plein, multicolore, saturé par toutes nos appartenances, nos mémoires, nos rencontres et les objets que nous côtoyons.

      Nous portons tous les noms du monde.

      2. L'état transparent (Nemo/Personne) : C'est la carte blanche, la vacuité intégrale, l'identité pure et sans patronyme.

      C'est l'universalité vide du sujet qui n'est « personne » pour pouvoir être n'importe qui.

      L'individu est cette « voix frémissante » qui oscille perpétuellement entre la plénitude maximale du monde et la transparence absolue du sujet pensant.

    1. Reviewer #3 (Public review):

      Reinwald et al. present the NoSeMaze, a semi-natural behavioral system designed to track social behaviors alongside reinforcement-learning in large groups of mice. Accumulating more than 4,000 days of behavioral monitoring, the authors demonstrate that social rank (determined by tube competitions) is a stable trait across shuffled cohorts and correlated with active chasing behaviors. The system also provides a solid platform for long-term measurements of reinforcement learning, including flexibility, response adaptation, and impulsiveness. Yet, the authors show that social ranking and chasing are mostly independent of these cognitive traits, and both seem mostly independent of oxytocin signaling in the AON.

      Strengths:

      (1) The neuroethological approach for automated tracking of several mice under semi-natural conditions is still rare in social behavioral research and should be encouraged.

      (2) The assessment of dominance by two independent measures, i.e., spontaneous tube competitions and proactive chasing, is innovative and valuable.

      (3) The integration of a long-term reinforcement-learning module into the semi-natural system provides novel opportunities to combine cognitive traits into social personality assessments.

      (4) The open-source system provides a valuable resource for the scientific community.

      Limitations:

      (1) Apparent ambiguity and inconsistency in age structure and cohort participation across rounds, raising concerns about uncontrolled confounds.

      (2) Chasing behavior appears more stable than tube-test competitions (Figure 4D vs. Figure 3D), which challenges the authors' decision to treat tube competitions as the primary basis for hierarchy determination.

      Major concerns:

      (1) Unclear and inconsistent handling of age groups and repeated sampling. The manuscript repeatedly refers to "younger" and "older" adults, but it is unclear whether age was ever controlled for or included in models. Some mice completed only one round, others 2-5 rounds, without explanation of the criteria or balancing.

      (2) Stability of chasing appears stronger than the stability of tube competitions. Figure 4D shows highly consistent chasing behavior across weeks, while Figure 3D shows weaker and more variable correlations for tube-based David scores. This is also evident from Figure 5A-B,D. Thus, it appears that chasing, which serves to quantify dominance in similar semi-natural setups, may be a more reliable and behaviorally meaningful measure of dominance than the incidental tube competitions.

      (3) Unbalanced participation across rounds compromises stability analyses. Stability analyses (e.g., ICCs, round-to-round correlations) assume comparable sampling across individuals. However, some mice contribute 1 round, others 2, 3, 4, and even 5 rounds. This imbalance may inflate stability estimates or confound group reshuffling effects, and the rationale for variable participation is not explained.

    1. Reviewer #1 (Public review):

      Summary:

      This manuscript uses serological data to quantify the effects of imprinting on subsequent influenza antibody responses. While this is an admirable goal, the HI dataset sounds impressive, and the authors developed a number of models, the manuscript came off as very dense and technical. One of the biggest pitfalls is that it is not easy to understand the lessons learned. The two Results section headers make clear statements - there was an imprinting signal in the HI titers, but much of this signal could also be seen in an imprinting-free simulation - and then the Discussion states a number of limitations. This is fine, but it leaves the reader wondering exactly how large an error would be introduced by ignoring imprinting effects altogether; alternatively, if imprinting is purposefully added, what would the expected effect size be? The comments below will provide some concrete steps to help clarify these points.

      Major comments:

      (1) Lines 107-133: The first Results section is a dense slog of information, and the reader is never given a good overview of what the imprinting coefficients exactly are. As the paper currently stands, if you do not start by reading the Methods, you will take away very little. I suggest adding a schematic for any of your models, showing what HI titers would be expected with/without imprinting effects. or age effects, or both, to tie in your modeling coefficients with quantities that all readers are familiar with.

      (1.1) Clarify what the imprinting coefficient (y-axis in Figure 1A) looks like in this schematic.

      (1.2) Another aspect that I missed: In addition to stating which models were best by BIC, what is the absolute effect size in the HI titers? During my initial reading, I had hoped that Figure 3 would answer this question, but it turned out to be just an overview of the dataset. I strongly suggest having such a figure to show the imprinting effect inferred by different models. What would the expected effect be if you kept someone's birth year constant but tuned their age? What if you kept their age at collection constant but tuned their birth year?

      (1.3) It would also help to explain in your schematic what the x-axis labels (H1, H2, H1/H3) would look like in these scenarios, and what imprinting relative to H3 means.

      (2) As mentioned above, it was hard to understand the takeaway messages, such as:

      (2.1) A similar question would be: If you model antibody titers without imprinting, how far off would you be from the actual measurements (2x off, 4x off...)? If you add the imprinting effect, how much closer do you get?

      (2.2) Are there specific age groups that require imprinting to be taken into consideration, since otherwise HI analyses will be markedly off?

      (2.3) Are there age groups where imprinting can be safely ignored?

      (3) HI titers against multiple H1 and H3 variants were measured, but it is unclear how these are used, and why titers against a single variant each season would not have worked equally well.

    1. Author response:

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

      Reviewer #1 (Public review):

      Thank you so much for your comprehensive and insightful assessment of our manuscript. We appreciate your recognition of the novelty of our experimental design and the utility of our computational framework for interpreting visual remapping across the lifespan and in clinical populations. We are very grateful for your suggestions regarding the narrative flow, which have helped us to improve the manuscript's focus and coherence. Our responses to your specific concerns are detailed below.

      (1) Relevance of the figure-copy results (pp. 13-15). Is it necessary to include the figure-copy task results within the main text? The manuscript already presents a clear and coherent narrative without this section. The figure-copy task represents a substantial shift from the LOCUS paradigm to an entirely different task that does not measure the same construct. Moreover, the ROCF findings are not fully consistent with the LOCUS results, which introduces confusion and weakens the manuscript's coherence. While I understand the authors' intention to assess the ecological validity of their model, this section does not effectively strengthen the manuscript and may be better removed or placed in the Supplementary Materials.

      We thank the reviewer  for their perspective regarding the narrative flow and the transition between the LOCUS paradigm and the ROCF results. However, we remain keen to retain these findings in the main text, as they provide critical ecological and clinical validation for the computational mechanisms identified in our study.

      We think these results strengthen the manuscript for the following main reasons:

      (1) The ROCF we used is a standard neuropsychological tool for identifying constructional apraxia. Our results bridge the gap between basic cognitive neuroscience and clinical application by demonstrating that specific remapping parameters—rather than general memory precision—predict real-world deficits in patients.

      (2) The finding that our winning model explains approximately 62% of the variance in ROCF copy scores across all diagnostic groups further indicates that these parameters from the LOCUS task represent core computational phenotypes that underpin complex, real-life visuospatial construction (copying drawings).

      (3) Previous research has often observed only a weak or indirect link between drawing ability and traditional working memory measures, such as digit span (Senese et al., 2020). This was previously attributed to “deictic” strategies—like frequent eye and hand movements—that minimise the need to hold large amounts of information in memory (Ballard et al., 1995; Cohen, 2005; Draschkow et al., 2021). While our study was not exclusively designed to catalogue all cognitive contributions to drawing, the findings provide significant and novel evidence indicating that transsaccadic integration is a critical driver of constructional (copying drawing) ability. By demonstrating this link, the results provide evidence to stimulate a new direction for future research, shifting the focus from general memory capacity toward the precision of spatial updating across eye movements.

      In summary, by including the ROCF results in the main text, we provide evidence for a functional role for spatial remapping that extends beyond perceptual stability into the domain of complex visuomotor control. We have expanded on these points throughout the revised manuscript:

      In the Introduction: p.2:

      “The clinical relevance of these spatial mechanisms is underscored by significant disruptions to visuospatial processing and constructional apraxia—a deficit in copying and drawing figures—observed in neurodegenerative conditions such as Alzheimer's disease (AD) and Parkinson's disease (PD).[20,21] This raises a crucial question: do clinical impairments in complex visuomotor tasks stem from specific failures in transsaccadic remapping? If so, the computational parameters that define normal spatial updating should also provide a mechanistic account of these clinical deficits, differentiating them from general age-related decline.”

      p.3: "Finally, by linking these mechanistic parameters to a standard clinical measure of constructional ability (the Rey-Osterrieth Complex Figure task), we demonstrate that transsaccadic updating represents a core computational phenotype underpinning real-world visuospatial construction in both health and neurodegeneration.

      In the Results:

      “To assess whether the mechanistic parameters derived from the LOCUS task represent core phenotypes of real-world visuospatial abilities, we also instructed all participants to complete the Rey-Osterrieth Complex Figure copy task (ROCF; Figure 7A) on an Android tablet using a digital pen (see examples in Figure 7B; all Copy data are available in the open dataset: https://osf.io/95ecp/). The ROCF is a gold-standard neuropsychological tool for identifying constructional apraxia.[29] Historically, drawing performance has shown only weak or indirect correlations with traditional working memory measures.[30] This disconnect has been attributed to active visual-sampling strategies—frequent eye movements that treat the environment as an external memory buffer, minimising the necessity of holding large volumes of information in internal working memory.[3–5]

      We hypothesised that drawing accuracy is primarily constrained by the precision of spatial updating across frequent saccades rather than raw memory capacity. To evaluate the ecological validity of the identified saccade-updating mechanism, we modelled individual ROCF copy scores across all four groups using the estimated (maximum a posteriori) parameters from the winning “Dual (Saccade) + Interference” model (Model 7; Figure 8) as regressors in a Bayesian linear model. Prior to inclusion, each regressor was normalised by dividing by the square root of its variance.

      This model successfully explained 61.99% of the variance in ROCF copy scores, indicating that these computational parameters are strong predictors of real-word constructional ability (Figure 8A). … This highlights the critical role of accurate remapping based on saccadic information; even if the core saccadic update mechanism is preserved across groups (as shown in previous analyses), the precision of this updating process is crucial for complex visuospatial tasks. Moreover, worse ROCF copy performance is associated particularly with higher initial angular encoding error. This indicates that imprecision in the initial registration of angular spatial information contributes to difficulties in accurately reproducing complex visual stimuli.”

      In the Discussion:

      “Importantly, our computational framework establishes a direct mechanistic link between trassaccadic updating and real-world constructional ability. Specifically, higher saccade and angular encoding errors contribute to poorer ROCF copy scores. By mapping these mechanistic estimates onto clinical scores, we found that the parameters derived from our winning model explain approximately 62% of the variance in constructional performance across groups. These findings suggest that the computational parameters identified in the LOCUS task represent core phenotypes of visuospatial ability, providing a mechanistic bridge between basic cognitive theory and clinical presentation.

