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  2. www.sciencedirect.com www.sciencedirect.com
    Chemotherapy enhances cancer vaccine efficacy and expands stem-like TCF1+CD8+ T cells
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    sciencedirect.com/science/article/pii/S1535610826004253
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    Caffeic acid phenethyl ester inhibits adipogenic differentiation in association with INSR suppression and AMPK activation
    10
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  4. drive.google.com drive.google.com
    someTitle
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    1. yasinwali08 05 Oct 2026
      Sam stated his theory of high school as, “The way I see it, theywere just giving a reason to keep a bunch of kids off the streetsuntil they’re eigh teen or whatever.”

      A bit cynical, but he did go to college so he must believe it helps.

    2. yasinwali08 05 Oct 2026
      The consensus among the stu-dents in the sample was that school is definitely not fun.

      Thats sad but true, if school is something you go just to endure it, somethings wrong.

    3. yasinwali08 05 Oct 2026
      “I want to at least get the associate,so if something happens . . . at least I will have the associate’sdegree. I’ll have something.

      A credential as a safety net is a pretty good defense.

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  5. escholarship.org escholarship.org
    Mirror, Mirror on the Wall: Preventing Shadow Boxing through Habituation - A Pilot Study
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    1. Tcampbell_ 05 Oct 2026
      In the wild, a number of species (e.g., Dusky Sunbird, Cinnyrus fuscus; pin-tailed whydah, Viduamacroura; White-bellied Sunbird, Cinnyris talatala) perceive their self-images as threatening conspecificsover multiple years (Roerig, 2013). For welfare purposes, mirrors have been provided in captive social species(e.g., as sensory or social enrichments). However, mirror exposure can lead to adverse consequences (increaseof submissive or aggressive behaviours; de Groot & Cheyne, 2016).

      I find it interesting how they mentioned another bird and its reaction to seeing self images and not the bird they were studying. Does the hornbill see its self-image the same was and is that why it is breaking windows and mirrors or is it just out of curiosity? (At least I dont remember reading that part)

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    escholarship.org/uc/item/68c9q9hq
  6. stylo.ecrituresnumeriques.ca stylo.ecrituresnumeriques.ca
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    1. MLib 05 Oct 2026
      Les choix initiaux de collecte, de catégorisation et de couverture sont intégrés dans le comportement du modèle, sans toujours être perceptibles dans les résultats.

      C'est bien ça que tu voulais dire juste au-dessus par "représentations internes" ?

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  7. uw.pressbooks.pub uw.pressbooks.pub
    Orthographic Projection
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    1. Synjynmw 05 Oct 2026
      When multiple edges project to the same line on the drawing, the line type is determined by the following precedence: (1) visible lines, (2) hidden lines, (3) center lines.

      This can be seen clearly in the last example, when selecting the correct orthographic views. It is important to remember what it would look like from above, as well as below, and how that might change any dotted lines that were visible from one perspective.

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  8. www.youtube.com www.youtube.com
    PROFS ABSENTS, PARCOURSUP, PRESSION : la parole aux LYCÉENS
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    1. tanemika 05 Oct 2026

      Document de Synthèse : Crise de l'Éducation, Mal-être Lycéen et Pistes de Refonte du Système Éducatif

      Résumé Exécutif

      Ce document propose une synthèse analytique des échanges issus de l'émission de France Culture consacrée à la crise du système éducatif français, à la parole des lycéens mobilisés et aux conclusions du rapport de l'Académie des Sciences co-dirigé par la mathématicienne Laure Saint-Raymond.

      La situation actuelle du milieu scolaire secondaire se caractérise par des tensions structurelles majeures : pénurie endémique d'enseignants, locaux détériorés, dégradation de la santé mentale des élèves (touchant environ 30 % d'entre eux) et surcharge des emplois du temps.

      Les réformes récentes du baccalauréat et le fonctionnement du dispositif Parcoursup ont instauré un climat de compétition perçu comme préjudiciable à la curiosité intellectuelle et à l'apprentissage réel, renforçant les inégalités sociales et territoriales, notamment dans des départements comme la Seine-Saint-Denis (93).

      Face à ces constatations, les mobilisations lycéennes de la fin septembre (environ 400 lycées perturbés le 29 septembre) mettent en lumière une volonté d'être entendus, malgré des épisodes de violences et de répression policière dénoncés par les différents acteurs.

      En parallèle, l'Académie des Sciences propose une vision à long terme articulée autour de cinq leviers fondamentaux, appelant à repenser la notion de temps scolaire, à concilier égalité et singularité des parcours, et à investir prioritairement dans le bâti, le personnel médico-social, la revalorisation enseignante et l'enseignement primaire.

      1. État des Lieux et Diagnostic du Terrain : La Crise du Quotidien

      Pénuries de Personnel et Vétusté des Infrastructures

      Les témoignages d'élèves du lycée Le Corbusier à Aubervilliers (Seine-Saint-Denis) ainsi que les rapports institutionnels mettent en évidence des défaillances quotidiennes :

      • Absence d'enseignants : Le manque de professeurs remplaçants est qualifié de problème endémique, particulièrement marqué dans l'académie de Créteil.

      Des situations extrêmes sont rapportées, telles que l'absence totale de professeur de philosophie durant une année entière d'épreuve du baccalauréat, ou des manques répétuits dès l'école primaire et le collège.

      • Surcharge des classes et locaux : Des effectifs dépassant régulièrement 30 à 35 élèves par classe s'accompagnent de dégradations matérielles graves (plafonds qui s'effondrent, installations sanitaires défectueuses).

      • Carence médico-sociale : Les personnels de santé (infirmières scolaires, psychologues) et d'encadrement (CPE) sont jugés très insuffisants ou totalement débordés.

      Cette carence est critique alors que la santé mentale concerne près d'un élève sur trois et que certains jeunes souffrent de pathologies physiques (problèmes respiratoires/pulmonaires).

      Inégalités Sociales et Territoriales

      La crise éducative accentue les fractures préexistantes :

      • L'écart territorial : Des différences flagrantes de conditions d'études sont constatées entre les lycées de Seine-Saint-Denis et d'autres établissements situés à quelques stations de métro dans Paris intra-muros.

      • Le rôle du milieu familial : L'absence de prise en compte des réalités sociales (parents ne maîtrisant pas le français, incapacité à aider pour les devoirs à la maison) brise la promesse républicaine d'égalité.

      L'école traite tous les élèves de manière identique alors que leurs situations de départ sont profondément inégales.

      • Mobilité sociale : Selon les données de l'OCDE, la France figure parmi les pays où l'ascenseur social et la mobilité intergénérationnelle sont les plus faibles.

      2. Impact des Réformes : Pression Scolaire, Parcoursup et Gestion du Temps

      [Réforme du Bac 2019] ──> [Contrôle Continu Permanent] ──> [Disparition du "Droit à l'Erreur"] │ [Emplois du temps lourds] ──> [Logique de Rang & Compétition] ◄────┘ (8h00 - 18h40 + devoirs) (Pression Parcoursup)

      Le Poids du Contrôle Continu et de Parcoursup

      L'instauration du contrôle continu dès la classe de Première modifie fondamentalement le rapport aux savoirs :

      • Disparition du droit à l'erreur : Les élèves expriment un sentiment de pression permanente où chaque évaluation compte pour le dossier Parcoursup, inhibant le plaisir d'apprendre au profit de la recherche de la note ultime et du classement.

      • Mise en compétition : Parcoursup met en rivalité les élèves à l'échelle nationale, pénalisant ceux issus de lycées moins cotés ou de séries technologiques et générales non favorisées.

      • Perte de sens : L'apprentissage n'est plus motivé par la curiosité ou l'acquisition de connaissances, mais par la maximisation du rang académique.

      Organisation des Emplois du Temps

      Les emplois du temps sont jugés inadaptés au rythme biologique des adolescents :

      • Journées à rallonge : Des amplitudes horaires allant de 8h00 à 18h40, suivies de 1 à 2 heures de devoirs à domicile, restreignent l'accès aux activités sportives et culturelles hors temps scolaire.

      • Emplois du temps "en gruyère" : La multiplication des enseignements de spécialité créés par la réforme de 2019 se heurte au manque de salles et de créneaux disponibles, générant de longs trous d'inactivité forcée dans la journée sans condensabilité des heures de cours.

      3. Synthèse du Rapport de l'Académie des Sciences : "L'École à bout de souffle"

      Le rapport co-dirigé par la mathématicienne Laure Saint-Raymond s'appuie sur une consultation élargie (ministère, Cour des comptes, collectivités territoriales, secteur médico-social, élèves et familles) et s'inscrit dans un temps long (institution appartisante de 360 ans).

      Les Paradoxes du Système Éducatif Français

      • Égalité contre Uniformité : Le rapport souligne la confusion entre l'égalité (offrir à tous les mêmes chances de s'épanouir selon leurs aspirations) et l'uniformité (imposer un parcours identique à tous).

      L'école doit pouvoir prendre en compte la singularité et les codes culturels de chaque élève sans renoncer à sa mission de cohésion sociale et de communauté.

      • Baisse de l'excellence et des performances : Les enquêtes PISA et les études nationales révèlent une baisse générale du niveau, particulièrement marquée en mathématiques, y compris chez les meilleurs élèves.

      Le rapport pointe des difficultés majeures de compréhension sur les textes longs et une perte globale d'attention et de motivation.

      Les 5 Leviers de Transformation Proposés

      • Reconnaître la singularité de l'élève : Accompagner la trajectoire propre de chacun par du tutorat et des parcours modulables, tout en maintenant l'exigence intellectuelle.

      • Favoriser l'émergence de collectifs : Créer une dynamique de projet et de travail en groupe au sein des établissements.

      • Relâcher les contraintes du temps : Assouplir la directivité des programmes pour laisser le temps d'assimiler, d'expérimenter et d'apprendre de ses erreurs.

      Redonner de la latitude aux enseignants.

      • Faire évoluer l'accompagnement et l'orientation : Varier les formats d'enseignement et offrir un soutien réel avant le choix des options pour éviter les choix par défaut ou purement stratégiques.

      • Développer le pouvoir d'agir : Accorder plus d'autonomie et de capacités d'initiative aux acteurs de terrain (élèves, enseignants, établissements).

      Priorités Budgétaires et Macro-Structurelles

      Pour refonder l'institution éducative, le rapport identifie quatre chantiers financiers prioritaires :

      • Revalorisation des enseignants : Augmenter la rémunération pour attractiver la profession et compenser la complexification de leurs missions.

      • Rénovation du bâti scolaire : Moderniser les deux tiers des bâtiments scolaires (transition énergétique, normes parasismiques/cycloniques, sécurité structurelle et réaménagement de l'espace pour les cours en petits groupes).

      • Renforcement des équipes médico-sociales : Recruter massivement des infirmières et psychologues scolaires.

      • Périscolaire et Primaire : Intégrer et harmoniser le périscolaire dès le primaire pour réduire les inégalités initiales.

      Le rapport rappelle que la France accuse un retard net de financement dans l'enseignement primaire par rapport à la moyenne des pays de l'OCDE.

      4. Tensions du Mouvement Lycéen et Dialogue Social

      Au cours des mobilisations de fin septembre (environ 400 lycées bloqués), la question du mode d'action et du dialogue social est ressortie avec acuité.

                    ```
              ┌─── Répression policière / Usage de la force
              │

      Tensions sur ─────┼─── Violences & Dégradations (Mortiers, poubelles brûlées) le terrain │ └─── Bilan officiel : 76 blessés (dont 18 personnels) ```

      Le Bilan Officiel des Violences

      Selon les déclarations du directeur général de l'enseignement scolaire Édouard Geffray (au 30 septembre) :

      • 76 personnes ont été blessées au sein des établissements scolaires depuis le début de la mobilisation, dont 18 personnels de l'Éducation nationale.

      • Des actes graves ont été répertoriés : agressions de proviseurs par tirs tendus de mortiers d'artifice, professeurs frappés ou pourchassés, et grave blessure à la main subie par un élève manipulant un mortier.

      La Position des Élèves Mobilisés

      • Répression contre Pacifisme : Les représentants lycéens (Antoine et Livie) rappellent que le durcissement du mouvement est une réaction à la répression policière subie lors des premières manifestations.

      Ils prônent la non-violence et la structuration syndicale pour porter pacifiquement leurs revendications.

      • Rupture du dialogue : Les élèves expriment un sentiment d'épuisement face à l'absence chronique d'écoute institutionnelle.

      Ils dénoncent le fait qu'il faille recourir au blocage pour que leurs conditions de travail élémentaires (locaux salubres, présence de professeurs) soient enfin prises en considération par la société.

      5. Synthèse des Citations Clés

      | Intervenant | Thématique | Citation Explicative / Propos Clé | | --- | --- | --- | | Livie (Élève de 1ère) | Perte de sens & Parcoursup | "On va à l'école pour être meilleur, mais pas pour agrandir notre éventail de connaissances. Ce fait de ne plus avoir droit à l'erreur, c'est un frein à l'éducation." | | Antoine (Élève de Tle) | Emploi du temps & Santé | "Commencer à 8h et finir à 18h40 plusieurs fois par semaine, ce n'est pas un rythme normal à suivre pour un adolescent en croissance ni pour l'apprentissage." | | Laure Saint-Raymond (Académie des Sciences) | Temps & Apprentissage | "Quand vous gavez les gens, ils ont juste envie de vomir. Si on veut allumer des étoiles dans les yeux des élèves, il faut leur laisser du temps gratuit et le droit de se tromper." | | Laure Saint-Raymond (Académie des Sciences) | Nature de l'École | "L'école est une école de la République, de la fraternité et de l'égalité, mais elle est devenue un système fondé sur la sélection et la compétition." | | Édouard Geffray (Rapporté) | Violences en marge | "Nous en sommes à 76 personnes blessées dans les établissements \[...\], on est quand même loin d'une mobilisation sereine." | | Livie (Élève de 1ère) | Sentiment d'abandon | "On est saoulés. On en a marre que ce soit tout le temps à nous de sonner la cloche et de dire 'regardez-nous, ça va pas', et qu'on nous réponde 'vous êtes jeunes, vous comprenez pas'." |

      lycée mobilisations 2026 France Culture Radio France éducation manque
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  9. www.biorxiv.org www.biorxiv.org
    OCRL regulates lysosomal function and endolysosomal homeostasis in Drosophila nephrocytes
    4
    1. EMBOpress 05 Oct 2026

      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

      • *

      __1. __General Statements [optional]

      This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

      • *

      We express our appreciation to all three reviewers for their detailed comments on our manuscript and for highlighting that the experiments are well designed, manuscript well written, noting the clarity of the data sets and methods of quantification and analysis.

      We thank reviewers for appreciating our work on LS using the Drosophila model and noting that the compartment-targeting approach is a potentially valuable approach. We are of the view that understanding a human disease is a challenging task that often requires the use of multiple model systems and cell types, each of which has its strengths and limitations. Sometimes, there are species and cell type specific differences in observations on cellular and molecular processes, and we believe such differences are just as informative in deciphering underlying as are commonalities across model systems.

      We are delighted that reviewers have noted conceptually important conclusions that arise from our study with implications both for understanding the sub-cellular mechanism of disease in LS but also for the understanding the overall wiring of the endo-lysosomal system (a primary role for lysosomes and feedback from late-endo-lysosomal homeostasis for regulating upstream endocytic function). Yet, we thank the reviewers for highlighting some aspects of our data that need to be strengthened to fully realize the potential of these conceptual advances. Below, we provide a pointwise response to reviewer comments and in almost all cases propose pragmatic experiments or analysis to address each point. We believe that when completed, the revised manuscript will be a substantive advance in the understanding of the cell biology of LS, in lysosomal biology and its role in the overall wiring of the endosomal system.

      __2. __Description of the planned revisions

      Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

      • *

      Reviewer #1

      In this study the authors explore the role of the Drosophila orthologue of the Lowe syndrome protein OCRL (called dOCRL) in nephrocytes, the functional equivalent of the mammalian kidney. Using genetic knockout and complementation approaches it is shown that dOCRL is required for the clearance of the toxic compound silver nitrate from nephrocytes. This effect is phenocopied by loss of Rab7 and can be rescued by a constitutively active form of Rab7, suggesting involvement of late endosomal or lysosomal compartments. This is supported by experiments showing disrupted lysosomal function upon dOCRL loss. Loss of dOCRL also causes defects earlier in the endocytic pathway, with reduced uptake of the endocytic tracers dextran and mBSA into nephrocytes, and a loss of early endosomal compartments. Interestingly, re-expression of dOCRL targeted to the lysosome rescues the defects in both endosome and lysosome function, suggesting that the defects observed are primarily due to dOCRL function at the lysosome.

      This is a very nicely done study. The data are clear and support the authors' conclusions. The means of analysis and the quantitation of the data are appropriate. There are a few things that could be addressed in a revised version which are indicated below:

      We thank Reviewer #1 for the positive assessment of our study and for the constructive suggestions

      1.) The ability of lysosome-targeted dOCRL to rescue the phenotypes is interesting. Can the authors exclude the possibility that there is residual cytosolic dOCRL in the cells that can transiently associate with other organelles e.g. early endosomes to drive the phenotypic rescue? There are experiments described in the discussion about targeting dOCRL to early endosomes not rescuing the phenotypes. Currently this is mentioned as data not shown. It should be included.

      Response: We thank the reviewer for this important comment. We have previously validated the lysosomal targeting sequence (LTS) in S2R+ cells by demonstrating its colocalization with established lysosomal markers, including LAMP1 and LysoTracker. These data confirm that the LTS directs transgenes specifically to the lysosomal compartment. We will include these validation data in the revised manuscript.

      To further exclude the possibility that dOCRL localisation to organelles other than lysosomes contributes to the observed phenotypic rescue, we will include rescue experiments using dOCRL::GFP construct specifically targeted to the early endosomal compartment. This early endosome compartment specific construct does not rescue the defects observed in dOCRLKO nephrocytes.

      In addition we will perform colocalization analyses of LTS::dOCRL::GFP with an early endosomal marker. These experiments will help confirm the specificity of LTS-mediated targeting and rule out significant localisation of the construct to the early endosomal compartment.

      2.) The results with AgNO3 are confusing. Uptake into nephrocytes is not affected by loss of dOCRL but is affected by other manipulations that disrupt endosomal trafficking (suppl table 1). Yet, loss of dOCRL affects cellular uptake of dextran and mBSA, which rely on endocytosis. More explanation needs to be provided on what we know about AgNO3 uptake into nephrocytes. Is it by endocytosis, as the table in the supplementary table would seem to indicate? In that case, why would AgNO3 uptake not be affected by dOCRL loss? Also, what underlies the clearance of AgNO3 taken up into nephrocytes? If it is already in the cells, then does it leave by exocytosis? Or does it enter and leave cells through transporters? How does dOCRL affect the clearance of AgNO3 from the cells i.e. through what mechanism? Filtration of AgNO3 is also mentioned but not fully explained. How does filtration and clearance of AgNO3 relate to the processes that occur in the mammalian kidney, where filtration of blood is performed by the glomerulus, while clearance is in the proximal tubules, which are the region affected in Lowe syndrome?

      Response: We thank the reviewer for this important comment. In the revised manuscript, we will provide a more detailed explanation of the basis of the AgNO₃ uptake and clearance assay, drawing both from our genetic screen and from previous studies of nephrocyte endocytosis. Our data support the conclusion that AgNO₃ uptake occurs through endocytic pathways. Specifically, inhibition of key endocytic regulators, including Rab5 and Shibire (Dynamin), completely abolishes AgNO₃ uptake in nephrocytes. In contrast, loss of dOCRL reduces AgNO₃ uptake but does not eliminate it entirely, indicating that endocytic activity is impaired but not fully blocked in the mutant.

      It is important to distinguish between the uptake assays used in this study. Dextran and mBSA uptake assays are ex vivo measurements that assess endocytic uptake over relatively short time periods. In contrast, the AgNO₃ uptake and clearance assay is an in vivo functional assay performed over approximately 80 hours of larval development, thereby capturing both uptake and long-term cargo processing and clearance.