      This relationship provides novel insights into the cognitive processes underlying drawing, specifically highlighting the role of transsaccadic working memoty.ry. Previous research has primarily focused on the roles of fine motor control and eye-hand coordination in this skill.[4,50–55] This is partly because of consistent failure to find a strong relation between traditional memory measures and copying abili [4,31] For instance, common measures of working memory, such as digit span and Corsi block tasks, do not directly predict ROCF copying performance.[31,56] Furthermore, in patients with constructional apraxia, these memory performance measures often remain relatively preserved despite significant drawing impairments.[56–58] In the literature, this lack of association has often been attributed to “deictic” visual-sampling strategies, characterised by frequent eye movements that treat the environment as an external memory buffer, thereby minimising the need to maintain a detailed internal representation.[4,59] In a real-world copying task, the ROCF requires a high volume of saccades, making it uniquely sensitive to the precision of the dynamic remapping signals identified here. Recent eye-tracking evidence confirms that patients with AD exhibit significantly more saccades and longer fixations during figure copying compared to controls, potentially as a compensatory response to trassaccadic working memory constraints.[56] This high-frequency sampling—averaging between 150 and 260 saccades for AD patients compared to approximately 100 for healthy controls—renders the task highly dependent on the precision of dynamic remapping signals.[56] To ensure this relationship was not driven by a general "g-factor" or non-spatial memory impairment, we further investigated the role of broader cognitive performance using the ACE-III Memory subscale. We found that the relationship between transsaccadic working memory and ROCF performance remains highly significant, even after controlling for age, education, and ACE-III Memory subscore. This suggests that transsaccadic updating may represent a discrete computational phenotype required for visuomotor control, rather than a non-specific proxy for global cognitive decline.

      In other words, even when visual information is readily available in the world, the act of copying depends critically on working memory across saccades. This reveals a fundamental computational trade-off: while active sampling strategies (characterised with frequent eye-hand movements) effectively reduce the load on capacity-limited working memory, they simultaneously increase the demand for precise spatial updating across eye movements. By treating the external world as an "outside" memory buffer, the brain minimises the volume of information it must hold internally, but it becomes entirely dependent on the reliability with which that information is remapped after each eye movement. This perspective aligns with, rather contradicts, the traditional view of active sampling, which posits that individuals adapt their gaze and memory strategies based on specific task demands.[3,60] Furthermore, this perspective provides a mechanistic framework for understanding constructional apraxia; in these clinical populations, the impairment may not lie in a reduced memory "span," but rather in the cumulative noise introduced by the constant spatial remapping required during the copying process.[58,61]

      Beyond constructional ability, these findings suggest that the primary evolutionary utility of high-resolution spatial remapping lies in the service of action rather than perception. While spatial remapping is often invoked to explain perceptual stability,[11–13,15] the necessity of high-resolution transsaccadic memory for basic visual perception is debated.[13,62–64] A prevailing view suggests that detailed internal models are unnecessary for perception, given the continuous availability of visual information in the external world.[13,44] Our findings support an alternative perspective, aligning with the proposal that high-resolution transsaccadic memory primarily serves action rather than perception.[13] This is consistent with the need for precise localisation in eye-hand coordination tasks such as pointing or grasping.[65] Even when unaware of intrasaccadic target displacements, individuals rapidly adjust their reaching movements, suggesting direct access of the motor system to remapping signals.66 Further support comes from evidence that pointing to remembered locations is biased by changes in eye position,[67] and that remapping neurons reside within the dorsal “action” visual pathway, rather than the ventral “perception” visual pathway.[13,68,69] By demonstrating a strong link between transsaccadic working memory and drawing (a complex fine motor skill), our findings suggest that precise visual working memory across eye movements plays an important role in complex fine motor control.”

      (2) Model fitting across age groups (p. 9).

      It is unclear whether it is appropriate to fit healthy young and healthy elderly participants' data to the same model simultaneously. If the goal of the model fitting is to account for behavioral performance across all conditions, combining these groups may be problematic, as the groups differ significantly in overall performance despite showing similar remapping costs. This suggests that model performance might differ meaningfully between age groups. For example, in Figure 4A, participants 22-42 (presumably the elderly group) show the best fit for the Dual (Saccade) model, implying that the Interference component may contribute less to explaining elderly performance.

      Furthermore, although the most complex model emerges as the best-fitting model, the manuscript should explain how model complexity is penalized or balanced in the model comparison procedure. Additionally, are Fixation Decay and Saccade Update necessarily alternative mechanisms? Could both contribute simultaneously to spatial memory representation? A model that includes both mechanisms-e.g., Dual (Fixation) + Dual (Saccade) + Interference-could be tested to determine whether it outperforms Model 7 to rule out the sole contribution of complexity.

      We thank you for the opportunity to expand upon and clarify our modelling approach. Our decision to use a common generative model for both young and older adults was grounded in the empirical finding that there was no significant interaction between age group and saccade condition for either location or colour memory. While older adults demonstrated lower baseline precision, the specific "saccade cost" remained remarkably consistent across cohorts. This was the justification we proceeded on to use of a common model to assess quantitative differences in parameter estimates while maintaining a consistent mechanistic framework for comparison.

      Moreover, our winning model nests simpler models as special cases, providing the flexibility to naturally accommodate groups where certain components—such as interference—might play a reduced role. This ultimately confirms that the mechanisms for age-related memory deficits in this task reflect more general decline rather than a qualitative failure of the saccadic remapping process.

      This approach is further supported by the properties of the Bayesian model selection (BMS) procedure we used, which inherently penalises the inclusion of unnecessary parameters. Unlike maximum likelihood methods, BMS compares marginal likelihoods, representing the evidence for a model integrated over its entire parameter space. This follows the principle of Bayesian Occam’s Razor, where a model is only favoured if the improvement in fit justifies the additional parameter space; redundant parameters instead "dilute" the probability mass and lower the model evidence.

      Consequently, we contend that a hybrid model combining fixation and saccade mechanisms is unnecessary, as we have already adjudicated between alternative mechanisms of equal complexity. Specifically, Model 6 (Dual Fixation + Interference) and Model 7 (Dual Saccade + Interference) possess an identical number of parameters. The fact that Model 7 emerged as the clear winner—providing substantial evidence against Model 6 with a Bayes Factor of 6.11—demonstrates that our model selection is driven by the specific mechanistic account of the data rather than a simple preference for complexity.

      We have revised the Results and Discussion sections of the manuscript to state these points more explicitly for readers and have included references to established literature regarding the robustness of marginal likelihoods in guarding against overfitting.

      In the Results,

      “By fitting these models to the trial-by-trial response data from all healthy participants (N=42), we adjudicated between competing mechanisms to determine which best explained participant performance (Figure 4). We used random-effects Bayesian model selection to identify the most plausible generative model. This process relies on the marginal likelihood (model evidence), which inherently balances model fit against complexity—a principle often referred to as Occam’s razor.[25–27] The analysis yielded a strong result: the “Dual (Saccade) + Interference” model (Model 7 in Table 1) emerged as the winning model, providing substantial evidence against the next best alternative with a Bayes Factor of 6.11.”

      In the Discussion:

      “Our framework employs Variational Laplace, a method used to recover computational phenotypes in clinical populations like those with substance use disorders,[34,35] and the models we fit using this procedure feature time-dependent parameterisation of variance—conceptually similar to the widely-used Hierarchical Gaussian Filter.[36–39] Importantly, the risk of overfitting is mitigated by the Bayesian Model Selection framework; by utilising the marginal likelihood for model comparison, the procedure inherently penalises excessive model complexity and promotes generalisability.[25–27,40] This generalisability was further evidenced by the model's ability to predict performance on the independent ROCF task, confirming that these parameters represent robust mechanistic phenotypes rather than idiosyncratic fits to the initial dataset.”

      Minor point: On p. 9, line 336, Figure 4A does not appear to include the red dashed vertical line that is mentioned as separating the age groups.

      Thank you for pointing out this inconsistency. We apologise for the oversight; upon further review, we concluded that the red dashed vertical line was unnecessary for the clear presentation of the data. We have therefore removed the line from Figure 4A and deleted the corresponding sentence in the figure caption.

      (3) Clarification of conceptual terminology.

      Some conceptual distinctions are unclear. For example, the relationship between "retinal memory" and "transsaccadic memory," as well as between "allocentric map" and "retinotopic representation," is not fully explained. Are these constructs related or distinct? Additionally, the manuscript uses terms such as "allocentric map," "retinotopic representation," and "reference frame" interchangeably, which creates ambiguity. It would be helpful for the authors to clarify the relationships among these terms and apply them consistently.

      Thank you for pointing this out. We have revised the manuscript to ensure that these terms are applied with greater precision and consistency. Our revisions standardise the terminology based on the following distinctions:

      Reference frames: We distinguish between the eye-centred reference frame (coordinate systems that shift with gaze) and the world-centred reference frame (coordinate systems anchored to the environment).

      Retinotopic representation vs. allocentric map: We clarify that retinotopic representations are encoded within an eye-centred reference frame and are updated with every ocular movement. Conversely, the allocentric map is anchored to stable environmental features, remaining invariant to the observer’s gaze direction or position.

      Retinotopic memory vs. transsaccadic memory: We have removed the term "retinal memory" to avoid ambiguity. We now consistently use retinotopic memory to describe the persistence of visual information in eye-centred coordinates within a single fixation. In contrast, transsaccadic memory refers to the higher-level integration of visual information across saccades, which involves the active updating or remapping of representations to maintain stability.

      To incorporate these clarifications, we have implemented the following changes:

      In the Introduction, the second paragraph has been entirely rewritten to establish these definitions at the outset, providing a clearer theoretical framework for the study.

      “Central to this enquiry is the nature of the coordinate system used for the brain's internal spatial representation. Does the brain maintain a single, world-centred (allocentric) map, or does it rely on a dynamic, eye-centred (retinotopic) representation?[11,13,15,16] In the latter system, retinotopic memory preserves spatial information within a fixation, whereas transsaccadic memory describes the active process of updating these representations across eye movements to achieve spatiotopic stability—the perception of a stable world despite eye movements.[11,16–18] If spatial stability is indeed reconstructed through such remapping, the mechanism remains unresolved: do we retain memories of absolute fixation locations, or do we reconstruct these positions from noisy memories of the intervening saccade vectors? We can test these hypotheses by analysing when and where memory errors occur. Assuming that memory precision declines over time,[19] the resulting error distributions should reveal the specific variables that are represented and updated across each saccade.”

      In the Results, the opening section of the Results has been reorganised to align with this terminology. We have ensured that the hypotheses and behavioural data—specifically the definition of "saccade cost"—are introduced using this consistent conceptual vocabulary to improve the overall coherence of the narrative.

      (4) Rationale for the selective disruption hypothesis (p. 4, lines 153-154). The authors hypothesize that "saccades would selectively disrupt location memory while leaving colour memory intact." Providing theoretical or empirical justification for this prediction would strengthen the argument.

      We have revised the Results to state the hypothesis more explicitly and expanded the Discussion to provide a robust theoretical and empirical rationale:

      In the Results,

      “This design allowed us to isolate and quantify the unique impact of saccades on spatial memory, enabling us to test competing hypotheses regarding spatial representation. If spatial memory were solely underpinned by an allocentric mechanism, precision should remain comparable across all conditions as the representation would be world-centred and unaffected by eye movements. Thus, performance in the no-saccade condition should be comparable to the two-saccade condition. Conversely, if spatial memory relies on a retinotopic representation requiring active updating across eye movements, the two-saccade condition was anticipated to be the most challenging due to cumulative decay in the memory traces used for stimulus reconstruction after each saccade.[22] Critically, we hypothesised that this saccade cost would be specific to the spatial domain; while location requires active remapping via noisy oculomotor signals, non-spatial features like colour are not inherently tied to coordinate transformations and should therefore remain stable (see more in Discussion below).

      Meanwhile, the no-saccade condition was expected to yield the most accurate localisation, relying solely on retinotopic information (retinotopic working memory). These predictions were confirmed in young healthy adults (N = 21, mean age = 24.1 years, ranged between 19 and 34). A repeated measures ANOVA revealed a significant main effect of saccades on location memory (F(2.2,43.9)=33.2, p<0.001, partial η²=0.62), indicating substantial impairment after eye movements (Figure 2A). In contrast, colour memory remained remarkably stable across all saccade conditions (Figure 2B; F(2.2, 44.7) = 0.68, p=0.53, partial η² =0.03).

      This “saccade cost”—the loss of memory precision following an eye movement—indicates that spatial representations require active updating across saccades rather than being maintained in a static, world-centred reference frame.

      Critically, our comparison between spatial and colour memory does not rely on the absolute magnitude of errors, which are measured in different units (degrees of visual angle vs. radians). Instead, we assessed the relative impact of the same saccadic demand on each feature within the same trial. While location recall showed a robust saccade cost, colour recall remained statistically unchanged. To ensure this null effect was not due to a lack of measurement sensitivity, we examined the recency effect; recall performance for the second item was predicted to be better than for the first stimulus in each condition.[23,24] As expected, colour memory for Item 2 was significantly more accurate than for Item 1 (F(1,20) = 6.52, p = 0.02, partial η² = 0.25), demonstrating that the task was sufficiently sensitive to detect standard working memory fluctuations despite the absence of a saccade-induced deficit.”

      In the Discussion, we now write that on p.18:

      “A clear finding was the specificity of the saccade cost to spatial features; it was not observed for non-spatial features like colour, even in neurodegenerative conditions. This discrepancy challenges notions of fixed visual working memory capacity unaffected by saccades.16,44–46 The differential impact on spatial versus non-spatial features in transsaccadic memory aligns with the established "what" and "where" pathways in visual processing.32,33 For objects to remain unified, object features must be bound to stable representations of location across saccades.19 One possibility is that remapping updates both features and location through a shared mechanism, predicting equal saccadic interference for both colour and location in the present study.

      However, our findings suggest otherwise. One potential concern is whether this dissociation simply reflects the inherent spatial noise introduced by fixational eye movements (FEMs), such as microssacades and drifts.47 Because locations are stored in a retinotopic frame, fixational instability necessarily shifts retinal coordinates over time. However, the "saccade cost" here was defined as the error increase relative to a no-saccade baseline of equal duration; because both conditions are subject to the same fixational drift, any FEM-induced noise is effectively subtracted out. Thus, despite the ballistic and non-Gaussian nature of FEMs,48 they cannot account for the fact the saccade cost in the spatial memory, but total absence in the colour domain. Another possibility is that this dissociation reflects differences in baseline task difficulty or dynamic range. Yet, the presence of a robust recency effect in colour memory (Figure 2B) confirms that our paradigm was sensitive to memory-dependent variance and was not limited by floor or ceiling effects.

      The fact that identical eye movements—executed simultaneously and with identical vectors—systematically degraded spatial precision while sparing colour suggests a feature-specific susceptibility to transsaccadic remapping. This supports the view that the computational process of updating an object’s location involves a vector-subtraction mechanism—incorporating noisy oculomotor commands (efference copies)—that introduces specific spatial variance. Because this remapping is a coordinate transformation, the resulting sensorimotor noise does not functionally propagate to non-spatial feature representations. Consequently, features like colour may be preserved or automatically remapped without the precision loss associated with spatial updating.11,49 Our paradigm thus provides a refined tool to investigate the architecture of transsaccadic working memory across distinct object features.”

      (5) Relationship between saccade cost and individual memory performance (p. 4, last paragraph).

      The authors report that larger saccades were associated with greater spatial memory disruption. It would be informative to examine whether individual differences in the magnitude of saccade cost correlate with participants' overall/baseline memory performance (e.g. their memory precision in the no-saccade condition). Such analyses might offer insights into how memory capacity/ability relates to resilience against saccade-induced updating.

      We have now conducted the correlation analysis to determine whether baseline memory capacity (no-saccade condition) predicts resilience to saccade-induced updating. The results indicate that these two factors are independent.

      To clarify the nature of the saccade-induced impairment, we have updated the text as follows:

      p.4: “This “saccade cost”—the loss of memory precision following an eye movement—indicates that spatial representations require active updating across saccades rather than being maintained in a static, world-centred reference frame.”

      p.5: “Further analysis examined whether individual differences in baseline memory precision (no-saccade condition) predicted resilience to saccadic disruption. Crucially, individual saccade costs (defined as the precision loss relative to baseline) did not correlate with baseline precision (rho = 0.20, p = 0.20). This suggests that the noise introduced by transsaccadic remapping acts as an independent, additive source of variance that is not modulated by an individual’s underlying memory capacity. These findings imply a functional dissociation between the mechanisms responsible for maintaining a representation and those involved in its coordinate transformation.”

      (6) Model fitting for the healthy elderly group to reveal memory-deficit factors (pp. 11-12). The manuscript discusses model-based insights into components that contribute to spatial memory deficits in AD and PD, but does not discuss components that contribute to spatial memory deficits in the healthy elderly group. Given that the EC group also shows impairments in certain parameters, explaining and discussing these outcomes of the EC group could provide additional insights into age-related memory decline, which would strengthen the study's broader conclusions.

      This is a very good point. We rewrote the corresponding results section (p.12-13):

      “Modelling reveals the sources of spatial memory deficits in healthy aging and neurodegeneration - To understand the source of the observed deficits, we applied the winning ‘Dual (Saccade) + Interference’ model the data from all participants (YC, EC, AD, and PD). By fitting the model to the entire dataset, we obtained estimates of the parameters for each individual, which then formed the basis for our group-level analysis. To formally test for group differences, we used Parametric Empirical Bayes (PEB), a hierarchical Bayesian approach that compares parameter estimates across groups while accounting for the uncertainty of each estimate [28]. This allowed us to identify which specific cognitive mechanisms, as formalised by the model parameters, were affected by age and disease.

      The Bayesian inversion used here allows us to quantify the posterior mode and variance for each parameter and the covariance for each parameter. From these, we can compute the probabilities that pairs of parameters differ from one another, which we report as P(A>B)—meaning the posterior probability that the parameter for group A was greater than that for group B.

      We first examined the specific parameters differentiating healthy elderly (EC) from young controls (YC) to isolate the factors contributing to non-pathological, age-related decline. The analysis revealed that healthy ageing is primarily characterised by a significant increase in Radial Decay (P(EC > YC) = 0.995), a heightened susceptibility to Interference (P(EC > YC) = 1.000), and a reduction in initial Angular Encoding precision (P(YC < EC) = 0.002; Figure 6). These results suggest that normal ageing degrades the fidelity of the initial memory trace and its resilience over time, while the core computational process of updating information across saccades remains intact.

      Beyond these baseline ageing effects, our clinical cohorts exhibited more severe and condition-dependent impairments. Radial decay showed a clear, graded impairment: AD patients had a greater decay rate than PD patients (P(AD > PD) = 1.000), who in turn were more impaired than the EC group (P(PD > EC) = 0.996). A similar graded pattern was observed for Interference, where AD patients were most susceptible (P(AD > PD) = 0.999), while the PD and EC groups did not significantly differ (P(PD > EC) = 0.532).

      Patients with AD also showed a tendency towards greater angular decay than controls (P(AD > EC) = 0.772), although this fell below the 95% probability threshold. This effect was influenced by a lower decay rate in the PD group compared to the EC group (P(PD < EC) = 0.037). In contrast, group differences in encoding were less pronounced. While YC exhibited significantly higher precision than all other groups, AD patients showed significantly higher angular encoding error than PD patients (P(AD > PD) = 0.985), though neither group differed significantly from the EC group.

      Crucially, parameters related to the saccade itself—saccade encoding and saccade decay—did not differentiate the groups. This indicates that neither healthy ageing nor the early stages of AD and PD significantly impair the fundamental machinery for transsaccadic remapping. Instead, the visuospatial deficits in these conditions arise from specific mechanistic failures: a faster decay of radial position information and increased susceptibility to interference, both of which are present in healthy ageing but significantly amplified by neurodegeneration.”

      In the Discussion, we added:

      “Although saccade updating was an essential component of the winning model, its two key parameters—initial encoding error and decay rate during maintenance—did not significantly differ across groups. This indicates that the core computational process of updating spatial information based on eye movements is largely preserved in healthy aging and neurodegeneration.

      Instead, group differences were driven by deficits in angular encoding error (precision of initial angle from fixation), angular decay, radial decay (decay in memory of distance from fixation), and interference susceptibility. This implies a functional and neuroanatomical dissociation: while the ventral stream (the “what” pathway) shows an age-related decline in the quality and stability of stored representations, the dorsal-stream (the “where” pathway) parietal-frontal circuits responsible for coordinate transformations remain functionally robust.[31–34] These spatial updating mechanisms appear resilient to the normal ageing trajectory and only break down when challenged by the specific pathological processes seen in Alzheimer’s or Parkinson’s disease.”

      (7) Presentation of saccade conditions in Figure 5 (p. 11). In Figure 5, it may be clearer to group the four saccade conditions together within each patient group. Since the main point is that saccadic interference on spatial memory remains robust across patient groups, grouping conditions by patient type rather than intermixing conditions would emphasize this interpretation.