      When AgNO₃ uptake is examined over shorter time intervals (10-15 hours), we observe reduced accumulation in dOCRLKO nephrocytes relative to controls, consistent with impaired endocytic uptake. However, as the assay progresses, defects in cargo clearance become increasingly apparent. After approximately 36 hours, AgNO₃ begins to accumulate in dOCRLKO nephrocytes, whereas it is efficiently cleared from control cells. Consequently, the mutant nephrocytes display higher AgNO₃ retention at later time points despite their reduced initial uptake. This observation is consistent with our mBSA pulse-chase experiments. While dOCRLKO nephrocytes show reduced cargo uptake at early time points, they exhibit increased retention of mBSA during extended chase periods (e.g., 40 minutes), supporting a defect in cargo degradation and/or clearance rather than enhanced uptake.

      To further strengthen these conclusions and clarify the interpretation of the AgNO₃ assay, we will include the short-term (10-hour) AgNO₃ uptake data in the revised manuscript. These additional data will help distinguish the effects of dOCRL loss on initial uptake from its effects on subsequent cargo processing and clearance.

      The precise mechanism by which AgNO₃ is cleared from nephrocytes remains incompletely understood and was one of the primary motivations for conducting our genetic screen. Given the possibility that clearance could occur through exocytosis, we included several candidate genes involved in late endosomal and lysosomal exocytic pathways in our screen. However, either because of insufficient RNAi-mediated knockdown efficiency or because depletion of these genes produced severe nephrocyte morphological defects, we were unable to definitively identify the molecular machinery responsible for AgNO₃ clearance. Nevertheless, our findings provide evidence that the late endolysosomal pathway plays an important role in this process. In particular, the ability of constitutively active Rab7 (Rab7CA) to rescue the dOCRLKO phenotype suggests that proper functioning of the late endosomal/lysosomal compartment is critical for AgNO₃ processing and clearance. While the exact mechanism remains to be established, our data are consistent with a model in which cargo clearance is mediated through late endolysosomal trafficking pathways.

      It is important to consider the unique physiology of Drosophila nephrocytes. Unlike the mammalian renal system, where filtration occurs at the glomerulus and reabsorption is performed by proximal tubule cells (PTCs), nephrocytes perform both of these functions within a single cell type. Hemolymph components are first filtered in a size-selective manner through the basement membrane and slit diaphragms and are subsequently internalised by endocytosis. The internalised cargo is then processed through the endolysosomal pathway, functionally resembling the cargo-handling and processing activities of mammalian PTCs. Because nephrocytes play a central role in maintaining hemolymph homeostasis and clearing circulating toxins, the AgNO₃ uptake and clearance assay provides a physiologically relevant functional readout of nephrocyte activity. We therefore employed this assay to assess nephrocyte function in the absence of dOCRL. The defects in AgNO₃ handling observed in dOCRLKO nephrocytes support the conclusion that dOCRL is required for efficient endolysosomal cargo processing and clearance, thereby contributing to the maintenance of nephrocyte homeostasis. We will expand this discussion in the revised manuscript to provide greater clarity regarding both the rationale for using the AgNO₃ assay and its inherent limitations.

      3.) The data overall are clear and convincing, but Fig 3C is not. There does not appear to be any reduction in mBSA signal in the dOCRL image shown.

      Response: We will replace the figure with the one representing mean values.

      4.) Discussion could be more expansive eg dOCRL versus mammalian OCRL-clathrin binding, localisation etc... Response: We thank the reviewer for their suggestion. We will include the comparison of Drosophila and mammalian OCRL in the discussion of our manuscript.

      5.) The figures need to appear in the correct order in the text. Currently they are presented in the text in a jumbled way. Response: We thank the reviewer for their suggestion. We will correct this in the revised manuscript.

      6.) References are missing in quite a few places in the text. There are numerous instances of statements being made without the references to support them. Response: We thank the reviewer for their suggestion. We will correct this in the revised manuscript.

      7.) There are a few minor grammatical errors. Response: We thank the reviewer for their suggestion. We will correct this in the revised manuscript.

      Reviewer #1 (Significance (Required)):

      This is a very nicely done study. The data are clear and support the authors' conclusions. The means of analysis and the quantitation of the data are appropriate. Response: We sincerely thank the reviewer for their positive evaluation of our manuscript and for their constructive feedback.

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

      Summary The authors establish germline and nephrocyte-specific dOCRL loss-of-function models in Drosophila. They report that dOCRL deficiency is associated with increased plasma-membrane PI(4,5)P₂, decreased plasma-membrane PI4P, reduced dextran and BSA uptake, alterations in early- and late-endosomal markers, lysosomal expansion/dysfunction, and delayed AgNO₃ clearance. They further claim that a late-endolysosomal-targeted dOCRL construct rescues AgNO₃ retention, Cathepsin L accumulation, Rabenosyn-5 staining, and BSA uptake, whereas a presumably phosphatase-dead construct fails to normalize Cathepsin L. The authors conclude that lysosomal OCRL activity is the primary requirement for endolysosomal homeostasis and that the endocytic abnormalities are secondary to lysosomal dysfunction. The Drosophila model and compartment-targeting approach are potentially valuable. However, the central conclusion is not adequately supported because the localization and biochemical activity of the targeted construct have not been established in nephrocytes, and several endocytic and lysosomal assays are interpreted beyond what they directly measure. In addition, the authors extend the relevance of these findings too far to the pathophysiology and potential treatment of Lowe syndrome in patients. The study is restricted to Drosophila nephrocytes, and no evidence is provided in mammalian proximal-tubule cells, patient-derived cells, or mammalian disease models that selective restoration of lysosomal OCRL activity is sufficient to rescue endocytosis. The conclusions should therefore remain specific to the Drosophila system, while their relevance to human Lowe syndrome requires validation in mammalian models. We thank the reviewer for their comments, constructive suggestions, and critical evaluation of our manuscript.

      Major comments

      1. Lysosomal specificity is not convincingly demonstrated. The LTS sequence is intended to target proteins to the cytosolic surface of late endosomes and lysosomes. However, its localization must be independently validated for each fusion protein and in each cell type. In Figure 5B, localization is assessed in S2R+ cells rather than nephrocytes, and by colocalization with LTS-mCherry, which contains the same targeting sequence. This approach is partly circular and does not independently establish that the labelled organelles are lysosomes. Furthermore, their morphology differs from that of Cathepsin L-, Rab7-, and LysoTracker-positive structures. The displayed LTS-dOCRL-GFP signal is not confined to LTS-mCherry-positive structures and includes substantial peripheral and non-overlapping signal. Figure 4 uses LTS-mCherry in nephrocytes, but this reporter is not colocalized with Cathepsin L, LysoTracker, or another independent lysosomal marker. The different patterns observed in Figures 4 and 5 are also difficult to compare because they were obtained in different cell types. Response:

      We thank the reviewer for this important suggestion and agree that further validation of the lysosomal targeting sequence (LTS) localisation would strengthen the study.

      We have validated the lysosomal localisation of the LTS sequence through colocalization analyses with the lysosomal markers LAMP1 and LysoTracker in Drosophila S2R+ cells. These data support the specificity of LTS-mediated lysosomal targeting, and we can incorporate these results into the revised manuscript.

      LTS colocalization with lysosomal markers in S2R+ cells

      __ ____ __

      In addition, we have performed colocalization studies of LTS::dOCRL::GFP with LTS::mCherry in other Drosophila cell types, including salivary gland cells and haemocytes, where we observed substantial overlap between the two markers. We can also include these data in the revised manuscript as further evidence supporting the localisation of the construct.

      Nephrocyte and hemocytes (Red arrows- hemocytes; Blue arrow- Nephrocyte)

      Salivary glands

      __ __

      In nephrocytes, however, visualisation of punctate LTS::dOCRL::GFP fluorescence is technically challenging because these cells contain a highly acidic lysosomal compartment, which likely compromises GFP fluorescence stability. To address this issue, larvae were fed chloroquine (3 mg/mL) to reduce lysosomal acidity, which enabled the detection of LTS::dOCRL::GFP signals that colocalised with LTS::mCherry. A similar reduction in GFP signal specifically in nephrocytes has been observed in our studies using the mCherry::GFP::Atg8a reporter and has also been reported previously in the literature (PMID: 35805186).

      Nephrocytes in chloroquine fed larvae

      To further address the reviewer's concern, we will perform additional colocalisation analyses of LTS::mCherry with an independent lysosomal marker in nephrocytes and include these data in the revised manuscript.

      Furthermore, to exclude the possibility that the LTS sequence targets organelles other than lysosomes, we will perform colocalization studies with markers of other endomembrane compartments, including early endosomes.

      Finally, to rule out the possibility that the observed phenotypic rescue arises from dOCRL function at the plasma membrane or early endosomes rather than lysosomes, we will include rescue experiments using dOCRL constructs specifically targeted to the plasma membrane and early endosomal compartments. These studies will help establish the compartment-specific contribution of dOCRL to the observed rescue phenotype.

      To address the reviewer’s suggestion regarding the localisation of LTS-dOCRL in nephrocytes relative to independent lysosomal, Rab7-positive, and early endosomal markers, we plan to generate an LTS::dOCRL::mCherry transgenic line and use it to examine the localisation of lysosome-targeted dOCRL relative to lysosomal, Rab7-positive late endosomal, and early endosomal compartments. This strategy is intended to overcome the loss of GFP fluorescence that we observed with the LTS::dOCRL::GFP construct in nephrocytes, likely due to the highly acidic nature of the lysosomal environment. This approach is necessary since immunostaining of endogenous dOCRL is currently not feasible, as the available Drosophila dOCRL antibody does not perform reliably for immunofluorescence applications.

      However, we acknowledge that there may be technical limitations with this approach. Specifically, we are uncertain whether the LTS::dOCRL::mCherry construct will be fully compatible with immunostaining procedures. Our current LTS::mCherry reporter exhibits poor signal retention during the immunostaining workflow, and reliable fluorescence can only be observed when samples are imaged immediately after fixation. Therefore, while we will pursue this strategy, the technical feasibility of performing extensive co-localisation analyses using the mCherry-tagged construct remains to be established.

      To complement these studies, we will independently perform colocalization experiments in S2R+ cells, where imaging and immunostaining conditions are more amenable to detailed subcellular localisation analyses. These experiments will allow us to assess the localisation of LTS::dOCRL relative to lysosomal, Rab7-positive, and early endosomal markers in an independent cellular context. Overall, we will make every effort to validate the localisation of lysosome-targeted dOCRL in nephrocytes using independent compartment-specific markers and will include any resulting data in the revised manuscript.

      Most importantly, the authors do not show: (i) direct measurement of lysosomal PI(4,5)P₂ in dOCRLKO nephrocytes; (ii) reduction of lysosomal PI(4,5)P₂ by LTS-dOCRL; or (iii) measurement of plasma-membrane PI(4,5)P₂ or PI4P following LTS-dOCRL expression. The last point is particularly important because the construct appears to display non-lysosomal signal, including apparent plasma-membrane localization and puncta that do not colocalize with LTS-mCherry. The authors should therefore quantify LTS-dOCRL localization in nephrocytes relative to independent lysosomal, Rab7-positive, and early-endosomal markers; determine whether LTS-dOCRL corrects plasma-membrane PI(4,5)P₂ and PI4P; and assess lysosomal PI(4,5)P₂ in control, dOCRLKO, and LTS-dOCRL-rescued nephrocytes.

      We thank the reviewer for this valuable suggestion. We will include measurements of plasma membrane PI(4,5)P₂ and PI4P levels following expression of LTS::dOCRL. These experiments will allow us to assess whether lysosome-targeted dOCRL influences phosphoinositide homeostasis at the plasma membrane and provide additional insight into the relationship between lysosomal dOCRL function and cellular phosphoinositide regulation. With respect to lysosomal PI4P and PI(4,5)P₂ levels, these experiments are currently technically challenging in the Drosophila nephrocyte system. Simultaneous expression of phosphoinositide biosensors, compartment-specific markers, Gal4 drivers, and LTS::dOCRL transgenes in either wild-type or dOCRLKO backgrounds requires the generation of flies carrying multiple transgenic elements. In many cases, these complex genetic combinations are difficult to obtain, or maintain limiting our ability to directly quantify lysosomal phosphoinositide pools in vivo at present.

      • *

      The reduction in plasma-membrane PI4P is a major and insufficiently validated finding. The marked reduction in plasma-membrane PI4P in dOCRLKO nephrocytes is potentially important but requires substantially stronger validation. Although OCRL hydrolyses PI(4,5)P₂ to generate PI4P, a reduction in the steady-state bulk plasma-membrane PI4P pool is not a commonly established consequence of OCRL loss. Direct analysis of plasma-membrane phosphoinositides in human platelets showed that OCRL inhibition did not reduce plasma-membrane PI4P in resting or activated platelets, and no change in plasma-membrane PI4P was detected in HEK293T cells. In contrast, inhibition of PI4KIIIα strongly depleted plasma-membrane PI4P, supporting PI4KA, rather than OCRL, as the principal determinant of this pool (Bura and Jurak Begonja, 2021; PMID: 34947862). Similarly, lipidomic analysis of Lowe syndrome patient-derived cells, performed by the same group as in the manuscript under review, did not reveal a consistent reduction in total PI4P/PIP (Akhtar et al., 2022; PMID: 35023542). The best-established phosphoinositide consequence of OCRL deficiency is instead the accumulation of PI(4,5)P₂ on early endosomes, clathrin-coated intermediates, lysosomes and, in some cell types, at the plasma membrane (Vicinanza et al., 2011; Nández et al., 2014). These studies did not identify depletion of bulk plasma-membrane PI4P as a characteristic phenotype of OCRL loss. The finding reported here could therefore represent a nephrocyte-specific effect, an indirect consequence of altered phosphoinositide metabolism, or a limitation of the probe-based measurement. This issue is mechanistically central because a substantial loss of plasma-membrane PI4P could itself affect membrane identity, PI(4,5)P₂ resynthesis, and endocytosis. Moreover, the authors do not determine whether expression of dOCRL or LTS::dOCRL restores plasma-membrane PI4P and PI(4,5)P₂.

      Response:

      We thank the reviewer for this valuable insight. We recognise that alterations in PI(4,5)P₂ levels are a well-established consequence of OCRL deficiency and have been extensively studied in multiple systems. In contrast, changes in PI4P levels following OCRL loss are less commonly reported and remain relatively unexplored in the literature.

      In the present study, we quantified PI4P levels in a renal cell type, Drosophila nephrocytes. The reduction in PI4P observed in dOCRLKO nephrocytes may represent a cell type specific consequence of OCRL loss and could reflect the unique phosphoinositide dynamics of highly endocytic renal cells.

      Supporting the possibility that OCRL can influence PI4P homeostasis, a previous study (PMID: 28871046) reported that depletion of OCRL results in reduced PI4P levels in primary cilia. These findings suggest that OCRL dependent regulation of phosphoinositide composition may extend beyond PI(4,5)P₂ and may vary according to subcellular compartment and cellular context. To further substantiate our findings, we will include a rescue experiment assessing PI4P levels in the dOCRLKO background in the revised manuscript.

      The AgNO₃ data do not currently provide a robust quantitative measure of clearance In Figure 1H, the percentage of dOCRLKO nephrocytes classified as containing high AgNO₃ is greater at 42 hours than at 36 hours. Differences in initial loading, imaging conditions, cell size, or thresholding could therefore produce an apparent increase at a later time point. For this reason, the authors should provide measurements of initial AgNO₃ accumulation. Furthermore, they should explain the use of different AgNO₃ concentrations-0.003% in Figure 1 and 0.0015% in Figures 2 and 5-as this limits direct comparison between experiments.

      Response:

      We measured AgNO₃ uptake in control and dOCRLKO nephrocytes concurrently with the clearance assays. In addition, we will include an experiment assessing AgNO₃ uptake at the initial time point to further substantiate these findings. The imaging conditions were kept the same throughout the experiment.

      The concentration of 0.003 was used during the assay standardisation phase, in which only control and dOCRLKO nephrocytes were analysed. At this concentration, AgNO₃ did not exhibit detectable toxicity. For subsequent experiments, we reduced the concentration to 0.0015 to facilitate more accurate comparisons between control and dOCRLKO nephrocytes, as well as across additional genotypes included in the study. Importantly, control and dOCRLKO nephrocytes were included in every experimental set alongside the other genotypes. This approach ensured direct internal comparisons across experiments and minimised variability arising from experimental conditions.

      In Figure 3, the authors show reduced dextran and BSA uptake, supporting an endocytic defect. However, lower Rabenosyn-5, 2xFYVE, and Rab7 fluorescence does not demonstrate fewer or smaller endosomes. These signals may instead reflect altered protein abundance, membrane recruitment, or phosphoinositide content. The authors should therefore avoid conclusions regarding reduced endosomal compartment size or preserved endosomal maturation unless organelle number and size are directly quantified.

      Similarly, Rab7CA rescue identifies Rab7 as a genetic suppressor but does not prove that endogenous Rab7 activity is reduced or that it causes the lysosomal defect. Increased Cathepsin L staining, although diffuse and not clearly localized to intracellular puncta, together with reduced Magic Red activity, suggests accumulation of poorly functional lysosomes rather than increased lysosomal capacity. However, the authors measured Cathepsin B activity using Magic Red substrate MR-(RR)2 , rather than Cathepsin L activity using the Magic Red substrate MR-FR2.

      The use of Cathepsin L staining as a readout of lysosomal dysfunction may therefore require further characterization. Finally, the authors should test whether Rab7CA restores lysosomal activity, rather than only Cathepsin L abundance.

      Response:

      By measuring the fluorescence intensity of these markers, we observed reduced labelling intensity for Rabenosyn-5, 2xFYVE, and Rab7. We will modify this statement into the revised manuscript. We have not yet analysed the size or number of these compartments, as reliable quantification is challenging because the cells are densely populated with endosomal structures throughout the cytoplasm.

      In dOCRLKO nephrocytes, we observed an expansion of the lysosomal compartment, as indicated by increased LTS::mCherry fluorescence. To independently validate this finding, we employed an additional lysosomal marker, Cathepsin L, which showed a similar increased signal.

      To determine whether the enlarged lysosomal compartment remained functionally active, we assessed lysosomal proteolytic activity using the Magic Red Cathepsin assay. We initially tested both the Magic Red Cathepsin L and Magic Red Cathepsin B kits. However, the Magic Red Cathepsin L reagent failed to generate a detectable signal even in control nephrocytes, suggesting a technical limitation of the kit under our experimental conditions. Consequently, we proceeded with the Magic Red Cathepsin B assay. Importantly, the manufacturer's documentation indicates that, although these substrates are designed to preferentially detect specific cathepsins, they can also be cleaved by other cathepsin family members, thereby serving as a broader readout of lysosomal cathepsin activity.

      We appreciate the reviewer's suggestion regarding the functional assessment of Rab7CA mediated rescue. To address this important point, we will perform the Magic Red Cathepsin assay in the Rab7CA rescue background and include these results in the revised manuscript.

      Figure 3C measures BSA accumulation after a 15-minute incubation, whereas Figure 4I uses an 8-minute pulse followed by a 40-minute chase, showing increased intracellular staining that is interpreted as reduced degradation. Although residual BSA is higher in dOCRLKO cells after the chase, the amount present immediately after the 8-minute pulse was not measured. Consequently, the fraction cleared or degraded cannot be calculated, and the data reported in Figure 4I cannot be directly correlated with those reported in Figure 3C. To support the conclusion of defective degradation, the authors should provide a quantitative assessment of BSA levels immediately after the 8-minute pulse and at multiple chase times, with residual fluorescence expressed relative to the genotype-specific starting signal.

      The uptake pathway used by BSA in Drosophila nephrocytes also requires clarification. Maleylated BSA is a scavenger-receptor ligand in other cell types, whereas protein uptake in nephrocytes is commonly associated with Cubilin/Amnionless-dependent uptake. The manuscript does not establish which receptor or pathway mediates BSA internalization in nephrocytes.