      There are several valid ways to present these plots, but we chose this format because it allows for a direct visual comparison of the post-hoc group differences within each specific task demand. This arrangement clearly illustrates the graded impairment from young controls through to patients with Alzheimer’s disease across every condition. This structure also directly mirrors our two-way ANOVA, which identified significant main effects for both Group and Condition, but crucially, no significant Group x Condition interaction. We felt that grouping the data by participant group would force readers to look across four separate clusters to compare the slopes, making the stability of the saccadic remapping mechanism much harder to grasp at a glance.

      Reviewer #1 (Recommendations for the authors):

      (1) Formatting of statistical parameters.

      The formatting of statistical symbols should be consistent throughout the manuscript. Some instances of F, p, and t are italicized, while others are not. All statistical symbols should be italicized.

      Thank you for pointing this out. We have audited the manuscript. While we have revised the text to address these instances throughout the Results and Methods sections, any remaining minor formatting inconsistencies will be corrected during the final typesetting stage.

      (2) Minor typographical issues.

      (a) Line 532: "are" should be "be."

      (b) Line 654: "cantered" should be "centered."

      (c) Line 213: In "(p(bonf) < 0.001, |t| {greater than or equal to} 5.94)," the t value should be reported with its degrees of freedom, and t should be reported before p. The same applies to line 215.

      Thank you for your careful reading. All corrected.

      Reviewer #2 (Public review):

      We thank you for your positive feedback regarding our eye-tracking methodology and computational approach. We appreciate your critical insights into the feature-specific disruption hypothesis and the task structure. We have substantially revised the results and discussion about the saccadic interference on colour memory. Below we will answer your suggestions point-by-point:

      Reviewer #2 (Recommendations for the authors):

      (1) The study treats colour and location errors as comparable when arguing that saccades selectively disrupt spatial but not colour memory. However, these measures are defined in entirely different units (degrees of visual angle vs radians on a colour wheel) and are not psychophysically or statistically calibrated. Baseline task difficulty, noise level, or dynamic range do not appear to be calibrated or matched across features. As a result, the null effect of saccades on colour could reflect lower sensitivity or ceiling effects rather than implicit feature-specific robustness.

      We agree that direct comparisons of absolute error magnitudes across different dimensions are not appropriate. Our argument for feature-specific disruption relies not on the scale of errors, but on the presence or absence of a saccade cost within identical trials. In our within-subject design, the same saccade vectors produced a systematic increase in location error while leaving colour error statistically unchanged. To address sensitivity, we observed that colour memory was sufficiently precise to show a significant recency effect (p = 0.02). To further quantify the evidence for the null effect, we performed Bayesian repeated measures ANOVAs, which yielded a BF10 = 0.22. This provides substantial evidence that saccades do not disrupt colour precision, regardless of baseline sensitivity.

      We have substantially revised this in Results, Methods and Discussion:

      In the Results:

      “This design allowed us to isolate and quantify the unique impact of saccades on spatial memory, enabling us to test competing hypotheses regarding spatial representation. If spatial memory were solely underpinned by an allocentric mechanism, precision should remain comparable across all conditions as the representation would be world-centred and unaffected by eye movements. Thus, performance in the no-saccade condition should be comparable to the two-saccade condition. Conversely, if spatial memory relies on a retinotopic representation requiring active updating across eye movements, the two-saccade condition was anticipated to be the most challenging due to cumulative decay in the memory traces used for stimulus reconstruction after each saccade.[22] Critically, we hypothesised that this saccade cost would be specific to the spatial domain; while location requires active remapping via noisy oculomotor signals, non-spatial features like colour are not inherently tied to coordinate transformations and should therefore remain stable (see more in Discussion below).

      Meanwhile, the no-saccade condition was expected to yield the most accurate localisation, relying solely on retinotopic information (retinotopic working memory). These predictions were confirmed in young healthy adults (N = 21, mean age = 24.1 years, ranged between 19 and 34). A repeated measures ANOVA revealed a significant main effect of saccades on location memory (F(2.2,43.9)=33.2, p<0.001, partial η²=0.62), indicating substantial impairment after eye movements (Figure 2A). In contrast, colour memory remained remarkably stable across all saccade conditions (Figure 2B; F(2.2, 44.7) = 0.68, p=0.53, partial η² =0.03).

      This “saccade cost”—the loss of memory precision following an eye movement—indicates that spatial representations require active updating across saccades rather than being maintained in a static, world-centred reference frame.

      Critically, our comparison between spatial and colour memory does not rely on the absolute magnitude of errors, which are measured in different units (degrees of visual angle vs. radians). Instead, we assessed the relative impact of the same saccadic demand on each feature within the same trial. While location recall showed a robust saccade cost, colour recall remained statistically unchanged. To ensure this null effect was not due to a lack of measurement sensitivity, we examined the recency effect; recall performance for the second item was predicted to be better than for the first stimulus in each condition.[23,24] As expected, colour memory for Item 2 was significantly more accurate than for Item 1 (F(1,20) = 6.52, p = 0.02, partial η² = 0.25), demonstrating that the task was sufficiently sensitive to detect standard working memory fluctuations despite the absence of a saccade-induced deficit.”

      In the Methods, at the beginning of “Statistical Analysis”, we added

      “Because location and colour recall involve different scales and units, all analyses were performed independently for each feature to avoid cross-dimensional magnitude comparisons.” (p25)

      In the Discussion, we added:

      “A potential concern is whether the observed dissociation between colour and location reflects differences in baseline task difficulty or dynamic range. Yet, the presence of a robust recency effect in colour memory (Figure 2B) confirms that our paradigm was sensitive to memory-dependent variance and was not limited by floor or ceiling effects.”

      (2) Colour and then location are probed serially, without a counter-balanced order. This fixed response order could introduce a systematic bias because location recall is consistently subject to longer memory retention intervals and cognitive interference from the colour decision. The observed dissociation-saccades impair location but not colour, and may therefore reflect task structure rather than implicit feature-specific differences in trans-saccadic memory.

      Thank you for the insightful observation regarding our fixed response order. We acknowledge that that a counterbalanced design is typically preferred to mitigate potential order effects. However, we chose this consistent sequence to ensure the task remained accessible for cognitively impaired patients (i.e., the Alzheimer’s disease (AD) and Parkinson’s disease (PD) cohorts). Conducting an eye-tracking memory task with cognitively impaired patients is challenging, as they may struggle with task engagement or forget complex instructions. During the design phase, we prioritised a consistent structure to reduce the cognitive load and task-switching demands that typically challenge these cohorts.

      Critically, because the saccade cost is a relative measure calculated by comparing conditions with identical timings, any bias from the fixed order is present in both the baseline and saccade trials. The disruption we report is therefore a specific effect of eye movements that goes beyond the noise introduced by the retention interval or the preceding colour report.

      We added the following text in the Methods – experimental procedure (p.22):

      “Recall was performed in a fixed order, with colour reported before location. This sequence was primarily chosen to minimise cognitive load and task-switching demands for the two neurological patient cohorts, ensuring the paradigm remained accessible for individuals with AD and PD. While this order results in a slightly longer retention interval for location recall, the saccade cost was identified by comparing location error across experimental conditions with similar timings but varying saccadic demands.”

      (3) Relatedly, because spatial representations are retinotopic, fixational eye movements (FEMs - microsaccades and drift) displace the retinal coordinates of encoded positions, increasing apparent spatial noise with time delays. Colour memory, however, is feature-based and unaffected by small retinal translations. Thus, any between-condition or between-group differences in FEMs could selectively inflate location error and the associated model parameters (encoding noise, decay, interference), while leaving colour error unchanged. Note that FEMs tend to be slightly ballistic [1,2], hence not well modelled with a Gaussian blur.

      This is a very insightful point. We have now addressed this in detail within the discussion:

      “However, our findings suggest otherwise. One potential concern is whether this dissociation simply reflects the inherent spatial noise introduced by fixational eye movements (FEMs), such as microssacades and drifts.[46] Because locations are stored in a retinotopic frame, fixational instability necessarily shifts retinal coordinates over time. However, the "saccade cost" here was defined as the error increase relative to a no-saccade baseline of equal duration; because both conditions are subject to the same fixational drift, any FEM-induced noise is effectively subtracted out. Thus, despite the ballistic and non-Gaussian nature of FEMs,n [47] they cannot account for the fact the saccade cost in the spatial memory, but total absence in the colour domain. Another possibility is that this dissociation reflects differences in baseline task difficulty or dynamic range. Yet, the presence of a robust recency effect in colour memory (Figure 2B) confirms that our paradigm was sensitive to memory-dependent variance and was not limited by floor or ceiling effects.”

      (4) There is no in silico demonstration that the modelling framework can recover the true generating model from synthetic data or recover accurate parameters under realistic noise levels, which can be challenging in generative models with a hierarchical structure (as per [3], for example). Figure 8b shows that the parameters possess substantial posterior covariance, which raises concerns as to whether they can be reliably disambiguate.

      Many thanks for this comment. We have added a simple recovery analysis as detailed below but are also keen to ensure we fully answer your question—which has more to do with empirical rather than simulated data—and make clear the rationale for this analysis in this instance.

      We added this in Supplementary Materials:

      “Model validation and recovery analysis

      The following section provides a detailed technical assessment of the model inversion scheme, focusing on the discriminability of the model space and the identifiability of individual parameters.

      Recovery analyses of this sort are typically used prior to collecting data to allow one to determine whether, in principle, the data are useful in disambiguating between hypotheses. In this sense, they have a role analogous to a classical power calculation. However, their utility is limited when used post-hoc when data have already been collected, as the question of whether the models can be disambiguated becomes one of whether non-trivial Bayes factors can be identified from those data.

      The reason for including a recovery analysis here is not to identify whether the model inversion scheme identifies a ‘true’ model. The concept of ‘true generative models’ commits to a strong philosophical position which is at odds with the ‘all models are wrong, but some are useful’ perspective held by many in statistics, e.g., (So, 2017). Of note, one can always confound a model recovery scheme by generating the same data in a simple way, and in (one of an infinite number of) more complex ways. A good model inversion scheme will always recover the simple model and therefore would appear to select the ‘wrong’ model in a recovery analysis. However, it is still the best explanation for the data. For these reasons, we do not necessarily expect ‘good’ recoverability in all parameter ranges. This is further confounded by the relationship between the models we have proposed—e.g., an interference model with very low interference will look almost identical to a model with no interference. The important question here is whether they can be disambiguated with real data.