      Response: We thank the reviewer for this valuable suggestion. Our initial analyses showed that mBSA uptake at the early time points (5, 10, and 15 minutes) was consistently reduced in dOCRLKO nephrocytes compared with controls, indicating an impairment in uptake. However, during the subsequent chase period of 40 minutes, we observed a stronger and more persistent mBSA signal in dOCRLKO nephrocytes relative to controls, suggesting reduced degradation.

      To improve the clarity of the data presentation, we will include the pulse-only uptake data (8-minute incubation) alongside the pulse-chase experiments performed at the different time points in the revised manuscript, as suggested.

      The mBSA uptake assay is mediated through Cubilin-dependent endocytosis. In support of this, we have observed that knockdown of Cubilin by RNAi completely abolishes mBSA uptake in nephrocytes, confirming the specificity of the assay. We will provide these data as supplementary evidence to further clarify the mechanistic basis of mBSA uptake.

      Statistical analyses should reflect the biological replicate structure Most imaging datasets contain many nephrocytes derived from a smaller number of animals and experimental trials. Individual cells from the same animal are not independent biological replicates. Several three-group experiments also appear to have been analysed using unpaired t-tests rather than a single ANOVA with appropriate multiple-comparison correction.

      The authors should reanalyse the data using animal- or experimental-trial means as the biological unit, or apply nested or mixed-effects models that account for cells nested within animals. N and n should be defined consistently in every figure legend. This should not require new experiments.

      Response: We thank the reviewer for this suggestion. We will reanalyse the experiments involving three-group comparisons using one-way ANOVA with appropriate multiple-comparison corrections. In addition, where measurements are obtained from multiple cells within the same animal, we will apply mixed-effects models that account for the nested data structure (cells nested within animals).

      Minor comments

      The authors should more clearly acknowledge the differences between the endocytic systems of Drosophila nephrocytes and mammalian proximal-tubule cells, rather than primarily emphasizing their similarities. Most notably, OCRL depletion affects dextran uptake in nephrocytes, whereas fluid-phase endocytosis is reportedly preserved in mammalian proximal-tubule cells. Another important and often underappreciated difference is the absence of the OCRL-related 5-phosphatase INPP5B in Drosophila. This may substantially limit the extent to which OCRL-depleted Drosophila nephrocytes can model the molecular and cellular pathophysiology of Lowe syndrome.

      Response: We thank the reviewer for their suggestion. We will include the comparison of Drosophila and mammalian OCRL, the limitations of the nephrocyte system in the discussion of our revised manuscript.

      The discordant results in Supplementary Table 1-complete loss of AgNO₃ uptake in the Trpml-null allele but no phenotype following Trpml RNAi-should be discussed.

      Response: One possible explanation for the observed results is the efficiency of the RNAi knockdown. While the Trpml null allele completely abolishes gene function and results in a complete loss of AgNO₃ uptake, the RNAi-mediated knockdown may not have been sufficient to reduce TRPML levels below the threshold required to produce a detectable phenotype. Consequently, residual TRPML activity in the RNAi background could account for the absence of an observable defect in AgNO₃ uptake.

      LTS-mCherry and Cathepsin L fluorescence should not be described as measures of lysosomal "size" or "expansion" unless organelle area, volume, or number is quantified. The early-endosome-targeted dOCRL experiment mentioned as "data not shown" is central to the proposed compartment-specific model. It should be presented, including localization and expression controls, or removed from the Discussion.

      Response: We will remove the lysosomal size comment from the manuscript. To strengthen the conclusions of the study, we will include the early-endosome-targeted dOCRL rescue experiment, as well as its localisation and expression control data, in the revised manuscript.

      The phosphatase-dead OCRL mutant should be assessed for functional activity by measuring PI(4,5)P₂ following mutant overexpression in OCRL-KO cells. Response: We will include this experiment in the revised manuscript.

      Reviewer #2 (Significance (Required)):

      The potentially important conceptual advance is the observation that a late-endolysosomal-targeted, catalytically active dOCRL construct can suppress not only lysosomal phenotypes but also early-endosomal marker abnormalities and endocytic cargo uptake in vivo. If the spatial specificity of the construct is demonstrated, this would support substantial feedback from late-endolysosomal homeostasis to upstream endocytic function. The role of OCRL at lysosomes and the lysosomal PI(4,5)P₂-TRPML1 pathway are already established in mammalian Lowe syndrome models. The principal potential advance of this study is therefore not the identification of lysosomal dysfunction itself, but the proposal that restoration of lysosomal OCRL activity is sufficient to rescue upstream endocytosis. At present, this conclusion remains uncertain because direct activity of the construct at the plasma membrane, early endosomes, or other non-lysosomal compartments has not been excluded. The data are compatible with an important lysosomal contribution but do not yet establish lysosomes as the unique or primary site of OCRL action. The work should be of interest to researchers studying phosphoinositides, membrane trafficking, lysosomal signalling, renal endocytosis, Drosophila nephrocytes, and Lowe/Dent disease mechanisms. Field of expertise: membrane trafficking, phosphoinositide metabolism, lysosomal biology, inherited renal tubulopathies

      Response: We think that experiments using 2xFYVE-dOCRL and dOCRL::CAAX, which selectively target dOCRL to the early endosomal and plasma membrane compartments, respectively, would be important for assessing the contribution of these compartments to the observed rescue phenotype. In response, we will include rescue experiments using these compartment-specific dOCRL constructs in the revised manuscript.

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

      __Summary: Progress over recent decades has established a critical role for OCRL in endosomal-lysosomal biology, yet its primary subcellular site of action has remained a central point of debate. In this manuscript, the authors utilize a Drosophila nephrocyte model of Lowe Syndrome to demonstrate that lysosome-specific expression of functional dOCRL (specifically requiring its 5-phosphatase enzymatic activity) is sufficient to rescue key cellular phenotypes. The core conceptual strength of this study lies in its proposal of a "lysosome-first" model suggesting that lysosomal dysfunction is the primary driver of disease pathology in dOCRL-depleted nephrocytes, and that upstream endocytic uptake defects occur secondary to this lysosomal failure. The manuscript is well-written, the experiment is well designed. The data are clear, with proper controls. The results nicely quantified. I only have relatively minor questions for the authors.

      We thank the reviewer for their comments, constructive suggestions, and critical evaluation of our manuscript.

      Minor comments: 1. The authors state that "dOCRL KO larvae showed lethality at every stage of development when grown on yeast medium"; does this imply that survival rates differ when they are grown on standard media without yeast paste?

      Response: We thank the reviewer for this important point. dOCRLKO larvae exhibit lethality at the third instar larval stage when maintained on standard fly food. However, when reared on yeast paste media, the larvae survive until the pupariation stage and display significantly improved overall health compared with those grown on standard media. To ensure consistency across experimental conditions, all genotypes included in this study were reared on yeast-paste media.

      In Figure 1C and 1E, since the cell sizes differ significantly, with mutants weighing only approx. 50% of controls, it would be beneficial to normalize for cell size or area before calculating the plasma membrane to cytoplasm (PM:Cytoplasm) ratio

      Response: We thank the reviewer for their suggestion. We quantified plasma membrane and cytoplasmic fluorescence intensities by drawing line ROIs specifically across the plasma membrane and within the cytosolic region, rather than measuring the intensity of the entire cell. This approach minimises the potential confounding effects arising from differences in cell size between control and mutant nephrocytes. It is the accepted method of quantifying the levels of protein probes that localize to membranes and report the level of a lipid.

      For the benefit of the reader, it would be helpful if the figures followed a stricter chronological order; for example, Supplementary Figure 5 is referenced immediately after Figure 1.

      Response: We thank the reviewer for pointing this out. We will correct the order of the figures in the revised manuscript to ensure that they are presented in the appropriate chronological sequence.

      Figures 5E and 5F show lower levels of Rabenosyn-5. What is the proposed mechanism for this reduction in early endosomal markers?

      Response: Does the reviewer mean the figures 3E and 3F. We have seen that depleting dOCRL leads to reduced endocytosis and reduced early endosomal compartment stained by Rabenosyn-5. The precise mechanism by which loss of dOCRL leads to reduced early endosomal levels is not yet fully understood. One possibility is that this is a secondary consequence of a broader imbalance within the endolysosomal system. Impaired endocytic uptake in dOCRL deficient nephrocytes could lead to reduced delivery of cargo and membrane into the early endosomal pathway, ultimately resulting in a decrease in early endosome formation or maintenance. A similar reduction in labelling intensity of EEA1 have also been observed in the zebrafish pronephric tubule upon loss of ocrl (PMID: 25838181).

      The rescue of the clearance defect by Rab7CA implies that the accumulation of PI(4,5)P₂ on late endolysosomes might interfere with Rab7 recruitment or activation, thereby stalling the fusion and maturation processes necessary for AgNO3 clearance. A possible explanation for why Rab7CA is sufficient to bypass the need for dOCRL activity during clearance should be highlighted in the Discussion section.

      Response: We thank the reviewer for this insightful suggestion and agree that discussing the potential mechanism underlying the Rab7CA mediated rescue would strengthen the manuscript. We will include this in our revised manuscript.

      Lines 341-343 do not appear to match the referenced figure (Supplementary Figure 2H), which shows results for the Hml-GAL4 driver rather than the Sns-GAL4 driver mentioned in the text.

      Response: We thank the reviewer for identifying this error. We will correct this mistake in the revised manuscript.

      There are a few typographical errors that need correction, such as the repetition of "that" in line 405.

      Response: We thank the reviewer for identifying this error. We will correct this mistake in the revised manuscript.

      Reviewer #3 (Significance (Required)):

      Overall, this study provides a significant shift in how we might view the progression of Lowe Syndrome. By identifying the lysosome as the critical hub for dOCRL function, it opens new therapeutic avenues focused on correcting lysosomal PI(4,5)P₂ levels or acidification rather than targeting early endocytic machinery. The data is robust, the model is appropriate, and the "lysosome-first" hypothesis is a major step forward for the field of endolysosomal homeostasis. While the data are robust and the model is appropriate, the observation that lysosomal dOCRL rescues early endocytic uptake remains "surprising" and suggests a powerful homeostatic feedback loop. How lysosomal dysfunction signals back to inhibit the initial steps of endocytosis? whether through mTORC1/TFEB pathways or the depletion of recycling membrane resources, remains an intriguing question that warrants future study. This study is relevant to broader cell biology community and have future translational values.

      Response: We thank Reviewer #3 for this thoughtful and constructive comment. At present, the mechanism by which lysosomal dysfunction feeds back to inhibit the early stages of endocytosis remains unclear. We agree that understanding this connection represents an important area for future investigation.

      Nevertheless, our observations suggest that such a feedback mechanism exists in nephrocytes. In addition to the dOCRL mutant phenotype described here, we have also observed that impairment of lysosomal function through depletion of the lysosomal TRPML channel also leads to a reduction in mBSA uptake, indicating defects in early endocytic events. These findings support the existence of a feedback loop between lysosomal activity and endocytic uptake that may function to maintain endolysosomal homeostasis.

      • *

      __3. __Description of the revisions that have already been incorporated in the transferred manuscript

      Not applicable

      • *

      __4. __Description of analyses that authors prefer not to carry out

      Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

      With respect to assessing lysosomal PI4P and PI(4,5)P₂ levels, these experiments are currently technically challenging in the Drosophila nephrocyte system. Simultaneous expression of phosphoinositide biosensors, compartment-specific markers, Gal4 drivers, and LTS::dOCRL transgenes in either wild-type or dOCRLKO backgrounds requires the generation of flies carrying multiple transgenic elements. In many cases, these complex genetic combinations are difficult to obtain, or maintain limiting our ability to directly quantify lysosomal phosphoinositide pools in vivo at present. We therefore believe that there are time, resource and technical limitations in addressing this one point.

      PeerReviewed
    2. EMBOpress 05 Oct 2026

      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

      Summary:

      Progress over recent decades has established a critical role for OCRL in endosomal-lysosomal biology, yet its primary subcellular site of action has remained a central point of debate. In this manuscript, the authors utilize a Drosophila nephrocyte model of Lowe Syndrome to demonstrate that lysosome-specific expression of functional dOCRL (specifically requiring its 5-phosphatase enzymatic activity) is sufficient to rescue key cellular phenotypes. The core conceptual strength of this study lies in its proposal of a "lysosome-first" model suggesting that lysosomal dysfunction is the primary driver of disease pathology in dOCRL-depleted nephrocytes, and that upstream endocytic uptake defects occur secondary to this lysosomal failure. The manuscript is well-written, the experiment is well designed. The data are clear, with proper controls. The results nicely quantified. I only have relatively minor questions for the authors.

      Minor comments:

      1. The authors state that "dOCRL KO larvae showed lethality at every stage of development when grown on yeast medium"; does this imply that survival rates differ when they are grown on standard media without yeast paste?
      2. In Figure 1C and 1E, since the cell sizes differ significantly, with mutants weighing only approx. 50% of controls, it would be beneficial to normalize for cell size or area before calculating the plasma membrane to cytoplasm (PM:Cytoplasm) ratio
      3. For the benefit of the reader, it would be helpful if the figures followed a stricter chronological order; for example, Supplementary Figure 5 is referenced immediately after Figure 1.
      4. Figures 5E and 5F show lower levels of Rabenosyn-5. What is the proposed mechanism for this reduction in early endosomal markers?
      5. The rescue of the clearance defect by Rab7CA implies that the accumulation of PI(4,5)P₂ on late endolysosomes might interfere with Rab7 recruitment or activation, thereby stalling the fusion and maturation processes necessary for AgNO3 clearance. A possible explanation for why Rab7CA is sufficient to bypass the need for dOCRL activity during clearance should be highlighted in the Discussion section.
      6. Lines 341-343 do not appear to match the referenced figure (Supplementary Figure 2H), which shows results for the Hml-GAL4 driver rather than the Sns-GAL4 driver mentioned in the text.
      7. There are a few typographical errors that need correction, such as the repetition of "that" in line 405.

      Significance

      Overall, this study provides a significant shift in how we might view the progression of Lowe Syndrome. By identifying the lysosome as the critical hub for dOCRL function, it opens new therapeutic avenues focused on correcting lysosomal PI(4,5)P₂ levels or acidification rather than targeting early endocytic machinery. The data is robust, the model is appropriate, and the "lysosome-first" hypothesis is a major step forward for the field of endolysosomal homeostasis. While the data are robust and the model is appropriate, the observation that lysosomal dOCRL rescues early endocytic uptake remains "surprising" and suggests a powerful homeostatic feedback loop. How lysosomal dysfunction signals back to inhibit the initial steps of endocytosis? whether through mTORC1/TFEB pathways or the depletion of recycling membrane resources, remains an intriguing question that warrants future study. This study is relevant to broader cell biology community and have future translational values.

      PeerReviewed
    3. EMBOpress 05 Oct 2026

      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

      Summary

      The authors establish germline and nephrocyte-specific dOCRL loss-of-function models in Drosophila. They report that dOCRL deficiency is associated with increased plasma-membrane PI(4,5)P₂, decreased plasma-membrane PI4P, reduced dextran and BSA uptake, alterations in early- and late-endosomal markers, lysosomal expansion/dysfunction, and delayed AgNO₃ clearance. They further claim that a late-endolysosomal-targeted dOCRL construct rescues AgNO₃ retention, Cathepsin L accumulation, Rabenosyn-5 staining, and BSA uptake, whereas a presumably phosphatase-dead construct fails to normalize Cathepsin L. The authors conclude that lysosomal OCRL activity is the primary requirement for endolysosomal homeostasis and that the endocytic abnormalities are secondary to lysosomal dysfunction.

      The Drosophila model and compartment-targeting approach are potentially valuable. However, the central conclusion is not adequately supported because the localization and biochemical activity of the targeted construct have not been established in nephrocytes, and several endocytic and lysosomal assays are interpreted beyond what they directly measure. In addition, the authors extend the relevance of these findings too far to the pathophysiology and potential treatment of Lowe syndrome in patients. The study is restricted to Drosophila nephrocytes, and no evidence is provided in mammalian proximal-tubule cells, patient-derived cells, or mammalian disease models that selective restoration of lysosomal OCRL activity is sufficient to rescue endocytosis. The conclusions should therefore remain specific to the Drosophila system, while their relevance to human Lowe syndrome requires validation in mammalian models.

      Major comments

      1. Lysosomal specificity is not convincingly demonstrated. The LTS sequence is intended to target proteins to the cytosolic surface of late endosomes and lysosomes. However, its localization must be independently validated for each fusion protein and in each cell type. In Figure 5B, localization is assessed in S2R+ cells rather than nephrocytes, and by colocalization with LTS-mCherry, which contains the same targeting sequence. This approach is partly circular and does not independently establish that the labelled organelles are lysosomes. Furthermore, their morphology differs from that of Cathepsin L-, Rab7-, and LysoTracker-positive structures. The displayed LTS-dOCRL-GFP signal is not confined to LTS-mCherry-positive structures and includes substantial peripheral and non-overlapping signal. Figure 4 uses LTS-mCherry in nephrocytes, but this reporter is not colocalized with Cathepsin L, LysoTracker, or another independent lysosomal marker. The different patterns observed in Figures 4 and 5 are also difficult to compare because they were obtained in different cell types. Most importantly, the authors do not show: (i) direct measurement of lysosomal PI(4,5)P₂ in dOCRLKO nephrocytes; (ii) reduction of lysosomal PI(4,5)P₂ by LTS-dOCRL; or (iii) measurement of plasma-membrane PI(4,5)P₂ or PI4P following LTS-dOCRL expression. The last point is particularly important because the construct appears to display non-lysosomal signal, including apparent plasma-membrane localization and puncta that do not colocalize with LTS-mCherry. The authors should therefore quantify LTS-dOCRL localization in nephrocytes relative to independent lysosomal, Rab7-positive, and early-endosomal markers; determine whether LTS-dOCRL corrects plasma-membrane PI(4,5)P₂ and PI4P; and assess lysosomal PI(4,5)P₂ in control, dOCRLKO, and LTS-dOCRL-rescued nephrocytes.
      2. The reduction in plasma-membrane PI4P is a major and insufficiently validated finding. The marked reduction in plasma-membrane PI4P in dOCRLKO nephrocytes is potentially important but requires substantially stronger validation. Although OCRL hydrolyses PI(4,5)P₂ to generate PI4P, a reduction in the steady-state bulk plasma-membrane PI4P pool is not a commonly established consequence of OCRL loss. Direct analysis of plasma-membrane phosphoinositides in human platelets showed that OCRL inhibition did not reduce plasma-membrane PI4P in resting or activated platelets, and no change in plasma-membrane PI4P was detected in HEK293T cells. In contrast, inhibition of PI4KIIIα strongly depleted plasma-membrane PI4P, supporting PI4KA, rather than OCRL, as the principal determinant of this pool (Bura and Jurak Begonja, 2021; PMID: 34947862). Similarly, lipidomic analysis of Lowe syndrome patient-derived cells, performed by the same group as in the manuscript under review, did not reveal a consistent reduction in total PI4P/PIP (Akhtar et al., 2022; PMID: 35023542). The best-established phosphoinositide consequence of OCRL deficiency is instead the accumulation of PI(4,5)P₂ on early endosomes, clathrin-coated intermediates, lysosomes and, in some cell types, at the plasma membrane (Vicinanza et al., 2011; Nández et al., 2014). These studies did not identify depletion of bulk plasma-membrane PI4P as a characteristic phenotype of OCRL loss. The finding reported here could therefore represent a nephrocyte-specific effect, an indirect consequence of altered phosphoinositide metabolism, or a limitation of the probe-based measurement. This issue is mechanistically central because a substantial loss of plasma-membrane PI4P could itself affect membrane identity, PI(4,5)P₂ resynthesis, and endocytosis. Moreover, the authors do not determine whether expression of dOCRL or LTS::dOCRL restores plasma-membrane PI4P and PI(4,5)P₂.
      3. The AgNO₃ data do not currently provide a robust quantitative measure of clearance In Figure 1H, the percentage of dOCRLKO nephrocytes classified as containing high AgNO₃ is greater at 42 hours than at 36 hours. Differences in initial loading, imaging conditions, cell size, or thresholding could therefore produce an apparent increase at a later time point. For this reason, the authors should provide measurements of initial AgNO₃ accumulation. Furthermore, they should explain the use of different AgNO₃ concentrations-0.003% in Figure 1 and 0.0015% in Figures 2 and 5-as this limits direct comparison between experiments.
      4. In Figure 3, the authors show reduced dextran and BSA uptake, supporting an endocytic defect. However, lower Rabenosyn-5, 2xFYVE, and Rab7 fluorescence does not demonstrate fewer or smaller endosomes. These signals may instead reflect altered protein abundance, membrane recruitment, or phosphoinositide content. The authors should therefore avoid conclusions regarding reduced endosomal compartment size or preserved endosomal maturation unless organelle number and size are directly quantified. Similarly, Rab7CA rescue identifies Rab7 as a genetic suppressor but does not prove that endogenous Rab7 activity is reduced or that it causes the lysosomal defect. Increased Cathepsin L staining, although diffuse and not clearly localized to intracellular puncta, together with reduced Magic Red activity, suggests accumulation of poorly functional lysosomes rather than increased lysosomal capacity. However, the authors measured Cathepsin B activity using Magic Red substrate MR-(RR)2 , rather than Cathepsin L activity using the Magic Red substrate MR-FR2. The use of Cathepsin L staining as a readout of lysosomal dysfunction may therefore require further characterization. Finally, the authors should test whether Rab7CA restores lysosomal activity, rather than only Cathepsin L abundance.
      5. Figure 3C measures BSA accumulation after a 15-minute incubation, whereas Figure 4I uses an 8-minute pulse followed by a 40-minute chase, showing increased intracellular staining that is interpreted as reduced degradation. Although residual BSA is higher in dOCRLKO cells after the chase, the amount present immediately after the 8-minute pulse was not measured. Consequently, the fraction cleared or degraded cannot be calculated, and the data reported in Figure 4I cannot be directly correlated with those reported in Figure 3C. To support the conclusion of defective degradation, the authors should provide a quantitative assessment of BSA levels immediately after the 8-minute pulse and at multiple chase times, with residual fluorescence expressed relative to the genotype-specific starting signal. The uptake pathway used by BSA in Drosophila nephrocytes also requires clarification. Maleylated BSA is a scavenger-receptor ligand in other cell types, whereas protein uptake in nephrocytes is commonly associated with Cubilin/Amnionless-dependent uptake. The manuscript does not establish which receptor or pathway mediates BSA internalization in nephrocytes.
      6. Statistical analyses should reflect the biological replicate structure Most imaging datasets contain many nephrocytes derived from a smaller number of animals and experimental trials. Individual cells from the same animal are not independent biological replicates. Several three-group experiments also appear to have been analysed using unpaired t-tests rather than a single ANOVA with appropriate multiple-comparison correction. The authors should reanalyse the data using animal- or experimental-trial means as the biological unit, or apply nested or mixed-effects models that account for cells nested within animals. N and n should be defined consistently in every figure legend. This should not require new experiments.

      Minor comments

      The authors should more clearly acknowledge the differences between the endocytic systems of Drosophila nephrocytes and mammalian proximal-tubule cells, rather than primarily emphasizing their similarities. Most notably, OCRL depletion affects dextran uptake in nephrocytes, whereas fluid-phase endocytosis is reportedly preserved in mammalian proximal-tubule cells. Another important and often underappreciated difference is the absence of the OCRL-related 5-phosphatase INPP5B in Drosophila. This may substantially limit the extent to which OCRL-depleted Drosophila nephrocytes can model the molecular and cellular pathophysiology of Lowe syndrome.

      The discordant results in Supplementary Table 1-complete loss of AgNO₃ uptake in the Trpml-null allele but no phenotype following Trpml RNAi-should be discussed. LTS-mCherry and Cathepsin L fluorescence should not be described as measures of lysosomal "size" or "expansion" unless organelle area, volume, or number is quantified.

      The early-endosome-targeted dOCRL experiment mentioned as "data not shown" is central to the proposed compartment-specific model. It should be presented, including localization and expression controls, or removed from the Discussion.

      The phosphatase-dead OCRL mutant should be assessed for functional activity by measuring PI(4,5)P₂ following mutant overexpression in OCRL-KO cells.

      Significance

      The potentially important conceptual advance is the observation that a late-endolysosomal-targeted, catalytically active dOCRL construct can suppress not only lysosomal phenotypes but also early-endosomal marker abnormalities and endocytic cargo uptake in vivo. If the spatial specificity of the construct is demonstrated, this would support substantial feedback from late-endolysosomal homeostasis to upstream endocytic function. The role of OCRL at lysosomes and the lysosomal PI(4,5)P₂-TRPML1 pathway are already established in mammalian Lowe syndrome models. The principal potential advance of this study is therefore not the identification of lysosomal dysfunction itself, but the proposal that restoration of lysosomal OCRL activity is sufficient to rescue upstream endocytosis.

      At present, this conclusion remains uncertain because direct activity of the construct at the plasma membrane, early endosomes, or other non-lysosomal compartments has not been excluded. The data are compatible with an important lysosomal contribution but do not yet establish lysosomes as the unique or primary site of OCRL action. The work should be of interest to researchers studying phosphoinositides, membrane trafficking, lysosomal signalling, renal endocytosis, Drosophila nephrocytes, and Lowe/Dent disease mechanisms.

      Field of expertise: membrane trafficking, phosphoinositide metabolism, lysosomal biology, inherited renal tubulopathies

      PeerReviewed
    4. EMBOpress 05 Oct 2026

      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

      In this study the authors explore the role of the Drosophila orthologue of the Lowe syndrome protein OCRL (called dOCRL) in nephrocytes, the functional equivalent of the mammalian kidney. Using genetic knockout and complementation approaches it is shown that dOCRL is required for the clearance of the toxic compound silver nitrate from nephrocytes. This effect is phenocopied by loss of Rab7 and can be rescued by a constitutively active form of Rab7, suggesting involvement of late endosomal or lysosomal compartments. This is supported by experiments showing disrupted lysosomal function upon dOCRL loss. Loss of dOCRL also causes defects earlier in the endocytic pathway, with reduced uptake of the endocytic tracers dextran and mBSA into nephrocytes, and a loss of early endosomal compartments. Interestingly, re-expression of dOCRL targeted to the lysosome rescues the defects in both endosome and lysosome function, suggesting that the defects observed are primarily due to dOCRL function at the lysosome.

      This is a very nicely done study. The data are clear and support the authors' conclusions. The means of analysis and the quantitation of the data are appropriate. There are a few things that could be addressed in a revised version which are indicated below:

      1. The ability of lysosome-targeted dOCRL to rescue the phenotypes is interesting. Can the authors exclude the possibility that there is residual cytosolic dOCRL in the cells that can transiently associate with other organelles e.g. early endosomes to drive the phenotypic rescue? There are experiments described in the discussion about targeting dOCRL to early endosomes not rescuing the phenotypes. Currently this is mentioned as data not shown. It should be included.
      2. The results with AgNO3 are confusing. Uptake into nephrocytes is not affected by loss of dOCRL but is affected by other manipulations that disrupt endosomal trafficking (suppl table 1). Yet, loss of dOCRL affects cellular uptake of dextran and mBSA, which rely on endocytosis. More explanation needs to be provided on what we know about AgNO3 uptake into nephrocytes. Is it by endocytosis, as the table in the supplementary table would seem to indicate? In that case, why would AgNO3 uptake not be affected by dOCRL loss? Also, what underlies the clearance of AgNO3 taken up into nephrocytes? If it is already in the cells, then does it leave by exocytosis? Or does it enter and leave cells through transporters? How does dOCRL affect the clearance of AgNO3 from the cells i.e. through what mechanism? Filtration of AgNO3 is also mentioned but not fully explained. How does filtration and clearance of AgNO3 relate to the processes that occur in the mammalian kidney, where filtration of blood is performed by the glomerulus, while clearance is in the proximal tubules, which are the region affected in Lowe syndrome?
      3. The data overall are clear and convincing, but Fig 3C is not. There does not appear to be any reduction in mBSA signal in the dOCRL image shown.
      4. Discussion could be more expansive eg dOCRL versus mammalian OCRL-clathrin binding, localisation etc...
      5. The figures need to appear in the correct order in the text. Currently they are presented in the text in a jumbled way.
      6. References are missing in quite a few places in the text. There are numerous instances of statements being made without the references to support them.
      7. There are a few minor grammatical errors.

      Significance

      This is a very nicely done study. The data are clear and support the authors' conclusions. The means of analysis and the quantitation of the data are appropriate.

      PeerReviewed
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    biorxiv.org/lookup/doi/10.64898/2025.12.03.692239
  10. www.hks.harvard.edu www.hks.harvard.edu
    How AI can empower city workers to make decisions while remaining accountable
    3
    1. KennyJam123 05 Oct 2026
      Even in two years, if incorporated thoughtfully, AI will help city leaders more quickly and comprehensively meet residents’ expectations in ways that build trust. And in so doing, government will become more conversational, personalized, and proactive.

      This shows that Ai is emerging strongly and quickly. Ai implementation in this way can give people more power to address concern at any givne time.

    3. KennyJam123 05 Oct 2026
      AI creates new forms of accountability because decisions become more transparent, traceable, and reviewable.

      While Ai is automating the repetitive work, if it fails or makes a mistake that worker is still held accoutable.

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    hks.harvard.edu/faculty-research/policy-topics/cities-communities/how-ai-can-empower-city-workers-make-decisions
  11. www.youtube.com www.youtube.com
    BAC : le lycée qui était le pire de France est devenu un modèle
    1
    1. tanemika 05 Oct 2026

      DOCUMENT DE BRIEFING : MODÈLES D'EXCELLENCE ET DE RACCROCHAGE SCOLAIRE DANS L'ENSEIGNEMENT SECONDAIRE

      Synthèse Exécutive

      Ce document de briefing analyse les trajectoires, les méthodologies pédagogiques et les leviers de réussite observés au sein de trois établissements scolaires franciliens : le lycée Mozart (Stains, Seine-Saint-Denis), le lycée Galilée (Gennevilliers, Hauts-de-Seine) et le Microlycée de Sénart (Évry/Sénart).

      Autrefois perçus comme en grande difficulté ou confrontés à des publics en rupture scolaire, ces établissements démontrent qu'une combinaison de rigueur disciplinaire, d'engagement enseignant, d'innovations pédagogiques et d'accompagnement sur mesure permet de renverser le déterminisme social et de réengager les élèves vers l'obtention du baccalauréat et l'accès aux études supérieures.

      Constats clés :

      • Redressement spectaculaire du Lycée Mozart (Stains) : Classé dernier lycée de France il y a 10 ans avec 49,7 % de réussite au baccalauréat (1 593ᵉ sur 1 593 au palmarès de L'Express), cet établissement de 870 élèves atteint désormais 88 % de réussite, dépassant la moyenne nationale.

      Il a été classé premier lycée public de France par un grand quotidien.

      • Pôle d'excellence en ZEP au Lycée Galilée (Gennevilliers) : En zone d'éducation prioritaire, la filière scientifique atteint 88 % de réussite au baccalauréat (3 points au-dessus de la moyenne nationale).

      • Efficacité des Microlycées contre le décrochage : Alors que 100 000 jeunes (9 % des élèves) quittent chaque année le système traditionnel sans diplôme, le Microlycée de Sénart réintègre 90 jeunes de 17 à 26 ans.

      70 % des élèves maintenus y décrochent leur baccalauréat.

      • Leviers communs d'efficacité : Un niveau d'exigence élevé combiné à une présence continue (devoirs surveillés chaque samedi, stages intensifs gratuits durant les vacances ou en immersion), un encadrement personnalisé et la refonte de la relation autoritaire classique au profit d'un contrat de confiance.

      1. Redressement et Rigueur : Le Cas du Lycée Mozart (Stains)

      1.1. Restauration de la discipline et lutte contre l'absentéisme

      Sous l'impulsion de Jamel Malek, responsable de la vie scolaire depuis 10 ans, le lycée Mozart a appliqué une politique d'intransigeance face aux retards et aux absences :

      • Fermeture des portes à 8h10 : Aucun retard n'est toléré.

      Les élèves retardataires sont envoyés en permanence afin de ne pas perturber les cours.

      • Réduction de l'absentéisme : Le taux d'absentéisme a été divisé par deux en une décennie.

      • Sanctions graduées et présence le samedi : Les retenues pour absences injustifiées (ex. sécher le dernier cours pour réviser une autre matière) vont de 1 heure en semaine à 4 heures le samedi matin, effectuées directement sous la surveillance du responsable de la vie scolaire.

      • Partenariat avec les familles : La sévérité est pensée comme un soutien aux familles souvent dépassées par la gestion de leurs enfants.

      1.2. Stratégies pédagogiques et partenariats d'excellence

      • Devoirs sur table (DST) hebdomadaires : Depuis 6 ans, aucun cours ordinaire n'est dispensé le samedi matin ; ce créneau est exclusivement réservé à des épreuves blanches hebdomadaires de 4 heures pour habituer les élèves à la pression des examens.

      • Stages de révision gratuits pendant les vacances : Durant les vacances de printemps, l'établissement reste ouvert.

      Des enseignants volontaires (tels que Jean-Luc Thomasie, professeur de physique-chimie depuis 23 ans) dispensent gratuitement des stages intensifs aux terminales, financés en heures supplémentaires.

      • Convention avec Sciences Po Paris : Depuis 10 ans, le lycée est partenaire de Sciences Po.

      Les élèves sont dispensés des épreuves écrites et préparent un oral spécifique via un atelier hebdomadaire d'actualité et d'expression orale (animé par Gillard Chambault, professeur d'histoire-géographie).

      En moyenne, deux élèves du lycée intègrent Sciences Po Paris chaque année, créant une dynamique collective qui tire l'ensemble de l'établissement vers le haut.

      • Inclusion absolue : Aucun élève n'est exclu pour insuffisance de résultats scolaires.

      2. Culture de l'Examen et Immersion : Le Cas du Lycée Galilée (Gennevilliers)

      2.1. Simulation en conditions réelles et suivi continu

      Au lycée Galilée, situé en Zone d'Éducation Prioritaire (ZEP), la préparation des 650 candidats au baccalauréat repose sur l'entraînement intensif :

      • Devoirs surveillés du samedi : Organisation régulière d'épreuves de 4 heures dans des espaces réaménagés (ex. la cafétéria) sous la supervision des enseignants (comme MM. Lou et Abadi), confrontant les élèves aux sujets récemment tombés dans les centres d'examen à l'étranger (ex. Liban).

      • Gestion du stress et soutien parental : Un fort suivi parental est observé chez les élèves visant des mentions (ex. Guillaume, encouragé par son père Clarence, gestionnaire de patrimoine ayant repris ses études à 40 ans).

      2.2. Le Stage de Ménilles : L'immersion pédagogique

      Chaque année depuis 30 ans, les élèves de terminale participent à un stage d'immersion de 5 jours au château de Ménilles, propriété de la municipalité de Jeunevilliers :

      • Organisation : Coût de 150 € par élève. 8 heures de cours par jour dispensées en demi-groupes (17 à 20 élèves).

      • Encadrement étendu : Préparation aux oraux de langues et de spécialités s'étendant de 20h30 à 23h00 avec des enseignants en poste ou des retraités volontaires (ex. Dominique Engel, ancienne professeure d'allemand).

      • Méthodes alternatives : Cours sur le terrain, comme les séances de SVT en plein air animées par Pierre-Yves Mercier (normalien et agrégé) pour appliquer concrètement les notions théoriques (ex. la pollinisation).

      3. Le Microlycée de Sénart : La Réintégration des Décrocheurs Scolaires

      3.1. Diagnostic du décrochage et profil du public

      Le Microlycée de Sénart est l'une des 61 structures publiques françaises dédiées aux jeunes en rupture scolaire.

      • Chiffres clés : Accueil de 90 élèves âgés de 17 à 26 ans, de la seconde à la terminale.

      La structure reçoit environ 40 candidatures par an pour seulement 20 places disponibles.

      • Facteurs de décrochage : Phobie scolaire, harcèlement en raison de l'orientation ou de l'apparence physique (ex. Lola, déscolarisée 2 ans suite à du harcèlement et un séjour en hôpital psychiatrique), dépression liée à des ruptures familiales (ex. Romain, ancien élève brillant arrêté pendant 2 ans), ou désocialisation et rythme nocturne (ex. Léo, déscolarisé pendant 2 ans).

      3.2. Dispositifs d'accompagnement et déconstruction du cadre traditionnel

      Le Microlycée applique un modèle pédagogique alternatif visant la réconciliation avec l'institution scolaire :

                    ```
              ┌──────────────────────────────────────────┐
              │    DÉCROCHAGE ET RUPTURE SCOLAIRE       │
              │ (Phobie, harcèlement, dépression, nuit) │
              └────────────────────┬─────────────────────┘
                                   │
                                   ▼
              ┌──────────────────────────────────────────┐
              │       MICROLYCÉE DE SÉNART : LEVER      │
              │          LES FREINS STRUCTURELS          │
              └────────────────────┬─────────────────────┘
                                   │
     ┌─────────────────────────────┼─────────────────────────────┐
     ▼                             ▼                             ▼

      ┌──────────────────┐ ┌──────────────────┐ ┌──────────────────┐ │ RITUALISATION │ │ PÉDAGOGIE ET │ │ GOUVERNANCE │ │ BIENVEILLANTE │ │ ÉVALUATION │ │ PARTAGÉE │ ├──────────────────┤ ├──────────────────┤ ├──────────────────┤ │• Réveil vocal │ │• Groupes réduits │ │• Responsabilité │ │ personnalisé │ │ (max 9 élèves) │ │ des élèves (ex. │ │• Tolérance des │ │• Tutoiement et │ │ gestion budget) │ │ retards sans │ │ prénoms │ │• Conseils de │ │ sanction │ │• Évaluation en % │ │ classe en « speed│ │• Pauses mixtes │ │ co-construite │ │ dating » │ └──────────────────┘ └──────────────────┘ └──────────────────┘ ```

      • Le réveil personnalisé : Une enseignante d'anglais (Christine) appelle chaque matin individuellement les élèves absents pour les encourager à venir en cours sans utiliser la menace.

      • Souplesse disciplinaire : Pas de sanctions d'exclusion pour retard.

      Dérogations accordées pour autoriser la cigarette durant les pauses ou l'écoute de musique sous casque pendant le travail individuel.

      • Effectifs réduits et proximité : Classes limitées à 9 élèves.

      Les élèves tutoient les professeurs et les appellent par leur prénom.

      • Évaluation co-construite : Remplacement des notes traditionnelles sur 20 par un pourcentage de réussite négocié et discuté entre l'élève et l'enseignant (ex. négociation d'une reprise de devoir non finalisé pour améliorer le score).

      • Conseils de classe en « speed dating » : L'élève participe activement à son propre conseil de classe.

      Il passe devant chaque professeur pour des entretiens individuels de 3 minutes afin de discuter et valider conjointement les appréciations.

      • Responsabilisation : Implication des élèves dans les tâches collectives (ex. Léo gérant le budget de 30 € par élève pour le ravitaillement des pauses-café communes).

      • Groupes de parole pour les parents : Réunions régulières organisées pour permettre aux parents d'élèves décrocheurs d'échanger sur leur détresse et de mesurer les progrès constatés.