      Instead, the value of a post-hoc recovery analysis here is to evaluate whether there was a sensible choice of model space—i.e., that it was not a priori guaranteed that a single model (and, specifically, the model we found to be the best explanation for the data) would explain the results of all others. To address this, for each model, we simulated 16 datasets, each of which relied upon parameters sampled from the model priors, which included examples of each of the experimental conditions. We then fit each of these datasets to each of the 7 models to construct the confusion matrix shown in the lower panel of Supplementary Figure 3, by accumulating evidence over each of the 16 participants generated according to each ‘true’ model (columns) for each of the possible explanatory models (rows). This shows that no one model, for the parameter ranges sampled here, explains all other datasets. Interestingly, our ‘winning’ model in the empirical analysis is not the best explanation for any of the datasets simulated (including its own). This is reassuring, in that it implies this model winning was not a foregone conclusion and is driven by the data—not just the choice of model space.”

      Your point about the posterior covariance is well founded. As we describe in Supplementary Materials, this is an inherent feature of inverse problems (analogous to EEG source localisation). However, the fact that our posterior densities move significantly away from the prior expectations demonstrates that the data are indeed informative. By adopting a Bayesian framework, we are able to explicitly quantify this uncertainty rather than ignoring it, providing a more transparent account of parameter identifiability. We have added the following in the same section of Supplementary Materials:

      “This problem is an inverse problem—inferring parameters from a non-linear model. We therefore expect a degree of posterior covariance between parameters and, consequently, that they cannot be disambiguated with complete certainty. While some degree of posterior covariance is inherent to inverse models—including established methods like EEG source localisation—the fact that many of the parameters are estimated with posterior densities that do not include their prior expectations implies the data are informative about these.

      The advantage of the Bayesian approach we have adopted here is that we can explicitly quantify posterior covariance between these parameters, and therefore the degree to which they can be disambiguated. While the posterior covariance matrices from empirical data are the relevant measure here, we can better understand the behaviour of the model inversion scheme in relation to the specific models used using the model recovery analysis reported in Supplementary figure 3.

      The middle panel of the figure is key, along with the correlation coefficients reported in the figure caption. Here, we see at least a weak positive correlation (in some cases much stronger) for almost all parameters and limited movement from prior expectations for those parameters that are less convincingly recovered. This reinforces that the ability of the scheme to recover parameters is best assessed in terms of the degree of movement of posterior from prior values following fitting to empirical data.”

      (5) The authors employ Bayes factors (BFs) to disambiguate models, but BFs would also strengthen the claims that location, but not colour, is impacted by saccades. Despite colour being a circular variable, colour error is analysed using ANOVA on linearised differences (radians). The authors should also arguably use circular statistics, such as the von Mises distribution, for the analysis of colour.

      Regarding the use of circular statistics, you are correct that such error distributions are not suitable for ANOVA, and it is better to use circular statistics. However, for the present dataset, we used the mean absolute angular error per condition (ranging from 0 to π radians), which represents the shortest distance on the colour wheel between the target and the response.

      This approach effectively linearises the measure by removing the 2π wrap-around boundary. because the observed errors were relatively small and did not cluster near the π boundary—even in the patient cohorts (Figure 5B)—the "wrap-around" effect of circular space is negligible. Moreover, by analysing the mean error across trials for each condition, rather than trial-wise data, we invoke the Central Limit Theorem. This ensures that the distribution of these means is approximately normal, satisfying the fundamental assumptions of ANOVA. Due to these reasons, we adopted simpler linear models. We confirmed that the data did not violate the assumptions of linear statistics. In this low-noise regime, linear and circular models converge on the same conclusions. This has been revised in Methods:

      “For colour memory, we calculated the absolute angular error, defined as the shortest distance on the colour wheel between the target and the reported colour (range 0 to π radians). For the primary statistical analyses, we utilised the mean absolute error per condition for each participant. By analysing these condition-wise means rather than trial-wise raw data, we invoke the Central Limit Theorem, which ensures that the sampling distribution of these means approximates normality. Because the absolute errors in this paradigm were relatively small and did not approach the π boundary (Figure 5B) even in the clinical cohorts, the data were treated as a continuous measure in our linear ANOVAs and regression models. Moreover, because location and colour recall involve different scales and units, all analyses were performed independently for each feature to avoid cross-dimensional magnitude comparisons.”

      We have also now integrated Bayesian repeated measures ANOVA throughout the manuscript. The Results section for the young healthy adults now reads (p. 4):

      “A repeated measures ANOVA revealed a significant main effect of saccades on location memory (F(3, 20) = 51.52, p < 0.001, partial η²=0.72), with Bayesian analysis providing decisive evidence for the inclusion of the saccade factor (BF<sub>incl</sub> = 3.52 x 10^13, P(incl|data) = 1.00). In contrast, colour memory remained remarkably stable across all saccade conditions (F(3, 20) = 0.57, p = 0.64, partial η² =0.03). This null effect was supported by Bayesian analysis, which provided moderate evidence in favour of the null hypothesis (BF<sub>01</sub> = 8.46, P(excl|data) = 0.89), indicating that the data were more than eight times more likely under the null model than a model including saccade-related impairment.”

      For elderly healthy adults:

      “In contrast, colour memory remained unaffected by saccade demands (F(3, 20) = 0.57, p = 0.65, partial η² =0.03), again supported by the Bayesian analysis: BF<sub>01</sub> = 8.68, P(excl|data) = 0.90.”

      For patient cohorts:

      “Bayesian repeated measures ANOVAs further supported this dissociation, providing moderate evidence for the null hypothesis in the AD group (BF<sub>01</sub> = 3.35, P(excl|data) = 0.77) and weak evidence in the PD group (BF<sub>01</sub> = 2.23, P(excl|data) = 0.69). This indicates that even in populations with established neurodegeneration, the detrimental impact of eye movements is specific to the spatial domain.”

      Related description is also updated in Methods – Statistical Analysis.

      Minor:

      (1) The modelling is described as computational but is arguably better characterised as a heuristic generative model at Marr's algorithmic level. It does not derive from normative computational principles or describe an implementation in neural circuits.

      We appreciate your perspective on the classification of our model within Marr’s hierarchy. We agree that our framework is best characterised as an algorithmic-level generative model. Our objective was to identify the mechanistic principles governing transsaccadic updating rather than to provide a normative derivation or a specific circuit-level implementation.

      To ensure readers do not over-interpret the term ‘computational’, we have added a clarifying statement in the Discussion acknowledging the algorithmic nature of the model. Interestingly, we note that a model predicated on this form of spatial diffusion implies a neural field representation with a spatial connectivity kernel whose limit approximates the second derivative of a Dirac delta function. While a formal neural field implementation is beyond the scope of the present work, our algorithmic results provide the necessary constraints for such future biophysical models.

      p.20: “While we describe the present framework as 'computational', it is more precisely characterised as an algorithmic-level generative model within Marr’s hierarchy. Our focus was on defining the rules of spatial integration and the sources of eye-movement-induced noise, rather than deriving these processes from normative principles or defining their specific neural implementation.”

      (2) I did not find a description of the recruitment and characterization of the AD and PD patients.

      Apologies for this omission. We have now included a detailed description of participant recruitment and clinical characterisation in the Methods section and also updated Table 2:

      “A total of 87 participants completed the study: 21 young healthy adults (YC), 21 older healthy adults (EC), 23 patients with Parkinson’s disease (PD), and 22 patients with Alzheimer’s disease (AD). Their demographic and clinical details are summarised in Table 2. Initially, 90 participants were recruited (22 YC, 21 EC, 25 PD, 22 AD); however, three individuals (1 YC and 2 PD) were excluded from all analyses due to technical issues during data acquisition.

      All participants were recruited locally in Oxford, UK. None were professional artists, had a history of psychiatric illness, or were taking psychoactive medications (excluding standard dopamine replacement therapy for PD patients). Young participants were recruited via the University of Oxford Department of Experimental Psychology recruitment system. Older healthy volunteers (all >50 years of age) were recruited from the Oxford Dementia and Ageing Research (OxDARE) database.

      Patients with PD were recruited from specialist clinics in Oxfordshire. All had a clinical diagnosis of idiopathic Parkinson's disease and no history of other major neurological or psychiatric conditions. While specific dosages of dopamine replacement therapy (e.g., levodopa equivalent doses) were not systematically recorded, all patients were tested while on their regular medication regimen ('ON' state).

      Patients with PD were recruited from clinics in the Oxfordshire area. All had a clinical diagnosis of idiopathic Parkinson’s disease and no history of other major neurological or psychiatric illnesses. While all patients were tested in their regular medication ‘ON’ state, the specific pharmacological profiles—including the exact types of medication (e.g., levodopa, dopamine agonists, or combinations) and dosages—were not systematically recorded. The disease duration and PD severity were also un-recorded for this study.

      Patients with AD were recruited from the Cognitive Disorders Clinic at the John Radcliffe Hospital, Oxford, UK. All AD participants presented with a progressive, multidomain, predominantly amnestic cognitive impairment. Clinical diagnoses were supported by structural MRI and FDG-PET imaging consistent with a clinical diagnosis of AD dementia (e.g., temporo-parietal atrophy and hypometabolism).69 All neuroimaging was reviewed independently by two senior neurologists (S.T. and M.H.).

      Global cognitive function was assessed using the Addenbrooke’s Cognitive Examination-III (ACE-III).70 All healthy participants scored above the standard cut-off of 88, with the exception of one elderly participant who scored 85. In the PD group, two participants scored below the cut-off (85 and 79). In the AD group, six participants scored above 88; these individuals were included based on robust clinical and radiological evidence of AD pathology rather than their ACE-III score alone.”

      (3) YA and OA patients appear to differ in gender distribution.

      We acknowledge the difference in gender distribution between the young (71.4% female) and older adult (57.1% female) cohorts. However, we do not anticipate that gender influences the fundamental computational mechanisms of retinotopic maintenance or transsaccadic remapping. These processes represent low-level visuospatial functions for which there is no established evidence of gender-specific differences in precision or coordinate transformation. We have ensured that the gender distribution for each cohort is clearly listed in the demographics table (Table 2) for full transparency.

      Thank you very much for very insightful feedback!

      Reviewer #3 (Public review):

      Thank you for the positive feedback regarding our inclusion of clinical groups and the identification of computational phenotypes that differentiate these cohorts.

      To address your concerns about the model, we have clarified our use of Bayesian Model Selection, which inherently penalises model complexity to ensure that our results are not driven solely by the number of parameters. We will also provide further evidence regarding model generalisability to address the concern of overfitting.

      Regarding the link with the ROCF, we have revised the manuscript to better highlight the specific relationship between our transsaccadic parameters and the ROCF data and better motivate the inclusion of these results in the main text.

      Below is our response to your suggestions point-by-point:

      (1) The models tested differ in terms of the number of parameters. In general, a larger number of parameters leads to a better goodness of fit. It is not clear how the difference in the number of parameters between the models was taken into account. It is not clear whether the modelling results could be influenced by overfitting (it is not clear how well the model can generalize to new observations).

      To ensure our results were not driven by the number of parameters, we utilised random-effects Bayesian Model Selection (BMS) to adjudicate between our candidate models. Unlike maximum likelihood methods, BMS relies on the marginal likelihood (model evidence), which inherently balances model fit against parsimony—a principle known as the Occam’s Razor (Rasmussen and Ghahramani, 2000). In this framework, a model is only preferred if the improvement in fit justifies the additional parameter space; redundant parameters actually lower model evidence by diluting the probability mass. We would be happy to point toward literature that discusses how these marginal likelihood approximations provide a more robust guard against overfitting than standard metrics like BIC or AIC (MacKay, 2003; Murray and Ghahramani, 2005; Penny, 2012).

      The fact that the "Dual (Saccade) + Interference" model (Model 7) emerged as the winner—with a Bayes Factor of 6.11 against the next best alternative—demonstrates that its complexity was statistically justified by its superior account of the trial-by-trial data.

      Furthermore, to address the risk of overfitting, we established the generalisability of these parameters by using them to predict performance on an independent clinical task. These parameters successfully explained ~62% of the variance in ROCF copy scores—a very distinct, real-world task--confirming that they represent robust computational phenotypes rather than idiosyncratic fits to the initial dataset.

      In the Results (p10):

      “We used random-effects Bayesian model selection to identify the most plausible generative model. This process relies on the marginal likelihood (model evidence), which inherently balances model fit against complexity—a principle often referred to as Occam’s razor.[25–27]”

      In the Discussion (p17):

      “Importantly, the risk of overfitting is mitigated by the Bayesian Model Selection framework; by utilising the marginal likelihood for model comparison, the procedure inherently penalises excessive model complexity and promotes generalisability.[25–27,42] This generalisability was further evidenced by the model's ability to predict performance on the independent ROCF task, confirming that these parameters represent robust mechanistic phenotypes rather than idiosyncratic fits to the initial dataset.”

      (2) Results specificity: it is not clear how specific the modelling results are with respect to constructional ability (measured via the Rey-Osterrieth Complex Figure test). As with any cognitive test, performance can also be influenced by general, non-specific abilities that contribute broadly to test success.

      We agree that constructional performance is influenced by both specific mechanistic constraints and general cognitive abilities. To isolate the unique contribution of transsaccadic updating, we therefore performed a partial correlation analysis across the entire sample. We examined the relationship between location error in the two-saccades condition (our primary behavioural measure of transsaccadic memory) and ROCF copy scores. Even after partialling out the effects of global cognitive status (ACE-III total score), age, and years of education, the correlation remained highly significant (rho = -0.39, p < 0.001).

      This suggests that our model captures a specific computational phenotype—the precision of spatial updating during active visual sampling—rather than acting as a proxy for non-specific cognitive decline. This mechanistic link explains why traditional working memory measures (e.g., digit span or Corsi blocks) frequently fail to predict drawing performance; unlike those tasks, figure copying requires thousands of saccades, making it uniquely sensitive to the precision of the dynamic remapping signals identified by our modelling framework.

      We added the following text in the Discussion (p19):

      “We also found that the relationship between transsaccadic working memory and ROCF performance remains highly significant (rho = -0.39, p < 0.001), even after controlling for age, education, and global cognitive status (ACE-III total score). Consequently, transsaccadic updating may represent a discrete computational phenotype required for visuomotor control, rather than a non-specific proxy for global cognitive decline.[57]”

      Reviewer #3 (Recommendations for the authors):

      (1) The authors mention in the introduction the following: "One key hypothesis is that we use working memory across visual fixations to update perception dynamically", citing the following manuscript:

      Harrison, W. J., Stead, I., Wallis, T. S. A., Bex, P. J. & Mattingley, J. B. A computational 906 account of transsaccadic attentional allocation based on visual gain fields. Proc. Natl. 907 Acad. Sci. U.S.A. 121, e2316608121 (2024).

      However, the manuscript above does not refer explicitly to the involvement of working memory in transaccadic integration of object location in space. Rather, it takes advantage of recent evidence showing how the true location of a visual object is represented in the activity of neurons in primary visual cortex ( A. P. Morris, B. Krekelberg, A stable visual world in primate primary visual cortex. Curr. Biol. 29, 1471-1480.e6 (2019) ). The model hypothesizes that true locations of objects are readily available, and then allocates attention in real-world coordinates, allowing efficient coordination of attention and saccadic eye movements.

      Thank you for clarification. As suggested, we have now included the citation of Morris & Krekelberg (2019) to acknowledge the evidence for stable object locations within the primary visual cortex.

      (2) The authors in the introduction and the title use the terms 'transaccadic memory' and 'spatial working memory'. However, it is not clear whether these can be used interchangeably or are reflecting different constructs.

      Classical measures of visuo-spatial working memory are derived from the Corsi task (or similar), where the location of multiple objects is displayed and subsequently remembered. In such tasks, eye movements and saccades are not generally considered, only memory performance, representing the visuo-spatial span.

      Transaccadic memory tasks are instead explicitly measuring the performance on remembered object locations of features across explicit eye movements, usually using a very limited number of objects (1 or 2, as is the case for the current manuscript).

      While the two constructs share some features, it is not clear whether they represent the same underlying ability or not, especially because in transaccadic tasks, participants are required to perform one or more saccades, thus representing a dual-task case.

      I think the relationship between 'transaccadic memory' and 'spatial working memory' should be clarified in the manuscript.

      Thank you. Yes, we have added this within the Methods - Measurement of saccade cost to clarify that spatial working memory is the broad cognitive construct responsible for short-term maintenance, whereas transsaccadic memory is the specific, dynamic process of remapping representations to maintain stability across eye movements.

      In Methods (p.22):

      “Within this framework, it is important to distinguish between the broad construct of spatial working memory and the specific process of transsaccadic memory. While spatial working memory refers to the general ability to maintain spatial information over short intervals, transsaccadic memory describes the dynamic updating of these representations—termed remapping—to ensure stability across eye movements. Unlike classical 'static' measures of spatial working memory, such as the Corsi block task which focuses on memory span, transsaccadic memory tasks explicitly require the integration of stored visual information with motor signals from intervening saccades. Our paradigm treats transsaccadic updating as a core computational process within spatial working memory, where eye-centred representations are actively reconstructed based on noisy memories of the intervening saccade vectors.”

      (3) In Figure 1, the second row indicates the presentation of item 2. Indeed, in the condition 'saccade-after-item-1', the target in the second row of Figure 1 is displaced, as expected. This clarifies the direction and amplitude of the first saccade requested. However, from Figure 1, it is hard to understand the amplitude and direction of the second requested saccade. I think the figure should be updated, giving a full description of the direction and amplitude of the second saccade as well ('saccade-after-item-2' and 'two-saccades' conditions).

      We agree that making the figure legend more self-contained is beneficial for the reader. While the specific physical parameters and the trial sequence for each condition are detailed in the Results and Methods sections, we have now updated the legend for Figure 1 to explicitly define these details. Specifically, we have clarified that the colour wheel itself served as the target for the second instructed saccade (i.e., the movement from the second fixation cross to the colour wheel location). We have also included the quantitative constraint that all saccade vectors were at least 8.5 degrees of visual angle in amplitude. Given the limited space within a figure legend, we hope these concise additions provide the transparency requested without interrupting the conceptual flow of the diagram.

      Updated Figure 1 legend:

      “Participants were asked to fixate a white cross, wherever it appeared. They had to remember the colour and location of a sequence of two briefly presented coloured squares (Item 1 and 2), each appearing within a white square frame. They then fixated a colour wheel wherever it appeared on the screen, which served as the target for the second instructed saccade (i.e., a movement from the second fixation cross to the colour wheel location). This cued recall of a specific square (Item 1 or Item 2 labelled within the colour wheel). Participants selected the remembered colour on the colour wheel which led to a square of that colour appearing on the screen. They then dragged this square to its remembered location on the screen. Saccadic demands were manipulated by varying the locations of the second frame and the colour wheel, resulting in four conditions in their reliance on retinotopic versus transsaccadic memory: (1) No-Saccade condition providing a baseline measure of within-fixation precision as no eye movements were required. (2) Saccade After Item 1; (3) Saccade After Item 2; (4) Saccades after both items (Two Saccades condition). In all conditions requiring eye movements, saccade vectors were constrained to a minimum amplitude of 8.5° (degrees of visual angle). While the No-Saccade condition isolates retinotopic working memory, conditions (2) to (4) collectively quantify the impact of varying saccadic demands and timings on the maintenance of spatial information, thereby assessing the efficacy of the transsaccadic updating process.”

      (4) The authors write: "Eye tracking analysis confirmed high compliance: participants correctly maintained fixation or executed saccades as instructed on the vast majority of trials (83% {plus minus} 14%). Non-compliant trials were excluded 136 from further analysis." 14% of excluded trials are a substantial fraction of trials, given the task requirements. Is this proportion of excluded trials different between experimental groups, and are experimental groups contributing equally to this proportion?

      We thank the reviewer for pointing this out, and we apologise for the confusion. The 83% trial number was actually across all four cohorts, and all conditions, and it was actually above 90% for YC, EC and even AD, but dropped to 60 ish in PD group.

      We now have conducted a full analysis of compliant trial counts using a mixed ANOVA (4 saccade conditions x 4 cohorts). This analysis revealed a main effect of group (F(3, 80) = 8.06, p < 0.001), which was driven by lower compliance in the PD cohort (mean approx. 25.4 trials per condition) compared to the AD, EC, and YC cohorts (means ranging from 35.8 to 38.9 trials per condition). Crucially, however, the interaction between group and condition was not statistically significant (p = 0.151). This indicates that the relative impact of saccade demands on trial retention was consistent across all four groups.

      Because our primary behavioural measure—the saccade cost—is a within-subject comparison of impairment across conditions, these differences in absolute trial numbers do not introduce a systematic bias into our findings. Furthermore, even with the higher attrition in the PD group, we retained a sufficient number of high-quality trials (minimum mean of ~23 trials in the most demanding condition) to support robust trial-by-trial parameter estimation and valid statistical inference. We have updated the Results and Methods to reflect these details.

      In Results (p4):

      “To mitigate potential confounds, we monitored eye position throughout the experiment. Eye-tracking analysis confirmed high compliance in healthy adults, who followed instructions on the vast majority of trials (Younger Adults: 97.2 ± 5.2 %; Older Adults: 91.3 ± 20.4 %). The mean difference between these groups was negligible, representing just 1.25 trials per condition, and was not statistically significant (t(80) = 0.16, p = 1.000; see more in Methods – Eyetracking data analysis). Non-compliant trials were excluded from all further analyses.”