      4. Tableau Comparatif des Modèles d'Établissements

      | Critère | Lycée Mozart (Stains) | Lycée Galilée (Gennevilliers) | Microlycée de Sénart | | --- | --- | --- | --- | | Public cible | 870 élèves de banlieue ( Seine-Saint-Denis ) | 650 candidats au BAC (ZEP, Hauts-de-Seine) | 90 décrocheurs scolaires (17-26 ans) | | Ancien statut / Problématique | Dernier lycée de France il y a 10 ans (49,7 % de réussite) | Zone d'Éducation Prioritaire (ZEP) | Rupture scolaire prolongée (1 à 3 ans) | | Résultats actuels | 88 % de réussite au BAC ; 1er lycée public de France (palmarès quotidien) | 88 % de réussite en Terminale S (+3 pts vs moyenne nationale) | 70 % de réussite au BAC (20 % d'abandon en cours d'année) | | Dispositif clé 1 | Fermeture stricte des portes à 8h10 & retenues le samedi | Devoirs surveillés de 4h le samedi matin | Classes à effectifs réduits (max 9 élèves) | | Dispositif clé 2 | Stages de révision gratuits pendant les vacances de printemps | Stage d'immersion de 5 jours au château de Ménilles | Conseils de classe interactifs en "speed dating" | | Partenariat / Spécificité | Convention Sciences Po Paris (admission sur oral) | Préparation intensive aux oraux de langues jusqu'à 23h | Évaluation co-construite en % (pas de note /20) | | Relation Pédagogique | Cadre strict, autorité bienveillante, zéro exclusion | Partenariat intensif profs-élèves, encadrement fort | Tutoiement, absence de sanction, réveil téléphonique |

      5. Synthèse des Parcours Indiviudels et Témoignages

      5.1. Élèves du cursus ordinaire (Mozart et Galilée)

      • Collins (20 ans, Terminale S, Mozart) : En échec l'année précédente, passé par la délinquance et la garde à vue dans sa cité à Bondy.

      Son père Moïse (60 ans, chauffeur de bus, arrêté l'école en 5ᵉ) se lève à 3h du matin et le pousse à obtenir le BAC pour éviter les travaux pénibles.

      Collins révise avec l'aide de ses camarades du quartier (ex. Edwin, étudiant en fac de maths).

      • Fati (17 ans, Terminale S, Mozart) : 11/20 de moyenne en physique.

      Ses parents d'origine turque ne parlent pas français à la maison (père dans le bâtiment, mère couturière ayant arrêté l'école en CM2).

      Arrivé à la maternelle sans parler français, Fati sert d'interprète à ses parents et vise une classe préparatoire puis une école d'ingénieurs.

      • Maxence (17 ans, Terminale L/ES, Mozart) : Issu de la classe moyenne (père inspecteur des finances, mère puéricultrice), il bénéficie du coaching Sciences Po pour devenir avocat d'affaires.

      • Guillaume et Lénaël (17 ans, Terminale ES, Galilée) : Visent la mention Très Bien et Sciences Po.

      Guillaume s'appuie sur le soutien strict de son père Clarence (gestionnaire de patrimoine) et sur des cours en ligne dispensés par des enseignants retraités sur YouTube.

      5.2. Élèves du Microlycée de Sénart

      • Romain (17 ans, Seconde) : Ancien élève brillant tombé en dépression après le divorce de ses parents.

      Après 2 ans d'interruption totale, il est devenu le premier élève de sa classe avec des taux de réussite de 75 % à 100 %.

      • Lola (17 ans, Seconde) : Déscolarisée pendant 2 ans suite à du harcèlement en collège lié à son homosexualité.

      Après un séjour en hôpital psychiatrique, elle a raccroché grâce au Microlycée.

      Malgré un niveau de 5ᵉ en langues, l'équipe enseignante lui accorde un passage conditionnel/repêchage pour soutenir son ambition de devenir avocate.

      • Léo (19 ans, Seconde) : Exclu de plusieurs lycées, il a vécu la nuit pendant un an.

      En réintégration, il assume désormais la fonction d'intendant pour le matériel de convivialité du lycée et compose des textes de musique.

      6. Quotes et Citations Clés

      « Si on les accepte à chaque fois qu'ils sont en retard, les professeurs ne pourront pas faire leur cours correctement.

      C'est sévère, mais on a de bons résultats par rapport à la présence en classe. » — Jamel Malek, responsable de la vie scolaire au lycée Mozart.

      « Les études c'est la clé, c'est la clé du succès, faut pas lâcher. Nous ici on lâche trop, on prend trop exemple sur les mauvaises personnes. » — Un habitant de la cité de Bondi, chauffeur de bus et ancien élève de bac pro.

      « Moi je me lève à 3h du matin pour aller bosser. Si lui il a son bac, ces heures-là il va pas les connaître. [...] Sa seule satisfaction, c'est d'avoir son bac et de voir le sourire de ses parents. » — Moïse, père de Collins (lycée Mozart).

      « Je pense qu'ils ont besoin d'un coup de pouce supplémentaire, ils n'ont pas forcément tout à leur disposition sur le plan financier pour se payer des professeurs particuliers. Nous, si on peut leur donner ça, c'est avec plaisir. » — Jean-Luc Thomasie, professeur de physique-chimie au lycée Mozart.

      « Le diplôme c'est vraiment important, c'est un passeport pour les entreprises. C'est la seule façon d'ouvrir les portes de l'avenir. » — La mère de Fati (traduite par son fils).

      « L'idée ce n'est pas que 4 ou 5 élèves réussissent Sciences Po... L'idée c'est qu'il y ait une dynamique collective, que l'ensemble du lycée soit tiré vers le haut. »

      — Gillard Chambault, professeur d'histoire-géographie au lycée Mozart.

      « Ici au Microlycée, l'évaluation ne doit pas être un couperet ou une note qui sanctionne, mais une note sur laquelle on peut s'appuyer, une note qui aide. Le vécu de la note classique est souvent très destructeur. » —

      Emmanuelle Catinois, professeure de français au Microlycée de Sénart.

      « Vraiment, vous nous avez sauvés.

      Le Romain de l'année dernière n'avait plus envie de rien...

      Aujourd'hui je retrouve un gamin de 17 ans bien dans ses baskets, qui adore venir. C'est le jour et la nuit, on respire enfin. » — Laurence, mère de Romain (Microlycée de Sénart).

      reportage lycée discipline comportement absentéisme 93 2026 IVAL accompagnement baccalauréat
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  12. www.biorxiv.org www.biorxiv.org
    Hippocampal and prefrontal contributions to memory-guided navigation depend on task epoch
    5
    1. Public_Reviews 05 Oct 2026

      eLife Assessment

      Dohi et al. asked what role the dorsal hippocampus and medial prefrontal cortex play during different temporal epochs of a memory-guided navigation task, a long-standing question for neuroscientists studying hippocampal-prefrontal contributions to working memory. This useful study used a delayed, cue-guided T-maze task in mice and reported impaired choice accuracy when silencing occurred early in the central-arm run but not during the delay. However, the evidence for the study's claim is incomplete in its current form: the silencing windows are not duration-matched across epochs, key negative findings rest on three to four mice without power analysis or reported effect sizes, no non-mnemonic control task distinguishes disrupted memory-guided behavior from a general action-selection deficit, and no neural recordings accompany the causal manipulations to verify the manipulations' assumed mechanistic effects. The reported perseveration also reflects an increased directional bias rather than repetition of the previous choice, and the language should be revised accordingly.

      Summary
    2. Public_Reviews 05 Oct 2026

      Reviewer #1 (Public review):

      Summary:

      In this study, the authors trained mice to perform a memory-guided navigation task, in which they must navigate to a previously cued arm after a delay period. They optogenetically inhibited the dorsal hippocampus or mPFC (targeting PL) in different task epochs. They found that for both regions, inactivating at the beginning of the navigation epoch impaired performance and induced mice to revert to habitual side biases. Inactivating during other epochs, including a delay period before the navigation phase, had little or no impact on behavior. The relationship between trial duration and behavioral performance was differentially impacted by hippocampal and mPFC inactivation, suggesting that the nature of the deficits was somewhat different.

      Strengths:

      The effects of perturbations are robust across animals and generally convincing. The lack of effect at some task epochs serves as a nice internal control. The finding that hippocampus and mPFC inactivation produced subtly different effects is interesting.

      Weaknesses:

      The simplicity of the behavior makes it difficult to resolve how exactly the hippocampus and mPFC contribute to working memory. Also, the language does not always reflect the trends in the data: the authors claim that optogenetic perturbations cause mice to repeat previous choices, but the data show that perturbations increase the likelihood of choosing a preferred side (which is left for most mice). A side bias is not the same as choice repetition. This has implications for interpreting the nature of the behavioral effects.

      Review 1
    3. Public_Reviews 05 Oct 2026

      Reviewer #2 (Public review):

      Summary:

      The study uses transient optogenetic silencing of the dorsal hippocampus or prefrontal cortex in mice using a delayed response task in a T-maze, with a temporal delay of 1s after a visual cue, followed by a central stem run period before choice execution. Silencing of either the dorsal hippocampus or the dorsal prefrontal cortex is executed for varying time periods during the stem/ central arm running epoch or the 1s temporal delay epoch by targeting either PV+ or Dlx interneurons in a block-wise or random-trial design. The main result reported is that silencing of either region during long periods of stem running impaired choice behavior, and silencing during the temporal delay period did not have an effect.

      Strengths:

      The major strength of the study is using the optogenetic silencing strategy to target different temporal periods of the task.

      Weaknesses:

      (1) A major weakness of the study is the lack of a balanced design with equal time periods of silencing during the stem running period and temporal delay period in many of the animals, which precludes any conclusion about distinct functional roles of the regions during these two phases of the task. The central question of this study is not new, with many previous studies investigating distinct and overlapping roles of hippocampus and prefrontal cortex in spatial working memory tasks and memory-guided navigation, using inactivation of one or both regions, crossed inactivation approaches, as well as targeting direct and indirect connections between the regions (PMIDs: 20074655, 9030646, 17045348, 30179661, 27511010, 10491611, 26017312, etc.), in addition to several physiology studies. A key extension for the current study would have been to show a distinction between roles in the temporal delay period after the cue and the stem-running working memory period. However, the inactivation period during stem running shows effects only for long inactivation periods, 2s initial periods and later ~1.6s periods (run after 0.8s, >2/3 total running periods), whereas the temporal delay period inactivation is 1s for the large majority of animals, which is a clear mismatch in inactivation periods, obviating this conclusion of distinction.

      (2) It is not clarified why such short delay periods were used compared to long periods of ~10s in T-maze spatial alternation tasks with delay, and whether the temporal delay period of 1 sec is strictly distinct from the spatiotemporal delay period during stem running in terms of short-term memory function. The choice of time windows for inactivation needs to be better justified, which currently appears to be rather random (2s initial running period, run after 0.8s, run after 1.6s; for an average reported running period of ~2.4-2.5s). The ideal design clearly would have been to use a 2s temporal delay period so that the inactivation time in this epoch matched the initial 2s run period. Only a subset of 4 animals were run with a longer temporal delay period, and too with only with hippocampal inactivation (Figure 4d). The main conclusion of distinction between temporal delay and stem running delay periods is therefore not adequately tested for the prefrontal cortex, and the statistics in terms of number of animals for this important control for hippocampal inactivation are also not comparable to the main experiment.

      (3) The mixture of PV-Cre animals (5 animals), Dlx targeting (2 animals), and one WT animal is also suggestive of a fragmented approach, and inactivation efficacy cannot be assumed to be similar for different animals. Importantly, there is no physiological evidence for confirmation of suppression in the optogenetic experiments, even in exemplar animals.

      Review 2
    4. Public_Reviews 05 Oct 2026

      Reviewer #3 (Public review):

      The authors sought to determine when the dorsal hippocampus and mPFC are causally required during a delayed spatial working memory task. Using temporally precise optogenetic silencing in mice performing a delayed cue-guided T-maze task, they tested the effects of transient perturbations during distinct behavioral epochs. Contrary to the common view that these regions are primarily required during the delay period to maintain working memory representations, they report that silencing during the delay had little effect on performance, whereas perturbation during the early phase of central-arm traversal consistently impaired performance. The authors conclude that hippocampal and prefrontal contributions to memory-guided behavior are dynamically engaged during active navigation rather than passive maintenance of information.

      The study has several strengths. The behavioral paradigm is carefully designed to dissociate cue, delay, and movement epochs, allowing temporally specific causal manipulations. The systematic comparison of multiple task epochs represents a major strength and provides compelling evidence that the behavioral effects of perturbation are epoch-dependent. The authors also include several important controls, including stimulation during multiple task phases, a longer-delay condition to dissociate task epoch from elapsed time since cue presentation, and analyses of movement trajectories and perseverative behavior that provide additional insight into the nature of the behavioral deficits. Together, these experiments convincingly demonstrate that transient dorsal hippocampal and mPFC perturbations have markedly different behavioral consequences depending on when they occur within a trial.

      The evidence is generally solid and supports the primary finding that perturbations during early navigation produce larger impairments than perturbations during the delay period. However, some aspects of the broader interpretation are less well supported. Most notably, the study lacks a non-memory control task, such as a visually guided version of the maze, making it difficult to determine whether the observed deficits specifically reflect disruption of memory-guided behavior or more general impairments in action selection, behavioral flexibility, or movement planning. The observed increase in perseverative responding and delayed commitment to a turn are consistent with either interpretation. In addition, several experimental conditions rely on relatively small numbers of animals, limiting confidence in some negative findings, particularly for the longer-delay and later-run manipulations. Finally, while the Discussion proposes that hippocampal-prefrontal circuits become engaged during the transformation of stored information into action, this mechanistic interpretation remains speculative because no neural recordings accompany the causal manipulations.

      Overall, the authors achieve their primary aim of demonstrating that the behavioral consequences of dorsal hippocampal and mPFC silencing depend strongly on task epoch. The data convincingly support the conclusion that these structures are more vulnerable to perturbation during early navigation than during the brief delay period used in this task. The broader conclusion that these findings redefine when hippocampal-prefrontal circuits support working memory should be interpreted more cautiously, as alternative explanations involving action selection or behavioral state remain plausible in the absence of additional control tasks.

      The findings are potentially important because they challenge the common assumption that hippocampal and prefrontal contributions to delayed-response tasks are centered on delay-period maintenance. Instead, the work supports the idea that these circuits may be recruited when remembered information is translated into goal-directed behavior. This framework is broadly consistent with recent distributed models of working memory and provides an interesting perspective that may help reconcile previous studies reporting effects during different task phases. The behavioral paradigm and temporally precise perturbation approach should also be useful for future studies aimed at dissecting the dynamic contributions of hippocampal-prefrontal circuits during memory-guided behavior.

      Review 3
    5. Public_Reviews 05 Oct 2026

      Author response:

      On the eLife Assessment. We appreciate the assessment’s recognition that this study addresses a long-standing question concerning hippocampal and prefrontal contributions to working memory. We think the broader significance lies in constraining how causal manipulations in working-memory tasks are interpreted. Working memory encompasses many processes distributed across a trial and showing that a region is required for a delayed-response task does not establish when its contribution is necessary. The observation that hippocampus and mPFC are required while navigating to a goal, but not during stationary delay, challenges a common assumption and, in our opinion, has implications across the broader field of working-memory research.

      We agree that the original manuscript did not adequately report effect sizes or convey the uncertainty associated with some smaller samples. However, the dataset does include duration-matched stationary and running conditions, including a hippocampal long-delay control matched to early-running stimulation in both duration and elapsed time after cue onset. The new effect-size and within-animal analyses support a robust hippocampal epoch difference, while the corresponding mPFC comparison is less precisely estimated and should be interpreted more cautiously.

      We therefore think the central finding remains well supported: hippocampal function, and potentially mPFC function, is required during the active navigation period of this memory-guided task but not detectably during the stationary delay. This does not establish the specific computation disrupted during running. Neural recordings would certainly provide further insight into the underlying mechanism, but their absence does not detract from the value of the behavioral result itself.

      Reviewer #1 (Public review):

      The simplicity of the behavior makes it difficult to resolve how exactly the hippocampus and mPFC contribute to working memory.

      The task was designed to combine the temporal precision of cue-based delayed-response paradigms with the behavioral richness of freely moving navigation. Few tasks combine a fixed cue-presentation period, an explicit delay, and a subsequent navigation phase involving extended running. This structure creates well-defined behavioral epochs that can be targeted with temporally precise perturbations, allowing us to ask when hippocampal and mPFC contributions are required within an ongoing memory-guided behavior. How these regions contribute is the harder question, and one we are pursuing next. Identifying when perturbations disrupt behavior is an important step toward understanding how these regions support memory-guided navigation

      Also, the language does not always reflect the trends in the data: the authors claim that optogenetic perturbations cause mice to repeat previous choices, but the data show that perturbations increase the likelihood of choosing a preferred side (which is left for most mice). A side bias is not the same as choice repetition. This has implications for interpreting the nature of the behavioral effects.

      We agree that our results do not clearly distinguish a directional bias from a tendency to repeat the previous choice. To examine whether mice consistently favored a particular direction, we compared their side preferences during silencing across sessions. Mice generally favored the same side across silencing conditions, although some switched direction in individual sessions (Author response image 1a). Within sessions, the preferred side was maintained from no-stimulation to stimulation trials in 13 of 19 cases and reversed in six (Author response image 1b). These observations are consistent with a directional preference that can sometimes reverse during stimulation, and cannot be disambiguated from perseveration. In the revision, we will describe the effect as increased directional bias and revise the language concerning choice repetition and perseveration throughout the manuscript.

      Author response image 1.

      Silencing increases directional bias. (a) Fraction of choices made to the right in each session, for every mouse (rows) and each silencing condition (symbols). Open symbols, no-stimulation trials; filled symbols, stimulation trials from the same session; blue and red denote a left or right preference during silencing. Filled symbols falling predominantly on the same side of 0.5 within a row indicate that a mouse generally favored the same direction across silencing conditions, although some mice switched direction. One session per mouse and condition, hippocampal silencing only; T2 and T6 did not perform the 0.8 s condition. (b) The same sessions expressed as signed bias, from no stimulation to silencing. Black lines mark the six sessions in which the preferred side reversed; grey lines the thirteen in which it was maintained.

      Reviewer #2 (Public review):

      A major weakness of the study is the lack of a balanced design with equal time periods of silencing during the stem running period and temporal delay period in many of the animals, which precludes any conclusion about distinct functional roles of the regions during these two phases of the task. The main conclusion of distinction between temporal delay and stem running delay periods is therefore not adequately tested for the prefrontal cortex, and the statistics in terms of number of animals for this important control for hippocampal inactivation are also not comparable to the main experiment.

      We agree that matching stimulation duration is an essential control and recognize that the relevant comparisons and statistics were not sufficiently clear in the original manuscript. Four hippocampal conditions used closely matched stimulation durations of 2 s: Cue+Delay, long delay, early running, and running with a 0.8 s onset (Author response image 2a). Crucially, the long-delay control matched both stimulation duration and elapsed time after cue onset to the early-run condition, while the mouse remained stationary.

      Author response image 2b–c shows the estimated impairment and its 95% confidence interval for each condition. In the hippocampal experiments, the duration-matched stationary conditions showed effects close to zero, whereas the running conditions showed large impairments. Despite the smaller sample, the upper confidence limit for the long-delay impairment was approximately 10 percentage points, substantially below the observed early-running impairment. These estimates establish that despite the smaller sample, the data support a lack of effect compared to early running.

      Author response image 2.

      Duration-matched stimulation produces different behavioral effects across task epochs. (a) Stimulation timing relative to cue onset. Numbers within bars indicate calculated median stimulation duration in seconds; black ticks indicate door opening. Bold labels identify conditions with 2 s stimulation. (b-c) Mean impairment in choice accuracy for hippocampal and mPFC manipulations. Impairment is accuracy during baseline minus accuracy with stimulation, in percentage points. Error bars show 95% confidence intervals across animals; numbers indicate mice. Open circles denote single-animal observations, and arrows indicate confidence intervals extending beyond the plotted range.

      For mPFC, the duration-matched Cue+Delay condition likewise showed an effect close to zero, whereas early-running stimulation produced substantial impairment. However, the long-delay condition included only one mouse. The later-running effects in both regions were also less precisely estimated. We will distinguish these limitations from the more informative stationary-condition results.