      In Methods (p27):

      “Eye-tracking analysis confirmed high compliance overall, with participants correctly maintaining fixation or executing saccades on the vast majority of trials (83% across all participants). A mixed ANOVA revealed a main effect of group on trial retention (F(3, 80) = 8.06, p < 0.001, partial η² = 0.23), primarily due to lower compliance in the PD cohort (YC: 97±4%; EC: 91±10%; AD: 95±5%; PD: 63±38%). Importantly, there was no significant interaction between group and saccade condition (F(3.36, 80) = 1.78, p = 0.15, partial η² = 0.008), suggesting that trial attrition was not disproportionately affected by specific task demands in any group.

      We acknowledge that this reduced trial count in the PD group represents a limitation for across-cohort comparison. However, the absolute number of compliant trials in PD group (mean approx. 25 per condition) remained sufficient for robust trial-by-trial parameter estimation. Furthermore, the lack of a significant group-by-condition interaction confirms that the results reported for this cohort remain valid and that our primary finding of a selective spatial memory deficit is robust to these differences in data retention.”

      (5) Modelling

      (a) Degrees of freedom, cross-validation, number of parameters.

      I appreciate the effort in introducing and testing different models. Models of increase in complexity and are based on different assumptions about the main drivers and mechanisms underlying the dependent variable. The models differ in the number of parameters. How are the differences in the number of parameters between models taken into account in the modelling analysis? Is there a cost associated with the extra parameters included in the more complex models?

      (b) Cross-validation and overfitting.

      Overfitting can occur when a model learns the training data but cannot generalize to novel datasets. Cross-validation is one approach that can be used to avoid overfitting. Was cross-validation (or other approaches) implemented in the fitting procedure against overfitting? Otherwise, the inference that can be derived from the modelled parameters can be limited.

      To address your concerns regarding model complexity and overfitting, we would like to clarify our use of Bayesian Model Selection (BMS). Unlike frequentist methods that often rely on cross-validation to assess generalisability, we used random-effects BMS based on the marginal likelihood (model evidence). This approach inherently implements Bayesian Occam’s Razor by integrating out the parameters. Under this framework, the use of the marginal likelihood for model selection provides a mathematically equivalent safeguard to frequentist cross-validation, as it evaluates the model's ability to generalise across the entire parameter space rather than just finding a maximum likelihood fit for the training data. Thus, models are penalised not just for the absolute number of parameters, but for their overall functional flexibility. A more complex model is only preferred if the improvement in model fit is substantial enough to outweigh this inherent penalty. The emergence of Model 7 as the winner (Bayes Factor = 6.11 against the next best alternative) confirms that its additional complexity is statistically justified.

      Furthermore, in this study we provided an external validation of these recovered parameters by demonstrating that they explain 62% of the variance in an independent, real-world, clinical task (ROCF copy). This empirical evidence confirms that our model captures robust mechanistic phenotypes rather than idiosyncratic noise. We have updated the Results and Discussion to explicitly state these.

      In Results: (p10)

      “We used random-effects Bayesian model selection to identify the most plausible generative model. This process relies on the marginal likelihood (model evidence), which inherently balances model fit against complexity—a principle often referred to as Occam’s razor.[26–28]”

      In Discussion: (p17)

      “Importantly, the risk of overfitting is mitigated by the Bayesian Model Selection framework; by utilising the marginal likelihood for model comparison, the procedure inherently penalises excessive model complexity and promotes generalisability.[26–28,43] This generalisability was further evidenced by the model's ability to predict performance on the independent ROCF task, confirming that these parameters represent robust mechanistic phenotypes rather than idiosyncratic fits to the initial dataset.”

      (6) n. of participants.

      (a) The authors write the following: "A total of healthy volunteers (21 young adults, mean age = 24.1 years; 21 older adults, mean age = 72.4 years) participated in this study. Their demographics are shown in Table 1. All participants were recruited locally in Oxford." However, Table 1 reports the data from more than 80 participants, divided into 4 groups. Details about the PD and AD groups are missing. Please clarify.

      We apologize for this lack of clarity in the text. We have rewrote and expand the “Participants” section and corrected Table 2 in the Methods section to reflect the correct number of participants.

      In Methods (p20):

      “A total of 87 participants completed the study: 21 young healthy adults (YC), 21 older healthy adults (EC), 23 patients with Parkinson’s disease (PD), and 22 patients with Alzheimer’s disease (AD). Their demographic and clinical details are summarised in Table 2. Initially, 90 participants were recruited (22 YC, 21 EC, 25 PD, 22 AD); however, three individuals (1 YC and 2 PD) were excluded from all analyses due to technical issues during data acquisition.

      All participants were recruited locally in Oxford, UK. None were professional artists, had a history of psychiatric illness, or were taking psychoactive medications (excluding standard dopamine replacement therapy for PD patients). Young participants were recruited via the University of Oxford Department of Experimental Psychology recruitment system. Older healthy volunteers (all >50 years of age) were recruited from the Oxford Dementia and Ageing Research (OxDARE) database.

      Patients with PD were recruited from specialist clinics in Oxfordshire. All had a clinical diagnosis of idiopathic Parkinson's disease and no history of other major neurological or psychiatric conditions. While specific dosages of dopamine replacement therapy (e.g., levodopa equivalent doses) were not systematically recorded, all patients were tested while on their regular medication regimen ('ON' state).

      Patients with PD were recruited from clinics in the Oxfordshire area. All had a clinical diagnosis of idiopathic Parkinson’s disease and no history of other major neurological or psychiatric illnesses. While all patients were tested in their regular medication ‘ON’ state, the specific pharmacological profiles—including the exact types of medication (e.g., levodopa, dopamine agonists, or combinations) and dosages—were not systematically recorded. The disease duration and PD severity were also un-recorded for this study.

      Patients with AD were recruited from the Cognitive Disorders Clinic at the John Radcliffe Hospital, Oxford, UK. All AD participants presented with a progressive, multidomain, predominantly amnestic cognitive impairment. Clinical diagnoses were supported by structural MRI and FDG-PET imaging consistent with a clinical diagnosis of AD dementia (e.g., temporo-parietal atrophy and hypometabolism).[70] All neuroimaging was reviewed independently by two senior neurologists (S.T. and M.H.).

      Global cognitive function was assessed using the Addenbrooke’s Cognitive Examination-III (ACE-III).[71] All healthy participants scored above the standard cut-off of 88, with the exception of one elderly participant who scored 85. In the PD group, two participants scored below the cut-off (85 and 79). In the AD group, six participants scored above 88; these individuals were included based on robust clinical and radiological evidence of AD pathology rather than their ACE-III score alone.”

      (b) As modelling results rely heavily on the quality of eye movements and eye traces, I believe it is necessary to report details about eye movement calibration quality and eye traces quality for the 4 experimental groups, as noisier data could be expected from naïve and possibly older participants, especially in case of clinical conditions. Potential differences in quality between groups should be discussed in light of the results obtained and whether these could contribute to the observed patterns.

      Thank you for pointing this out. We have revised the Methods about how calibration was done:

      (p27) “Prior to the experiment, a standard nine-point calibration and validation procedure was performed. Participants were instructed to fixate a small black circle with a white centre (0.5 degrees) as it appeared sequentially at nine points forming a 3 x 3 grid across the screen. Calibration was accepted only if the mean validation error was below 0.5 degrees and the maximum error at any single point was below 1.0 degree. If these criteria were not met, or if the experimenter noticed significant gaze drift between blocks, the calibration procedure was repeated. This calibration ensured high spatial accuracy across the entire display area, facilitating the precise monitoring of fixations on item frames and saccadic movements to the response colour wheel.”

      Moreover, as detailed in our response to Point 4, while the PD group exhibited lower compliance, there was no interaction between group and saccade condition for compliance (p = 0.151). This confirms that any noise or trial attrition was distributed evenly across experimental conditions. Consequently, the observed "saccade cost" (the difference in error between conditions) is not an artefact of unequal noise but represents a genuine mechanistic impairment in spatial updating. We have updated the Methods to clarify this distinction.

      Furthermore, our Bayesian framework explicitly estimates precision (random noise) as a distinct parameter from updating cost (saccade cost). This allows the model to partition the variance: even if a clinical group is "noisier" overall, this is captured by the precision parameter, ensuring it does not inflate the specific estimate of saccade-driven memory impairment.

      (7) Figure 5. I suggest reporting these results using boxplots instead of barplots, as the former gives a better overview of the distributions.

      We appreciate the suggestion to use boxplots to better illustrate data distributions. However, we have chosen to retain the current bar plot format due to the visual and statistical complexity of our 4 x 4 x 2 experimental design. Figure 5 represents 16 distinct distributions across four groups and four conditions for both location and colour measures; employing boxplots/violins for this density of data would significantly increase visual clutter and make the figure difficult to parse.

      Furthermore, the primary objective of this figure is to reflect the statistical analysis and illustrate group differences in overall performance and highlight the specific finding that patients with AD were significantly more impaired across all conditions compared to YC, EC, and PD groups. Our statistical focus remains on the mean effects—specifically the significant main effect of group (F(3, 318) = 59.71, p < 0.001) and the critical null-interaction between group and condition (p = 0.90). The error measure most relevant to these comparisons is the standard error of the mean (SEM), rather than the interquartile range (IQR). We think that bar plots provide the most straightforward and scannable representation of these mean differences and the consistent pattern of decay across cohorts for the final manuscript layout.

      To address the reviewer’s request for distributional transparency, we have provided a version of Figure 5 using grouped boxplots in the supplementary material (Supplementary figure 2). We note, however, that the spread of raw data points in these plots does not directly reflect the variance associated with our within-subject statistical comparisons.

      (8) Results specificity, trans-saccadic integration and ROCF. The authors demonstrate that the derived model parameters account for a significant amount of variability in ROCF performance across the experimental groups tested (Figure 8A). However, it remains unclear how specific the modelling results are with respect to the ROCF.

      The ROCF is generally interpreted as a measure of constructional ability. Nevertheless, as with any cognitive test, performance can also be influenced by more general, non-specific abilities that contribute broadly to test success. To more clearly link the specificity between modelling results and constructional ability, it would be helpful to include a test measure for which the model parameters would not be expected to explain performance, for example, a verbal working memory task.

      I am not necessarily suggesting that new data should be collected. However, I believe that the issue of specificity should be acknowledged and discussed as a potential limitation in the current context.

      We appreciate this important point regarding the discriminant validity of our findings. We agree that cognitive performance in clinical populations is often influenced by a general "g-factor" or non-specific executive decline. However, we chose the ROCF Copy task specifically because it is a hallmark clinical measure of constructional ability that effectively serves as a real-world transsaccadic task, requiring participants to integrate spatial information across hundreds of saccades between the model figure and the drawing surface.

      To address the reviewer’s concern regarding specificity, we leveraged the fact that all participants completed the ACE-III, which includes a dedicated verbal memory component (the ACE Memory subscale). We conducted a partial correlation analysis and found that the relationship between transsaccadic working memory and ROCF copy performance remains highly significant (rho = -0.46, p < 0.001), even after controlling for age, education, and the ACE-III Memory subscale score. This suggests that the link between transsaccadic updating and constructional ability is mechanistically specific rather than a byproduct of global cognitive impairment. We have substantially revised the Discussion to highlight this link and the supporting statistical evidence.

      We first updated the last paragraph of Introduction:

      “Finally, by linking these mechanistic parameters to a standard clinical measure of constructional ability (the Rey-Osterrieth Complex Figure task), we demonstrate that transsaccadic updating represents a core computational phenotype underpinning real-world visuospatial construction in both health and neurodegeneration.”

      The new section in Discussion highlighting the ROCF copy link:

      “Importantly, our computational framework establishes a direct mechanistic link between trassaccadic updating and real-world constructional ability. Specifically, higher saccade and angular encoding errors contribute to poorer ROCF copy scores. By mapping these mechanistic estimates onto clinical scores, we found that the parameters derived from our winning model explain approximately 62% of the variance in constructional performance across groups. These findings suggest that the computational parameters identified in the LOCUS task represent core phenotypes of visuospatial ability, providing a mechanistic bridge between basic cognitive theory and clinical presentation.

      This relationship provides novel insights into the cognitive processes underlying drawing, specifically highlighting the role of transsaccadic working memory. Previous research has primarily focused on the roles of fine motor control and eye-hand coordination in this skill.[4,50–55] This is partly because of consistent failure to find a strong relation between traditional memory measures and copying ability.[4,31] For instance, common measures of working memory, such as digit span and Corsi block tasks, do not directly predict ROCF copying performance.[31,56] Furthermore, in patients with constructional apraxia, these memory performance often remain relatively preserved despite significant drawing impairments.[56–58] In literature, this lack of association has often been attributed to “deictic” visual-sampling strategies, characterised by frequent eye movements that treat the environment as an external memory buffer, thereby minimising the need to maintain a detailed internal representation.[4,59] In a real-world copying task, the ROCF requires a high volume of saccades, making it uniquely sensitive to the precision of the dynamic remapping signals identified here. Recent eye-tracking evidence confirms that patients with AD exhibit significantly more saccades and longer fixations during figure copying compared to controls, potentially as a compensatory response to trassaccadic working memory constraints.[56] This high-frequency sampling—averaging between 150 and 260 saccades for AD patients compared to approximately 100 for healthy controls—renders the task highly dependent on the precision of dynamic remapping signals.[56] We also found that the relationship between transsaccadic working memory and ROCF performance remains highly significant (rho = -0.46, p < 0.001), even after controlling for age, education, and ACE-III Memory subscore. Consequently, transsaccadic updating may represent a discrete computational phenotype required for visuomotor control, rather than a non-specific proxy for global cognitive decline.[58]

      In other words, even when visual information is readily available in the world, the act of drawing performance depends critically on working memory across saccades. This reveals a fundamental computational trade-off: while active sampling strategies (characterised with frequent eye-hand movements) effectively reduce the load on capacity-limited working memory, they simultaneously increase the demand for precise spatial updating across eye movements. By treating the external world as an "outside" memory buffer, the brain minimises the volume of information it must hold internally, but it becomes entirely dependent on the reliability with which that information is remapped after each eye movement. This perspective aligns with, rather contradicts, the traditional view of active sampling, which posits that individuals adapt their gaze and memory strategies based on specific task demands.[3,60] Furthermore, this perspective provides a mechanistic framework for understanding constructional apraxia; in these clinical populations, the impairment may not lie in a reduced memory "span," but rather in the cumulative noise introduced by the constant spatial remapping required during the copying process.[58,61]

      Beyond constructional ability, these findings suggest that the primary evolutionary utility of high-resolution spatial remapping lies in the service of action rather than perception. While spatial remapping is often invoked to explain perceptual stability,[11–13,15] the necessity of high-resolution transsaccadic memory for basic visual perception is debated.[13,62–64] A prevailing view suggests that detailed internal models are unnecessary for perception, given the continuous availability of visual information in the external world.[13,44] Our findings support an alternative perspective, aligning with the proposal that high-resolution transsaccadic memory primarily serves action rather than perception.[13] This is consistent with the need for precise localisation in eye-hand coordination tasks such as pointing or grasping.[65] Even when unaware of intrasaccadic target displacements, individuals rapidly adjust their reaching movements, suggesting direct access of the motor system to remapping signals.[66] Further support comes from evidence that pointing to remembered locations is biased by changes in eye position,[67] and that remapping neurons reside within the dorsal “action” visual pathway, rather than the ventral “perception” visual pathway.[13,68,69] By demonstrating a strong link between transsaccadic working memory and drawing (a complex fine motor skill), our findings suggest that precise visual working memory across eye movements plays an important role in complex fine motor control.”

      We are deeply grateful to the reviewers for their meticulous reading of our manuscript and for the constructive feedback provided throughout this process. Your insights have significantly enhanced the clarity and rigour of our work.

      In addition to the changes requested by the reviewers, we wish to acknowledge a reporting error identified during the revision process. In the original Results section, the repeated measures ANOVA statistics for YC included Greenhouse-Geisser corrections, and the between-subjects degrees of freedom were incorrectly reported as within-subjects residuals. Upon re-evaluation of the data, we confirmed that the assumption of sphericity was not violated; therefore, we have removed the unnecessary Greenhouse-Geisser corrections and corrected the degrees of freedom throughout the Results and Methods sections. We have ensured that these statistical updates are reflected accurately in the revised manuscript and that they do not alter the significance or interpretation of any of our primary findings.

      We hope that these revisions address all the concerns raised and provide a more robust account of our findings. We look forward to your further assessment of our work.

    1. Consolidation de la Co-éducation autour de l'Enfant et de l'Adolescent : Synthèse et Orientations

      Résumé Analytique

      La co-éducation ne doit pas être perçue comme un simple souhait ou un idéal lointain, mais comme une réalité de fait.

      Tout enfant ou adolescent évolue au sein d'un écosystème d'éducateurs multiples (famille, école, structures de loisirs, médias).

      L'enjeu majeur n'est pas de fusionner ces rôles, mais de consolider cet écosystème en préservant la spécificité de chaque acteur tout en assurant une cohérence globale.

      Cette cohérence repose sur des projets communs, des comportements éducatifs équilibrés — évitant l'aléa et la rigidité — et une collaboration étroite face aux défis sociétaux modernes, tels que la gestion des outils numériques.

      1. La Co-éducation : Un Écosystème de Fait

      La co-éducation est une réalité intrinsèque au développement de l'enfant. Dès lors qu'un individu sort de son isolement, il est exposé à une multiplicité d'influences éducatives qui constituent son environnement quotidien.

      Une pluralité d'acteurs : L'éducation n'est pas le seul fait des parents.

      Elle inclut l'école, les clubs de loisirs, la famille élargie, les amis, ainsi que les influences médiatiques (télévision, internet, réseaux sociaux).

      La notion d'écosystème : Cet ensemble d'interactions forme un cadre dans lequel l'enfant évolue.

      Les différents éducateurs s'y complètent de manière de facto, exerçant chacun une influence sur la construction du sujet.

      2. Le Principe de Spécificité des Rôles

      Un pilier fondamental de la co-éducation réussie est le respect des domaines de compétence et des vocations de chaque acteur. La collaboration ne doit pas mener à une confusion des rôles.

      Le maintien des identités : Chaque éducateur doit garder sa spécificité. Les parents n'ont pas vocation à devenir des enseignants, et les enseignants ne doivent pas s'immiscer dans la sphère privée familiale.

      Différenciation des méthodes : Un animateur de loisirs peut aborder des concepts académiques (comme la proportionnalité), mais il doit le faire selon des modalités propres à son cadre, et non en reproduisant strictement les méthodes scolaires.

      La complémentarité plutôt que l'imitation : L'objectif de la rencontre entre adultes n'est pas de chercher à se ressembler ou à agir de manière identique, mais d'organiser une complémentarité efficace.

      3. Leviers de Cohérence Éducative

      Si la spécificité est de mise, elle ne doit pas conduire à l'incohérence.

      Le document souligne deux moyens principaux pour harmoniser l'action des adultes :

      La mise en œuvre de projets communs

      La cohérence peut naître d'actions concrètes impliquant simultanément plusieurs sphères de la vie de l'enfant.

      Exemple : Les classes découvertes ou les sorties scolaires qui associent parents, enseignants et animateurs du périscolaire autour d'un objectif unique.

      L'harmonisation des comportements éducatifs

      Il s'agit de tisser un système au service du développement de l'enfant pour l'aider à comprendre le monde et à devenir un sujet autonome.

      Un système éducatif sain se définit par sa structure :

      | Type de milieu | Caractéristiques | Impact sur l'enfant | | --- | --- | --- | | Milieu Aléatoire | Imprévisible. Les réactions des adultes (sanction ou félicitation) ne sont pas constantes. | L'enfant ne peut pas anticiper les conséquences de ses actes. | | Milieu Rigide | Règles définies à l'avance, immuables et non discutables. Tout est enfermé dans des normes strictes. | Absence de dialogue et de remise en question. | | Milieu Équilibré | Présence d'un cadre sécurisant, mais flexible. Les règles peuvent faire l'objet de discussions selon les événements. | Favorise l'émergence de la réflexivité et du dialogue entre enfant et adulte. |

      4. Un Défi Partagé : La Gestion du Numérique

      La co-éducation est particulièrement sollicitée face aux problématiques sociétales complexes, l'utilisation du téléphone portable et des outils numériques en étant l'exemple le plus prégnant.

      L'impossibilité d'une solution isolée : Ni les parents, ni les enseignants, ni les éducateurs spécialisés ne peuvent légiférer ou résoudre seuls la question des écrans.

      La nécessité d'une "législation" cohérente : Les adultes ont tout intérêt à se concerter pour adopter des comportements et des règles cohérents autour de cet objet.

      La solution réside dans la concertation et l'établissement d'une ligne de conduite partagée au sein de l'écosystème.

      Conclusion

      La consolidation de la co-éducation repose sur un paradoxe constructif : travailler ensemble tout en restant différent.

      La rencontre entre les adultes n'est pas une fin en soi, mais un moyen de structurer un environnement prévisible et réflexif pour l'enfant.

      En instaurant un dialogue constant et en s'accordant sur des comportements cohérents face aux enjeux modernes, les éducateurs favorisent un écosystème propice à l'autonomie et au développement global de l'enfant et de l'adolescent.

    1. he 2000–01 California energy crisis gave impetus to the next wave of pilots with time-varying rates. In addition toTOU rates, they featured dynamic pricing designs

      There is a distinction between "dynamic rates" and "TOU rates." Dynamic rates change fluidly, while TOU rates are set months or years in advance. TOU rates are the modern standard for pricing in CA and the US more generally.

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