      To directly test whether the duration-matched effects differed across epochs, we next compared impairment within the same mice, including only animals tested in both conditions (Author response image 3). For the hippocampus, every mouse showed greater impairment during early running than during either duration-matched stationary condition, and the confidence intervals for both paired differences excluded zero. These within-animal comparisons support an epoch-dependent effect that cannot be explained by stimulation duration alone. The mPFC comparison showed the same direction of effect, although the confidence interval for the Cue+Delay versus running difference narrowly included zero. We will therefore distinguish the stronger evidence for the hippocampal epoch difference from the more limited evidence for mPFC.

      Author response image 3.

      Within-animal comparisons of duration-matched stimulation effects. (a–b) Impairment during Cue+Delay or long-delay stimulation compared with early-running stimulation for hippocampal (a) and mPFC (b) manipulations. Points represent individual mice, lines connect observations from the same mouse, and black bars indicate mean. Annotations report the mean paired difference in impairment (running minus stationary), and its 95% confidence interval calculated using the t distribution. Positive differences indicate greater impairment during running. The single-mouse mPFC long-delay comparison is descriptive. Blue indicates stationary epochs and orange indicates running.

      The central question of this study is not new, with many previous studies investigating distinct and overlapping roles of hippocampus and prefrontal cortex in spatial working memory tasks and memory-guided navigation, using inactivation of one or both regions, crossed inactivation approaches, as well as targeting direct and indirect connections between the regions (PMIDs: 20074655, 9030646, 17045348, 30179661, 27511010, 10491611, 26017312, etc.), in addition to several physiology studies.

      We agree that hippocampal and prefrontal contributions to spatial working memory have been extensively studied. That’s precisely why we find these results impactful when placed in the rich context of the field. The requirement for these regions in delayed working memory tasks has often been interpreted in terms of their contributions during the delay period. However, a requirement during a delay-based task does not itself demonstrate a requirement during the delay. Previous manipulations have not isolated delay periods of waiting from the subsequent navigation within a trial.

      Our experiments extend this work by separately targeting cue presentation, the delay, and different portions of navigation within the same task. This allows us to test whether the behavioral consequences of perturbation depend on the particular epoch in which it occurs. This distinction is important: knowing that a region is required for a memory-guided task does not establish when its contribution is needed. Identifying those periods constrains how we interpret the deficits produced by longer-lasting inactivation. We believe that this is an important result that should be considered when interpreting these broader findings. We will ensure that the appropriate literature and discussion are included in the revision.

      It is not clarified why such short delay periods were used compared to long periods of ~10s in T-maze spatial alternation tasks with delay, and whether the temporal delay period of 1 sec is strictly distinct from the spatiotemporal delay period during stem running in terms of short-term memory function.

      The 1 s delay was chosen to maintain reliable task performance, as some mice could not perform the task with longer delays. Indeed, one reason the longer-delay condition includes fewer animals is that two mice could not perform reliably with the 3 s delay (one additional mouse was not tested in this condition). Delays on this timescale have also been used in rodent cued delayed-response tasks, including a 0.5 s delay in Kopec et al. (2015), a 1.3 s delay in Guo et al. (2014), and a 1.2 s delay in Inagaki et al. (2019). Like these tasks, our paradigm requires mice to remember an externally presented cue specifying the upcoming response, rather than their own previous arm choice as in spatial alternation. It therefore combines spatial navigation with a cued delayed-response requirement, and the delay durations tolerated in alternation tasks are not necessarily directly comparable. We will clarify this rationale in the manuscript.

      We agree that the stationary delay and the subsequent run both require retention of information after cue offset. Our experiments distinguish these behavioral epochs, but do not establish that they involve separate short-term memory processes. The different effects of perturbation suggest that the contribution of these regions changes as the animal moves from waiting to navigating. Determining what accounts for this change is an important direction for future work.

      The choice of time windows for inactivation needs to be better justified, which currently appears to be rather random (2s initial running period, run after 0.8s, run after 1.6s; for an average reported running period of ~2.4-2.5s).

      We sought to target different portions of the central-arm run. Given the typical traversal time of approximately 2.4–2.5 s, stimulation onsets at 0, 0.8, and 1.6 s sampled the beginning, middle, and later portions of the run. Our setup allowed precise control of stimulation timing, and, as shown in Figure 4b, these onset times correspond approximately to the start, middle, and end of the central arm. Each condition used a nominal 2 s stimulation window, truncated if the mouse reached the choice point sooner. The windows therefore overlap, with the later-onset condition generally producing shorter stimulation. We will clarify this rationale and the distinction between stimulation onset and duration in the revised manuscript.

      The mixture of PV-Cre animals (5 animals), Dlx targeting (2 animals), and one WT animal is also suggestive of a fragmented approach, and inactivation efficacy cannot be assumed to be similar for different animals. Importantly, there is no physiological evidence for confirmation of suppression in the optogenetic experiments, even in exemplar animals.

      The Dlx animals were included to improve regional specificity through local viral expression and to test whether the behavioral effects were consistent across targeting approaches. Both approaches produced comparable impairments during running, supporting their inclusion in the same analysis. We will clarify this rationale in the manuscript.

      Optogenetic activation of inhibitory interneurons is an established approach for suppressing local principal-cell activity, with physiological validation in previous studies (Guo et al., 2014; Li et al., 2019, Zutshi et al., 2022). In our experiments, running-period stimulation produced robust behavioral impairments that were consistent across animals and targeting approaches. Furthermore, comparisons across epochs were performed within animals, using the same preparation and stimulation parameters. Differences in efficacy between animals therefore cannot readily account for the observed epoch dependence. Although direct recordings would establish the magnitude and spatial extent of suppression in our preparation, the central behavioral finding is supported by these within-animal comparisons.

      Reviewer #3 (Public review):

      The findings are potentially important because they challenge the common assumption that hippocampal and prefrontal contributions to delayed-response tasks are centered on delay-period maintenance.

      We thank the reviewer for describing our findings as “potentially important” and for highlighting their implications for how hippocampal and prefrontal contributions to delayed-response tasks are understood. We appreciate the constructive suggestions and address the public comments below.

      Most notably, the study lacks a non-memory control task, such as a visually guided version of the maze, making it difficult to determine whether the observed deficits specifically reflect disruption of memory-guided behavior or more general impairments in action selection, behavioral flexibility, or movement planning. The observed increase in perseverative responding and delayed commitment to a turn are consistent with either interpretation.

      We agree that leaving the cue on throughout the trial would provide an important control for distinguishing memory-specific effects from broader effects on action selection or movement planning. The senior author is currently setting up a new laboratory, so implementing this control may take some time. We hope to include it in the revised manuscript.

      The running-period deficit indicates a disruption of processes that enable the animal to act on a remembered cue. Whether this reflects disruption of memory itself, movement planning, or another component of translating the cue into a choice requires further clarification but does not detract from the observed dependence on task epoch. Uncertainty about the mechanism of the running-period deficit also does not change the observation that the same manipulation produced no detectable impairment during the stationary delay, when the cue was absent and still had to be remembered. This will be clearly discussed in the revision.

      In addition, several experimental conditions rely on relatively small numbers of animals, limiting confidence in some negative findings, particularly for the longer-delay and later-run manipulations.

      We agree that small sample sizes limit the interpretation of some negative findings. We now report animal-level effect estimates and 95% confidence intervals for each condition (Author response image 2b–c; Author response table 1).

      Of the nine conditions with no detectable impairment and more than one mouse, seven had confidence intervals that excluded effects as large as the observed mean early-running impairment in the same region. This included the hippocampal long-delay condition, despite its smaller sample. These results argue against similarly large impairments in these conditions, although smaller effects remain possible. The two later-run conditions remained too uncertain to exclude such impairments. These and the two single-animal conditions are marked in Author response table 1 and will be interpreted cautiously.

      Author response table 1.

      Animal-level impairment estimates and uncertainty

      Finally, while the Discussion proposes that hippocampal-prefrontal circuits become engaged during the transformation of stored information into action, this mechanistic interpretation remains speculative because no neural recordings accompany the causal manipulations.

      We will clarify in the Discussion that the proposed transformation of stored information into action remains speculative. Nevertheless, we believe this is an exciting possibility raised by our findings that warrants further investigation. Neural recordings would help test this interpretation but are beyond the scope of the current paper. We plan to explore this question in future work.

      References

      Guo ZV, Li N, Huber D, Ophir E, Gutnisky D, Ting JT, Feng G, Svoboda K (2014). Flow of cortical activity underlying a tactile decision in mice. Neuron 81:179–94. doi:10.1016/j.neuron.2013.10.020. PMID 24361077.

      Kopec CD, Erlich JC, Brunton BW, Deisseroth K, Brody CD (2015). Cortical and subcortical contributions to short-term memory for orienting movements. Neuron 88:367–77. doi:10.1016/j.neuron.2015.08.033. PMID 26439529.

      Inagaki HK, Fontolan L, Romani S, Svoboda K (2019). Discrete attractor dynamics underlies persistent activity in the frontal cortex. Nature 566:212–217. doi:10.1038/s41586-019-0919-7. PMID 30728503.

      Li N, Chen S, Guo ZV, Chen H, Huo Y, Inagaki HK, Chen G, Davis C, Hansel D, Guo C, Svoboda K (2019). Spatiotemporal constraints on optogenetic inactivation in cortical circuits. eLife 8:e48622. doi:10.7554/eLife.48622. PMID 31736463.

      Zutshi I, Valero M, Fernández-Ruiz A, Buzsáki G (2022). Extrinsic control and intrinsic computation in the hippocampal CA1 circuit. Neuron 110:658–673.e5. doi:10.1016/j.neuron.2021.11.015. PMID 34890566.

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    Genome-wide mapping of Cas9-induced sister chromatid exchange across single and over 200 genomic targets
    5
    1. Public_Reviews 05 Oct 2026

      eLife Assessment

      Using sci-L3-Strand-seq, this study shows genome-wide, single-cell evidence that DSBs triggered by CRSIPR/Cas9 are frequently resolved through sister chromatid exchange (SCE), which is undetectable with conventional whole genome sequencing approaches. This important work provides helpful metrics quantifying the occurrence of SCE events at targeted and repetitive genomic loci that are of particular interest to those working in gene editing, DNA repair, genome instability, or repetitive genomic loci. The analysis of reciprocal daughter-cell pairs provides compelling evidence for SCE events, providing evidence consistent with CDK1-TTF2-TRAIP mediated cell-cycle regulated CMG helicase disassembly and fork cleavage at unreplicated regions.

      Summary
    2. Public_Reviews 05 Oct 2026

      Reviewer #1 (Public review):

      [Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

      Summary:

      This manuscript uses sci-L3-Strand-seq to map sister chromatid exchange events following CRISPR/Cas9-induced DNA damage. Because exchanges between identical sister chromatids are largely invisible to conventional sequencing, the study addresses an important blind spot in the assessment of genome editing outcomes. The authors compare single-locus Cas9 cleavage, simultaneous targeting of 237 repetitive genomic sites, and Cas9 nickase variants. They further use reciprocal daughter-cell pair analysis to ask whether Cas9-associated SCEs are copy-neutral or linked to larger structural alterations. Overall, this is a valuable study that introduces an important additional layer to the analysis of CRISPR/Cas9 repair outcomes. The central finding that Cas9-induced DSBs can trigger frequent local SCE is well supported and likely to be of broad interest.

      Strengths:

      The major strength of the manuscript is the application of a strand-resolved, single-cell method to a question that is difficult to address with standard genome sequencing. The evidence that a single Cas9-induced DSB can trigger strong local SCE is compelling in concept and supported by multiple guide RNAs targeting distinct loci. The reported on-target SCE frequencies, reaching up to 41%, suggest that inter-sister exchange is a substantial and underappreciated outcome of Cas9 cleavage.

      Of particular interest is the comparison between single-site and multi-site targeting. The finding that 237 programmed Cas9 targets produce only mild bulk enrichment of on-target SCE but stronger enrichment in a subpopulation of cells with elevated SCE burden is interesting and may have wider biological implications, particularly if the findings extend beyond Cas9-induced SCE to spontaneous SCEs.

      The reciprocal daughter-cell pair analysis is another notable feature of the study. The observation that some Cas9-associated SCEs are accompanied by structural alterations could challenge the assumption that SCE after a programmed break reflects error-free homologous recombination.

      Review 1
    3. Public_Reviews 05 Oct 2026

      Reviewer #2 (Public review):

      Summary:

      In this short paper, a clever single-cell Strand-seq method was used to study the number and location of sister chromatid exchange events (SCEs) in cells after CRISPR/Cas9-induced DNA double-strand breaks (DSBs). Unique as well as multiple genomic loci were targeted. Cas9-induced cuts at unique genomic locations led to statistical enrichment of SCEs at the target site, whereas Cas9 targeted at repetitive targets revealed only mild enrichment of on-target SCEs unless analysis was restricted to a subset of cells with >8 SCEs per cell. Interestingly, reciprocal daughter-cell pair analysis revealed large-scale structural alterations on some chromosomes. Whereas disruption of DNA repair genes, including LIG3, LIG4, XRCC1, and XRCC4, did not measurably alter SCE frequency per cell within 24 hrs, consistent with delayed functional loss following editing and selection against essential genes. Together, these findings demonstrate that Cas9-induced DSBs are potent local triggers of SCE at unique loci and can be associated with structural alterations, highlighting the influence of lesion type and genomic context on recombination outcomes during genome editing.

      Strengths:

      The data in this paper represent a very rich resource of how parental DNA template strands are distributed in paired daughter cells after various treatments. Abnormalities observed in only one of such paired daughter cells provide a novel and exciting approach to study mechanisms of DNA instability and DNA repair at a genome-wide level in general and following Cas9-induced DSB in particular.

      Weaknesses:

      The effect of Cas9-induced DSBs in the cells that are used will depend on the cell cycle stage of the cells that are targeted, as well as the number of times cuts are made. The latter could happen before, during, and after DNA repair reactions on one or both alleles in a diploid cell. As a result, it is very difficult to extrapolate the mechanisms of DNA instability and DNA repair from the observed genomic rearrangements. Novel approaches are needed to limit the number and timing of Cas9-induced breaks to overcome some of these limitations.

      Review 2
    4. Public_Reviews 05 Oct 2026

      Reviewer #3 (Public review):

      Chovanec and Yin used their newly developed sci-L3-Strand-seq powerful method to characterize SCE after Cas9 cleavage in a human cell line, using either a single target site or an element repeated 237 times in the genome. SCE are often neglected in DNA repair analyses since they are « genetically silent ». Interestingly, the authors found enrichment of SCE at unique Cas9 sites, but only a modest enrichment of SCE when Cas9 targets 237 sites in the genome. The genetic control of SCE formation at Cas9 sites is not deliberately addressed in this paper. However, the authors found that targeted SCE seem to be enriched in a subpopulation of cells, particularly « permissive » for SCE, but the determinants of such a population are unknown. Finally, the power of the sci-L3-Strand-seq allowed the authors to characterize a specific type of SCE based on the analysis of reciprocal daughter-cell pairs' genomes that is associated with a specific type of chromosomal rearrangement compatible with the ones observed in HR defective BRCA1/2 deficient cells.

      Review 3
    5. Public_Reviews 05 Oct 2026

      Author response:

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

      eLife Assessment

      Using sci-L3-Strand-seq, this study shows genome-wide, single-cell evidence that DSBs triggered by CRISPR/Cas9 are frequently resolved through sister chromatid exchange (SCE), which is undetectable with conventional whole-genome sequencing approaches. This important work provides helpful metrics quantifying the occurrence of SCE events at targeted and repetitive genomic loci that are of particular interest to those working in gene editing, DNA repair, genome instability, or repetitive genomic loci. However, the evidence supporting the proposed involvement of under-replicated region/replication termination-zone resolution and TRAIP/URR-like pathways is currently incomplete and could be strengthened with an increased number of reciprocal daughter-cell pairs and by genetic or molecular perturbation, or alternatively, this can be addressed by changing the discussion.

      We appreciate the editor’s and the reviewers’ recognition of Cas9-induced SCE as an important previously invisible repair outcome and of RDCP analysis as a notable feature of the study. As proposed, we incorporated the two recent Science studies and now tone down the Discussion of our RDCP observations as consistent with, rather than definitive evidence for, the TRAIP-dependent pathway. We also detail how our single-cell genomic observations complement and extend these two studies in terms of the biological significance of the CDK1-TTF2-TRAIP axis.

      Specifically, these changes are in:

      Discussion. We changed

      “Two recent studies revealed how the CDK1-TTF2-TRAIP axis is cell-cycle regulated to trigger mitotic CMG helicase disassembly and fork cleavage: one study showed a two-fold SCE reduction in mouse ES cells (Fujisawa and Labib, 2026), while the other showed that disrupting the TRAIP-TTF2 interaction reduced common fragile site deletions (Can et al., 2026). Our observation of the "WWC-or-WCC/deletion pair" signature in wild-type cells provides, to our knowledge, the first genetic evidence of linking a deletion with SCE and revealing both W and C unreplicated template strands present in the reciprocal daughter cell, consistent with this mechanism at single-cell genomic resolution (illustrated in Fig.3), although this is limited by the observation of only one RDCP.”

      While each study highlights the biological significance of the CDK1-TTF2-TRAIP axis individually for SCE and deletions, we show the coupling relationship (and further evidence of unreplicated template strands) in reciprocal daughter cells.

      Fig.3. Title and legends. We changed

      “Haplotype-aware analysis of observed RDCP (Pair 4, chr1) shows SV patterns at the SCE junctions consistent with the predicted RDCP signature of SCE mediated by URRs or replication termination zones (green shaded area), although the lagging strands, rather than the leading strands, must be resolved to generate these mitotic breaks.”

      Reviewer #1 (Public review):

      Summary:

      This manuscript uses sci-L3-Strand-seq to map sister chromatid exchange events following CRISPR/Cas9-induced DNA damage. Because exchanges between identical sister chromatids are largely invisible to conventional sequencing, the study addresses an important blind spot in the assessment of genome editing outcomes. The authors compare single-locus Cas9 cleavage, simultaneous targeting of 237 repetitive genomic sites, and Cas9 nickase variants. They further use reciprocal daughter-cell pair analysis to ask whether Cas9-associated SCEs are copy-neutral or linked to larger structural alterations. Overall, this is a valuable study that introduces an important additional layer to the analysis of CRISPR/Cas9 repair outcomes. The central finding that Cas9-induced DSBs can trigger frequent local SCE is well supported and likely to be of broad interest. The evidence for structural complexity associated with some induced SCEs is intriguing, but the mechanistic interpretation should either be tested directly or presented more cautiously.

      Strengths:

      The major strength of the manuscript is the application of a strand-resolved, single-cell method to a question that is difficult to address with standard genome sequencing. The evidence that a single Cas9induced DSB can trigger strong local SCE is compelling in concept and supported by multiple guide RNAs targeting distinct loci. The reported on-target SCE frequencies, reaching up to 41%, suggest that inter-sister exchange is a substantial and underappreciated outcome of Cas9 cleavage.

      Of particular interest is the comparison between single-site and multi-site targeting. The finding that 237 programmed Cas9 targets produce only mild bulk enrichment of on-target SCE but stronger enrichment in a subpopulation of cells with elevated SCE burden is interesting and may have wider biological implications, particularly if the findings extend beyond Cas9-induced SCE to spontaneous SCEs. Given that potential, the current manuscript would benefit greatly from any experiments characterizing this sub-population: are these cells in a particular cell cycle state, experiencing changes in gene expression, or do they have other unique biological properties?

      The reciprocal daughter-cell pair analysis is another notable feature of the study. The observation that some Cas9-associated SCEs are accompanied by structural alterations could challenge the assumption that SCE after a programmed break reflects error-free homologous recombination.

      Thank you very much for this assessment.

      Weaknesses:

      The number of informative RDCPs is limited, and the mechanistic interpretation of the "WWC-orWCC/deletion" signature is more suggestive than definitive. In particular, the manuscript invokes (even though only in the Discussion section) URR or replication-termination-zone resolution and discusses TRAIP-dependent CMG unloading, nuclease cleavage, and polymerase theta-mediated joining, but these pathway components are not directly tested herein. A more conservative conclusion that some Cas9-associated SCEs coincide with structural alterations is more appropriate, particularly in the Discussion and Conclusion. For example, the statement that this work provides "direct genetic evidence" for a URR-type mechanism is overstated unless supported by additional experiments or a more extensive analysis of alternative models. Similarly, while the authors explain the limitations of acute Cas9 disruption of LIG3, LIG4, XRCC1, and XRCC4, the manuscript should clarify what biological questions this experiment can and cannot answer.

      Please see response to the eLife Assessment as this is a common point raised by multiple reviewers.

      Additionally, we clarified what the DNA repair gene targeting experiment can and cannot answer (delayed protein loss, essential-gene selection) by adding the following text in the “Disruption of DNA repair genes at the cut site did not measurably alter SCE frequency per cell” section:

      “One additional complication is that, although Cas9 RNP achieves >90% knockout efficiency in bulk assays and is therefore used as a substitute for siRNA, sorting BrdU-labeled cells in the subsequent G1 enriches for cells that escaped frameshift editing, particularly for essential genes; thus, 100% knockout in 90% of cells is not equivalent to 90% knockdown in every cell, representing a unique challenge for single-cell assays.”

      Reviewer #1 (Recommendations for the authors):

      (1) Temper the mechanistic claims about URR/TRAIP-type resolution.

      The RDCP data support the conclusion that some Cas9-associated SCEs are accompanied by structural alterations and may arise through non-classical mechanisms. However, claims about TRAIPdependent CMG unloading, URR resolution, or polymerase theta-mediated joining should be framed as a model unless directly tested.

      We cited mechanistic dissection of the CDK1-TTF-2-TRAIP axis, which was published since the review of the paper. While each study highlights the biological significance of the CDK1-TTF2-TRAIP axis individually for SCE and deletions, we show the coupling relationship (and further evidence of unreplicated template strands) in reciprocal daughter cells but qualified that this observation is in only one RDCP. We further tempered our claims by changing “direct evidence” to “consistent with” as we did not perturb genes involved in these processes.

      (2) Clarify the impact of the small RDCP sample size.

      The manuscript would be strengthened by explicitly stating how many total RDCPs were analyzed, how many SCE events were informative, and how much confidence can be placed in the estimated fraction of SCEs associated with SVs. A short table summarizing RDCP counts, SCE counts, copy-neutral events, and SV-associated events would be helpful.

      A total of 15 RDCPs were recovered from close to 4,000 single cells analyzed across all conditions. We added Tab.S3 detailing SCEs in all 15 RDCPs in addition to SCE and SV breakdown in Tab.S2 (originally Tab.S1). The new Tab.S3 is cited in the “RDCP analysis reveals large-scale SVs on chromosomes with induced SCE, as well as structural alterations at Cas9-induced SCE junctions” section.

      (3) Provide more detail on the "rescued" SCE calls.

      Because the central conclusions rely on SCE detection, the criteria for breakpoint R-based calls versus rescued calls should be explained clearly in the main text or methods. It would be useful to know how sensitive the main conclusions are to the inclusion or exclusion of rescued calls. Is this laid out in greater detail in an additional manuscript?

      We previously included an “On-target SCE identification” section in the Methods, where we described in detail the rescue of on-target SCEs missed by the initial breakpoint R calls. We also depicted calls and calls+rescues for all the conditions in Fig.S1B.

      We agree with the reviewer and now expanded the description of rescued SCE calls in the main text (under the “A single Cas9 DSB induces potent local SCE” section). In brief, 50-92% of SCEs (typically >70%) across the four single-targeting sites were directly called rather than rescued, with the exception of LIG4, where only 30% were direct calls. This is because LIG4 is located only 6 Mb from the telomere and is therefore particularly prone to missed breakpointR calls in low-coverage cells. On-target rescue at individual sites is self-contained in this manuscript because our lab primarily focuses on spontaneous SCE, for which there are no expected SCE sites. However, the rescue methodology was previously implemented in the original development of sci-L3-Strand-seq to identify SCEs at centromeres.

      (4) Clarify the biological interpretation of the repetitive-target enrichment.

      The high-SCE subset analysis is interesting, but the manuscript should explain whether these cells have evidence of higher RNP uptake, altered cell-cycle state, greater DNA damage, or lower sequencing quality. If these possibilities cannot be distinguished, the text should state this clearly.

      We agree with the reviewer. The high SCE subset does not have lower sequencing quality by coverage or background (0.3% coverage for high-SCE vs. 0.28% coverage overall, and 3% background for both high-SCE and overall). However, our current data do not allow us to distinguish among biological explanations for the elevated SCE. The original manuscript acknowledged this limitation (“Whether this reflects a cell-cycle state more permissive to both cutting and recombination, or stochastic variation in RNP uptake coupled with a recombination-prone chromatin environment, remains to be determined.”). To make this limitation more explicit and to address the possibility of sequencing quality raised by the reviewer, we have revised the text as follows: “The high-SCE subset did not show evidence of lower sequencing quality, based on either sequencing coverage (p=0.17) or background SCE levels (p=0.13). However, we cannot distinguish whether the elevated on-target SCE reflects a cell-cycle state more permissive to both cutting and recombination, or stochastic variation in RNP uptake coupled with a recombination-prone chromatin environment.”

      This question may be better explored by future co-assays with sci-L3-Strand-seq; currently we cannot enrich for cells with high SCEs to characterize the molecular features of this subset of the cells using other omics approaches.

      (5) Reconsider the framing of the DNA repair gene targeting experiment.

      The current data do not strongly test whether LIG3, LIG4, XRCC1, or XRCC4 regulate Cas9-induced SCE, because functional protein loss is delayed and essential-gene targeting introduces selection. This section may be better framed as a negative/control observation rather than as a pathway analysis.

      We agree and please refer to Public Reviews for a single-cell assay-specific explanation.

      (6) Consider including some additional control experiments, for example, Cas9 without sgRNA, nontargeting sgRNA, or mock-transfected cells to make sure that some phenotypes (for example, cell-cycle arrest) directly result from DNA cleavage rather than from the transfection procedure.

      We thank the reviewer for this suggestion. We have carefully considered these additional controls but have chosen not to add further experiments. Our existing Cas9 nickase experiments provide a control that directly addresses whether the observed arrest is attributable to DSB formation rather than RNP delivery/transfection. Both the D10A and H840A Cas9 nickases were delivered under the same experimental conditions as wild-type Cas9, but neither produced the cell-cycle arrest observed following DSB induction by wild-type Cas9. Thus, these experiments control for Cas9 RNP delivery while altering the nature of the DNA lesion and support the interpretation that the observed arrest is associated specifically with Cas9-induced DSBs rather than the transfection procedure itself.

      (7) Figure 1: the fonts should be increased. The majority of the labels are impossible to read in a printed copy of this manuscript.

      We thank the review for pointing this out. We enlarged Fig.1 fonts.

      (8) Figure 1C. The pileup plots should be described and interpreted in a clear way. In its present form, it is unclear how the interpretations and conclusions are made.

      We added explanation of the pileup analysis immediately following mentioning the Fig.1C pileup: “We next examined … SCEs using genome-wide pileup analysis (Fig.1C, Fig.S1B), in which we plot the total number of SCEs detected across all single cells within each 1 Mb window.”

      Reviewer #2 (Public review):

      Summary:

      In this short paper, a clever single-cell Strand-seq method was used to study the number and location of sister chromatid exchange events (SCEs) in cells after CRISPR/Cas9-induced DNA double-strand breaks (DSBs). Unique as well as multiple genomic loci were targeted. Cas9-induced cuts at unique genomic locations led to statistical enrichment of SCEs at the target site, whereas Cas9 targeted at repetitive targets revealed only mild enrichment of on-target SCEs unless analysis was restricted to a subset of cells with >8 SCEs per cell. Interestingly, reciprocal daughter-cell pair analysis revealed largescale structural alterations on some chromosomes. Whereas disruption of DNA repair genes, including LIG3, LIG4, XRCC1, and XRCC4, did not measurably alter SCE frequency per cell within 24 hrs, consistent with delayed functional loss following editing and selection against essential genes. Together, these findings demonstrate that Cas9-induced DSBs are potent local triggers of SCE at unique loci and can be associated with structural alterations, highlighting the influence of lesion type and genomic context on recombination outcomes during genome editing.

      Strengths:

      The data in this paper represent a very rich resource of how parental DNA template strands are distributed in paired daughter cells after various treatments. Abnormalities observed in only one of such paired daughter cells provide a novel and exciting approach to study mechanisms of DNA instability and DNA repair at a genome-wide level in general and following Cas9-induced DSB in particular.

      Thank you very much for this assessment.

      Weaknesses:

      The effect of Cas9-induced DSBs in the cells that are used will depend on the cell cycle stage of the cells that are targeted, as well as the number of times cuts are made. The latter could happen before, during, and after DNA repair reactions on one or both alleles in a diploid cell. As a result, it is very difficult to extrapolate the mechanisms of DNA instability and DNA repair from the observed genomic rearrangements. Novel approaches are needed to limit the number and timing of Cas9-induced breaks to overcome some of these limitations. The language and logic in the paper can be improved, and some of the claims seem incorrect. For example, the abstract reads "A single Cas9 cut at a unique genomic locus led to strong local enrichment of SCE at the break site, reaching up to 41% in the same cell cycle and 17% in the subsequent division, indicating that DSB repair frequently engages non-local inter-sister repair." The evidence that only a single Cas9 cut was made is lacking (see my earlier comment); it is not clear how local enrichment or non-local inter-sister repair are defined.

      We agree with the limitations that Cas9-induced DSBs can be dependent on the cell cycle stage and the number of times cuts are made. We also agree that novel approaches are needed to limit the number and timing of Cas9-induced breaks to overcome these limitations, perhaps by using vfCas9 but more importantly, if new approaches to turn off Cas9 are developed. We have added a brief discussion on this limitation (in the Limitation section) and the resulting constraints on extrapolating mechanisms of DNA instability and repair from the observed genomic rearrangements. We thank the reviewer for pointing out the distinction between “a single Cas9 cut” vs. "Cas9 targeting of a single genomic locus." We went through the manuscript and revised where cutting only once was implied. We also explicitly acknowledge the possibility of multiple rounds of cutting at the same sites.

      We thank the reviewer for pointing out that “non-local repair” is a non-standard term. We use it operationally to distinguish repair confined to the broken chromatid (e.g., fill-in synthesis or end joining in cis) from repair involving exchange between sister chromatids. We have added a schematic (Fig. S1A) illustrating this distinction and cited this figure immediately before where we operationally defined SCE as a “reciprocal strand switch between sister chromatids, without implying a single mechanistic pathway.” This distinction is important because, particularly for two-ended Cas9 DSBs, an SCE-like outcome could potentially arise through either HR-mediated crossover or NHEJ of DNA ends across sister chromatids; the latter may involve different genetic requirements from classical NHEJ at least in end-tethering. We have revised the manuscript to define “non-local repair” explicitly at its first use.

      Reviewer #2 (Recommendations for the authors):

      References to relevant earlier studies using Strand-seq to study SCEs are missing (PMID: 27185886 and PMID: 29348659).

      We thank the reviewer for pointing this out. We added these references in the 3rd paragraph of the Introduction where we briefly review Strand-seq methods.

      Reviewer #3 (Public review):

      Summary:

      Chovanec and Yin used their newly developed sci-L3-Strand-seq powerful method to characterize SCE after Cas9 cleavage in a human cell line, using either a single target site or an element repeated 237 times in the genome. SCE are often neglected in DNA repair analyses since they are « genetically silent ». Interestingly, the authors found enrichment of SCE at unique Cas9 sites, but only a modest enrichment of SCE when Cas9 targets 237 sites in the genome. The genetic control of SCE formation at Cas9 sites is not deliberately addressed in this paper. However, the authors found that targeted SCE seem to be enriched in a subpopulation of cells, particularly « permissive » for SCE, but the determinants of such a population are unknown. Finally, the power of the sci-L3-Strand-seq allowed the authors to characterize a specific type of SCE based on the analysis of reciprocal daughter-cell pairs' genomes that is associated with a specific type of chromosomal rearrangement compatible with the ones observed in HR defective BRCA1/2 deficient cells.

      Strengths:

      This is an interesting paper that molecularly explores sister chromatid exchanges, which represent an important challenge in molecular biology since they are genetically silent.

      Thank you very much for this assessment.

      Weaknesses:

      A complexity of the current paper is that it heavily relies on a recently published paper (Chovanec et al 2026, NAR) describing the powerful but complex technique sci-L3-Strand-seq. Knowledge of this paper is a prerequisite to understanding the current manuscript because no reminder is provided. In addition, the current manuscript presents the use of the sci-L3-Strand-seq technique in the study of SCE after Cas9-induced DSBs, while a companion study is referred to several times for containing results about SCE in XRCC1 KO. At some point, one questions the relevance of splitting the use of sci-L3-Strandseq in different papers instead of making a single integrated one.

      We appreciate this concern. The original sci-L3-Strand-seq study is an extensive methodology paper that establishes and validates various computational framework, whereas the companion study focuses on the genetic regulation of spontaneous SCE. The experimental designs and biological questions of the companion study and the present work are therefore distinct, although we draw on selected results from the companion study where they provide useful comparisons and contrasts between spontaneous and Cas9-induced SCE. The present study addresses a distinct biological question, the response to Cas9-induced DSBs, and we therefore believe that combining these studies would make the resulting manuscript unnecessarily broad and obscure their different biological questions.

      We nevertheless agree that the present manuscript should be understandable without requiring detailed knowledge of either paper. We have therefore added a brief description of the sci-L3-Strandseq approach (3rd paragraph of Introduction, Fig.S1A legends, and Fig.1B legends) and clarified the relevant methodological concepts where they are first introduced. We hope to improve the self-contained nature of the manuscript so that readers need not consult the earlier NAR papers, and the companion preprint to understand the key results.

      Reviewer #3 (Recommendations for the authors):

      (1) Abstract

      "Identical sisters ": redundant

      "non-local" inter-sister repair: the meaning is not clear. Do the authors refer only to "inter-sister" and therefore "non-local" is redundant, or do they imply something specific by "non-local", in which case it needs to be clarified?

      "237 repetitive targets": at least a slight description of this target is needed. Is it a "random" repeat, a satellite sequence, a sequence related to a transposable element ?...

      We agree with the reviewer that “identical sisters” is technically redundant. However, we have retained “identical” here to emphasize the distinction between sister chromatids vs. homolog, as SCE is sometimes misconstrued as exchange between homologs and as potentially causing loss of heterozygosity. We prefer the slight redundancy here for conceptual clarity.

      We thank the reviewer for pointing out that the meaning of “non-local” was unclear. As discussed in our response to Reviewer #2, we use “non-local repair” operationally to distinguish repair confined to the broken chromatid in cis from repair involving exchange between sister chromatids. We have added a schematic (Fig.S1A) illustrating this distinction and explicitly define the term at its first use in the revised manuscript. Please see our response to Reviewer #2 above for the detailed rationale.

      We thank the reviewer for asking us to clarify the nature of the 237 repetitive targets. The sgRNA targets an Alu sequence and was selected from a larger screen of >20,000 sgRNAs targeting repetitive sequences occurring at >200 genomic sites. In that screen, cellular toxicity did not simply scale with the number of predicted target sites; we therefore selected this sgRNA because its intermediate phenotype allowed us to introduce a large number of programmed DSBs without either minimal perturbation or excessive loss of cells. Thus, the 237-site guide was not an arbitrarily selected Alu-targeting sgRNA. The full repetitive-element screen is beyond the scope of the present study, but we have clarified in the Abstract that these 237 sites are Alu targets and added a brief description of the guide selection in the Methods.

      (2) Introduction:

      "non-local outcome / non-local repair processes": The use of "non-local" is not standard and is obscure for the reader. Specify if it has any meaning or remove it.

      Please see our response above to both Reviewers #2 and #3 regarding our definition and use of “nonlocal repair”.

      The authors mention that replication through a DSB generates four broken ends. However, in case the DSB is reached by one replication fork before the converging one, there are only three broken ends for at least the time required for the converging fork to reach the DSB from the other side. This may influence the repair outcome.

      We agree with the reviewer. If one replication fork encounters the DSB before the converging fork, a transient three-ended intermediate can exist before the second fork reaches the break. This temporal asymmetry could influence repair pathway choice, including engagement of HR, end joining, or BIR-like repair. Our assay captures the resulting SCE outcome but cannot distinguish the order in which replication forks encounter the DSB or the repair pathway engaged at these intermediate stages. We have revised the text (2nd paragraph of the Introduction) to clarify that four broken ends represent the eventual configuration after replication through the DSB, rather than necessarily a simultaneous intermediate.

      (3) Results

      Cell cycle arrest experiment: it seems that a control condition with no Cas9 is missing to conclude better about what looks like a G2-M arrest, but that is not clearly mentioned.

      Please see our response to Reviewer #1, Recommendation 6, regarding additional controls for the cell-cycle arrest experiment. Briefly, the D10A and H840A Cas9 nickases were delivered under the same experimental conditions as wild-type Cas9 but did not produce the cell-cycle arrest observed following DSB induction, providing an internal control for RNP delivery/transfection and supporting the association of the arrest with Cas9-induced DSBs. We would also like to clarify that the observed cell-cycle arrest is primarily a G1/S, rather than G2/M, basing on the FACS signal (see revised Fig.1B legend). This is consistent with the strong G1/S checkpoint in mammalian cells and the predominantly G1 cell-cycle distribution of BJ-5ta cells.

      Note that the font size in Figure 1 is too small for readability.

      We have enlarged the font sizes throughout Figure 1 to improve readability.

      Figure 1B, D10A and H840A conditions:

      The authors mention that nicks can be converted into DSBs through the passage of the replication fork, but do not see any cell cycle defect in the conditions tested. Is it possible that the absence of effect results from the fact that the analysis is done prior to nicks being converted into DSBs? This remark notably applies to the 237 target sites experiment. It seems that controlling for cell cycle delays for longer times is needed to conclude clearly about this aspect. In case a clear absence of cell cycle delay is observed in the 237 target sites in the Cas9 nicking condition, this would suggest that replication born DSBs behave differently from "classical" two-ended DSBs and do not trigger cell cycle arrest.

      We agree that the timing of nick conversion during replication could contribute to the absence of a detectable cell-cycle delay under the conditions examined. However, extending the duration of Cas9 nickase treatment or labeling would not necessarily resolve this question, because persistent Cas9 activity permits repeated rounds of nicking across successive cell cycles, making it difficult to relate a later cell-cycle phenotype to a defined replication-born lesion. More generally, we believe that the relationship between replication-associated nicks, SCE formation, and cell-cycle progression is better addressed in the context of spontaneous SCE, which is the focus of our companion study. The present study is focused on SCE following programmed Cas9-induced DSBs, and analysis of replication-born nick lesions would require precise temporal control (ideally vfCas9 nickases that can be turned off) of individual nicking events relative to replication, for which an appropriate experimental system is not currently available to us.

      Figure 1C should mention somewhere the genomic location of the four targets to clearly show that they correspond to the four major SCE peaks. In addition, there is no legend for the vertical pink stripes. Finally, it might be wise to keep the same y-axis scale for better comparisons.

      Figure 1 overall: it might be wise to clearly show a no Cas9 condition to clearly set the SCE baseline and show that it is independent of Cas9 induction. As of now, it is not clear whether the non-targeted SCE comes from a specific cleavage of Cas9 or not. Such an aspect could benefit from putting Figure S1C in the main Figure 1. Alternatively, results from Chovanec et al 2026 (NAR) should be better restated because the reader does not necessarily have them in mind.

      We thank the reviewer for these suggestions. The expected Cas9 target positions were already indicated by vertical bars in Fig. 1C; however, we agree that this was not sufficiently clear. We have therefore revised the figure legend to explain that the vertical bars indicate the expected Cas9 target positions. The Chovanec et al. (2026, NAR) study focused entirely on spontaneous SCE, which we simultaneously map here as the background signal, rather than the on-target SCEs induced by Cas9. We hope that explicitly identifying the target locations in the revised legend makes this distinction clear and ensures that prior knowledge of the NAR study is not necessary to interpret Fig. 1C.

      We have retained the individual y-axis scales because the magnitude of SCE enrichment differs substantially among conditions. Using a common y-axis scale would make several of the on-target SCE peaks difficult to visualize.

      The section « Disruption of DNA repair genes at the cut site did not measurably alter SCE frequency per cell » is questionable in the results section for the following reasons:

      (i) The DNA repair genes are used here as target sites for Cas9 cleavage, but are not the object of the study, but may be the object of a companion paper. This aspect is slightly misleading.

      (ii) As first mentioned in this section, there is evidence strongly suggesting that inactivating DNA repair genes will not affect SCE, and this is what the authors observed.

      (iii) As an alternative, one could put the emphasis on the fact that the effect of Cas9-mediated inactivation of DNA repair genes (ie LIG3) starts to be detectable only in the washout condition ie after at least one cell cycle. But in this case, this is addressing the role of DNA repair genes in Cas9-induced SCE, which is not the point of the current paper.

      We thank the reviewer for this comment and agree that the original framing of this section could give the impression that these experiments were intended to test the functions of the targeted DNA repair genes in SCE. This was not our intent. Rather, these genes provided defined genomic target sites for Cas9 cleavage, and the primary purpose of the experiment was to characterize SCE associated with Cas9-induced DSBs at these loci.

      As discussed in our response to the Editor Assessment, there are important limitations to using these experiments to infer the consequences of loss of the targeted proteins, including the delay between Cas9 cleavage and depletion of pre-existing protein and, and particularly in the single-cell assay, selection for cells that escape disruptive editing at essential genes. We have added text to the Results explicitly describing these limitations.

      We therefore agree with the reviewer that the delayed effects observed under the washout condition should not be interpreted here as establishing a role for individual DNA repair genes in Cas9-induced SCE. We have revised the section title to “Cas9 targeting of DNA repair gene loci did not immediately alter overall SCE frequency per cell” to clarify the scope of this experiment and to avoid implying that testing the functions of the targeted DNA repair genes is a major objective of the present study.

      The conditions in Table 1 need to be homogenized and better explained:

      - 237 sites and 237 cuts are used: homogenize?

      We thank the reviewer for spotting this. We revised both to be “237 sites”.

      - May explain better the rationale for putting BrdU simultaneously with Cas9 or after 24 h and a wash.

      For the single-targeting sites, we observed more SCE when BrdU was added simultaneously with the Cas9 for the same 24 hours, compared to adding BrdU in the subsequent division after a wash. Therefore, for the 237 sites, we analyzed both conditions.

      - Typo in the text: 237cuts_24ws_40BrdU instead of 237cuts_24ws_BrdU

      We apologize for the lack of clarity in these labels and have substantially revised the Table 1 legend. In brief, the “40” is not a typo. In the 237 sites experiments, wild-type Cas9 considerably prolonged the cell cycle. Therefore, rather than labeling with BrdU for 24 hours as in the other conditions, we extended BrdU labelling to 40 hours in the last two conditions to allow more cells to progress into the subsequent G1 for successful Strand-seq analysis. We clarified that “237 sites 24 + 16hrs BrdU” refers to the condition in which Cas9 RNP and BrdU were added simultaneously. After 24 hours of Cas9 RNP treatment, BrdU labeling was continued for an additional 16 hours (a total of 40 hours of BrdU). The “237sites 24ws40BrdU” condition is the corresponding washout condition, in which Cas9 RNP was removed after 24 hours and cells were then labeled with BrdU for 40 hours post-washout.

      - 237 cuts: Are some sites more enriched in SCE than others?

      Yes, some sites showed greater SCE enrichment than others. We tested whether this variation correlated with chromatin accessibility but found no significant association. This was not unexpected, as the sgRNAs predominantly target Alu elements.

      - Table 1: There is a difference between 237 sites 24ws24BrdU and 237cuts24ws40BrdU, with a significant enrichment of on-target SCE for the latter condition only. Could the increase in SCE rise even more with longer BrdU exposure? In other words, does the low enrichment in SCE at target sites in the 237 sites experiment result from a non-optimal timing for the analysis?

      Yes, this is possible. We did not systematically test additional treatment or labeling durations. A 24-hour Cas9 RNP treatment is typically used for Cas9 RNP-mediated knockout experiments, and we therefore initially used this duration to assess gene-editing outcomes. For Strand-seq, BrdU labeling is ideally limited to approximately one cell division. Because BJ-5ta cells have an approximately 24-hour cell cycle, extending BrdU labeling substantially beyond 40 hours could allow some cells to undergo a second round of replication and become double-labelled. We therefore did not extend BrdU labeling beyond 40 hours. Thus, the lower enrichment in the 24ws24BrdU condition may in part reflect the timing of the assay.

      - Figure 2 / RDCP analysis:

      Interpretation of this figure relies exclusively on the 2026 NAR paper from the authors. This, at least, should be mentioned to help the reader understand it. Once the legend restates, this figure misses clear identification of the SCE and other genomic rearrangements. For readability, maybe the full genome should be kept for the supplementary data, and only the rearranged chromosomes should be kept in the main figure so that the rearrangements are clearly visible and annotated.

      We thank the reviewer for this suggestion. To make the Strand-seq plots interpretable without relying on our 2026 NAR paper, we have added an explanation of Strand-seq orientation in the third paragraph of the Introduction and in Fig.S1A. We have revised Fig.2 legends to improve readability. We have retained the whole-genome view because Strand-seq data are conventionally presented in this format and it provides important genome-wide context for interpreting the observed events. The rearranged chromosomes and events were annotated in Fig.S3.

      (4) Discussion

      - Most DSB never formed or did not produce SCE: how to understand this better? What would be the argument in favor of one or the other possibility?

      We agree that these are two possible explanations that cannot be distinguished by the current experiment. The absence of an SCE at a targeted site could reflect either inefficient DSB formation or repair of a DSB through a pathway that does not generate an SCE. Distinguishing these possibilities would require direct measurement of cutting efficiency at individual target sites, which was beyond the scope of this study.

      - The conclusion about the effect of the Cas9 nickases needs to be toned down as long as the proper timing for SCE analysis has not been performed (see comment above).

      We agree and have toned down this conclusion by specifying that no significant on-target SCE enrichment was detected under the conditions tested and acknowledging that we cannot exclude SCE formation at other time points (Discuss, first paragraph).

      - As much as possible, avoid the use of non-conventional acronyms like URR.

      We agree and have reduced the use of non-conventional acronyms where possible. We have retained URR (under-replicated region), as the term appears seven times throughout the manuscript, but have ensured that it is clearly defined at first use.

      - The discussion about the RDCP analysis in the second paragraph of the discussion should refer to Figure 3.

      Thank you for pointing this out. We added this reference to Fig.3

      AuthorResponse
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  14. press.rebus.community press.rebus.community
    Evolution To College: Becoming A College Student | Blueprint for Success in College and Career
    1
    1. mg0222 05 Oct 2026
      A student can choose to attend classes part-time or full-time. College class times try to accommodate a variety of student needs and may occur during the day, evening, online, or a combination of classroom and online (hybrid).

      Most college tend to make it available to all students to make sure their schedule clashes with their real life time.

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  15. hapgood.us hapgood.us
    SIFT (The Four Moves)
    5
    1. Edric 05 Oct 2026
      When it comes to claims, a key piece of context includes whether they are broadly accepted or rejected or something in-between. By scanning for other coverage you can see what the expert consensus is on a claim, learn the history around it, and ultimately land on a better source.

      Analyzing expert opinion rather than focusing on a source may be more time efficeint and smarter through researching

    2. Edric 05 Oct 2026
      so you can see it in it’s original context and get a sense if the version you saw was accurately presented.

      Tracing is important as you are able to understand and read the way the author originally intended not how the person who used them as a source intepreted their findings and conclusion.

    3. Edric 05 Oct 2026
      In these cases we’ll have you trace the claim, quote, or media back to the source, so you can see it in it’s original context and get a sense if the version you saw was accurately presented.

      This can be useful to my research as I can retrace the original source and perhaps interperet a different way.

    4. Edric 05 Oct 2026
      Taking sixty seconds to figure out where media is from before reading will help you decide if it is worth your time, and if it is, help you to better understand its significance and trustworthiness.

      Looking into the origin of the source is necessary to make the judgement of if the source is credible or not.

    5. Edric 05 Oct 2026
      Ask yourself whether you know the website or source of the information, and what the reputation of both the claim and the website is. If you don’t have that information, use the other moves to get a sense of what you’re looking at.

      Checking the source before actually consuming the knowledge and material in it would be useful in protecting yourself from bias claims of others.

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    hapgood.us/2019/06/19/sift-the-four-moves/
  16. doc-0k-3o-apps-viewer.googleusercontent.com doc-0k-3o-apps-viewer.googleusercontent.com
    International Political Sociology as a Mode of Critique: Fracturing Totalities
    15
    2. EnlightenMinty 05 Oct 2026
      To work against totalizing pulls within each approach andacross them—for example by seeking to build a synthesis that incorporates severalapproaches—can then mainly be done by including multiple approaches and fore-grounding the limits and tensions between them (

      THIS IS HOW TO DO STRUCTURAL ANALYSIS WITHOUT TOTALISIM

    5. EnlightenMinty 05 Oct 2026
      YouTube video of torturingdevices operate as fragments that set series of actual practices (e.g., series of whattorturers do) and virtual sense making (e.g., formalized prohibitions of torture) atplay in thought that raises questions about familiar modes of criticizing torture.

      Eg of fragment

    7. EnlightenMinty 05 Oct 2026
      His critique of historicism and of subjectivism is crucial to under-stand how, in his later writings, it was possible to decouple structure from totality.

      Althusser moved towards Decoupling structure and totalit

    8. EnlightenMinty 05 Oct 2026
      By staging the need for “sovereignty” in this way, Dean is not fracturingsovereignty through Foucaultian dispositifs that foreground relationality in hetero-geneity but rather reintroduces sovereignty as a master rationale that defines inter-national relations.

      Good constrasting example of fracturing vs master-narrative totalising

    9. EnlightenMinty 05 Oct 2026
      Breadsworth argues that a renewed critique of actual (and histori-cal) forms of domination has to answer to the challenge of “shaping the world asworld” in which any local processes are to be understood under a global framing ofthe economic and the political.

      A totalising narrative example

    12. EnlightenMinty 05 Oct 2026
      Its aim is not to delete or fragment critique but torevisit criticality through a distinct mode of doing IPS that reconceptualizes the re-lation between fragment and structure in ways that explicitly work against returns ofgrand narratives and presumed wholes.

      Drawing a lot on postmodernism and poststructuralism

    13. EnlightenMinty 05 Oct 2026
      Keeping the main thrust of deconstructing the international, 3 we propose a revis-iting of criticality through fracturing analytics

      i.e analyse the international phenomenon via a fracturing lens, not a totalising one. A fracturing lens most importantly treats macro level structures and structures in general as provisional and fluid, i.e they can be changed by different phenomenon.

      More importantly, the emphasis is on structural analysis as seeing structures emerge from fragments, not seeing fragments as instances of already existing whole structures.

    15. EnlightenMinty 05 Oct 2026
      We are par-ticularly concerned with how an intensified interest in the local, micro, and situatedis understood to be insufficient for critical knowledge, and how this assumption ofinsufficiency inscribes horizons of totalization into international political sociology

      That is, they are concerned about how smaller level of analysis might lead to totalisation — that is, they might lead to explanation of the smaller phenomenon be treated as instances of macro level explanations, flattening and putting a lens on it

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  17. github.bmc.com github.bmc.com
    Authorizing Snowflake Ingestion with Okta OAuth
    1
    1. sgritsan 05 Oct 2026
      Open https://developer.okta.com/signup/. Create an account and a free Okta organization. Sign in to the Okta Admin Console.

      we should assume the reader has his Okta account set

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    github.bmc.com/pages/CTO-BIL/beaconSaasPreview-flare-documentation/Authorize_Snowflake_Ingestion_With_Okta_OAuth.htm
  18. rws511.pbworks.com rws511.pbworks.com
    ThompsonF2022.pdf
    6
    1. cierraM 05 Oct 2026
      tudents write essays that are substantially longerand have better organization and content than when they're writingfor their teacher.

      students experience audience effect

    2. kisurii_soups 05 Oct 2026
      e "audience effect"-the shift in ourperformaqce when we know people are watching.

      It's a real thing ! When it comes to writing or even posting on social media on an account no one yet knows or follows, at some point you will have viewers and you will be seen whether you intend to or not when posting

    4. kisurii_soups 05 Oct 2026

      I lowkey kinda wished I lived through this era! The thought of writing letters to someone, waiting for their replies and being able to put so much thought into them felt so special and heartwarming.

    6. kisurii_soups 05 Oct 2026

      It's honestly crazy how much people still continue write and the different kinds of people you constantly see on writing platforms. While AI may be taking over a lot of things, I feel like AI can never truly take away the art of people's voices and their right to speak, which I find truly amazing

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    rws511.pbworks.com/w/file/fetch/150110607/Thompson_Pub_ThinkingF2022.pdf
  19. github.bmc.com github.bmc.com
    Creating a Snowflake Programmatic Access Token
    12
    12. sgritsan 05 Oct 2026
      Create a Snowflake trial account for evaluation and testing: Go to https://signup.snowflake.com/. Choose Start for Free (30-day trial). To complete the registration process, enter email, name, and company. Verify email if asked. Select the cloud provider, region, and edition. Create an administrator account. Open Snowsight after account creation.

      We should assume the reader has his Snowflake account set up

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  20. github.bmc.com github.bmc.com
    Authorizing Kafka Ingestion with Entra ID OAuth
    1
    1. sgritsan 05 Oct 2026
      Open Azure Free Account or Azure Portal. Sign in with a Microsoft account, work account, or school account. Create a free Azure subscription or use an existing subscription. Open the Microsoft Entra admin center or navigate to Azure Portal → Microsoft Entra ID. Confirm that a tenant is available. Record the Tenant ID Your Organization ID—the tenant slug that identifies your BMC Beacon environment. Pass the same value to run.sh as --tenant-id. For SaaS, this is typically the hostname segment from your tenant URL. (Directory ID). This value is used later as AZURE_TENANT_ID. Verify that the account has permissions to: Create app registrations. Grant admin consent. Most enterprise environments already have a Microsoft Entra ID tenant. Create a new tenant only for evaluation or development environments. Tenant InformationLink copied to clipboard Record the following value for later Kafka OAuth configuration: Variable Source AZURE_TENANT_ID Microsoft Entra ID Directory (tenant) ID. Registering the Kafka API Application Create an application that represents the Kafka cluster. This application is used as the OAuth resource and does not require a client secret: Open the Microsoft Entra admin center. Select Applications > App registrations. Select New registration. In Name, enter beacon-kafka. Under Supported account types, select Accounts in this organizational directory only (Single tenant). Leave Redirect URI blank. Select Register. On the Overview page, record: Application (client) ID as KAFKA_APP_CLIENT_ID Directory (tenant) ID as AZURE_TENANT_ID Open Expose an API. In Application ID URI, enter: CopyCopied to clipboard set api://beacon-kafka Click Save. Open Manifest. Set: CopyCopied to clipboard "requestedAccessTokenVersion": 2 Click Save. Open App roles and then select Create app role. Configure the role:: Display name: Kafka Connect Allowed member types: Applications Value: Kafka.Connect Enable the role and select Apply. If requestedAccessTokenVersion is left null or set to 1, tokens are issued as v1 and the broker expected issuer rejects them. Registering the Kafka API Application Create an application that represents the Kafka cluster. This application acts as the OAuth resource for Kafka and does not require a client secret. Open the Microsoft Entra admin center. Select Applications > App registrations. Select New registration. In Name, enter beacon-kafka. Open Certificates & secrets → New client secret. Copy the Value (not the Secret ID) → AZURE_CLIENT_SECRET. Leave Redirect URI blank. Select Register. On the Overview page, record the following values: Application (client) ID as KAFKA_APP_CLIENT_ID Directory (tenant) ID as AZURE_TENANT_ID Open Expose an API. In Application ID URI, enter: CopyCopied to clipboard api://beacon-kafka Select Save. Open Manifest. Set: CopyCopied to clipboard "requestedAccessTokenVersion": 2 Save the manifest. Open App roles and then selectOpen App roles. Configure the role with the following values: Field Val Display name Kafka Connect Allowed member types Applications Value Kafka Connect Enable the role. Select Apply.

      We should assume the reader has Azure application configured. List in the prerequisites: Tenant_id client_id client_secret application id uri

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  21. github.bmc.com github.bmc.com
    Authorizing Databricks Ingestion with OAuth
    1
    1. sgritsan 05 Oct 2026
      Grant the service principal access to Unity Catalog resources.:

      Make a notion that sql is for example only. You can start sql part from -- Verify section. Use placehoders: -- Catalog GRANT USE CATALOG ON CATALOG <catalog_name> TO <client_id>;

      -- Schema GRANT USE SCHEMA ON SCHEMA <schema_name> TO <client_id>;

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  22. www.youtube.com www.youtube.com
    Лучшие вопросы с собеседований по PostgreSQL | Потренируйся - YouTube
    1
    1. devilboi 05 Oct 2026

      Индексы отдельная структура данных для быстро поиска строк. по хешу, используя b-tree, gist - для координат.

      explain показывает примерное время выполнение запросы explain analyze запускает запрос и показывает реальное время выполнение запроса.

      триггер - механизм, который после каких-то действий запускает другие действий. допустим при select он может после запустить команду для добавление в бд данных.

      пострег использует индексы тогда когда эта эффективно. если запрос возвращает все строки, то планировщик запросов понимаю, что в нём нету смысла.

      Primary-key - индефикатор сущности. foreighKey - Это ключ индификатор другой сущности.

      в postgresSql есть планироващик задач pg_cron.

      4 вида изолиции.

      read commited базовая даёт транзакция читать только за комиченные изменения

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    youtube.com/watch
  23. github.bmc.com github.bmc.com
    Creating a Databricks Personal Access Token
    1
    1. sgritsan 05 Oct 2026
      Go to https://www.databricks.com/try-databricks (or https://login.databricks.com and select Get started). Choose Get started with Community Edition or a free trial (wording varies): Community Edition: Free personal sandbox with limited features. Free trial: Full Databricks cloud workspace for a limited period. Prefer this option for Unity Catalog and Beacon ingestion The process of extracting metadata from data sources and loading it into BMC Beacon.. Sign up with email (or Google or Microsoft) and verify your email if asked. Complete the account wizard: company or personal details; cloud provider (AWS, Azure, or GCP) if on a trial; accept the terms. Wait until the workspace is ready, then open it. Confirm you land on a URL like https://<workspace-id>.cloud.databricks.com. Copy the workspace URL for ingestion The process of extracting metadata from data sources and loading it into BMC Beacon..

      We should assume the reader has his databricks account already set

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  24. github.bmc.com github.bmc.com
    Creating a Tableau Personal Access Token
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    github.bmc.com/pages/CTO-BIL/beaconSaasPreview-flare-documentation/Create_A_Tableau_Personal_Access_Token.htm
  25. openstax.org openstax.org
    1.1 What Is Sociology? - Introduction to Sociology 3e | OpenStax
    7
    1. Lorena.monroy 05 Oct 2026
      action. A written language system consists of symbols that refer to spoken sound. Taken together, these symbols convey specific meanings. The English language uses a combination of twenty-six letters to create words.

      language is something we use every day to communicate. I learned language mostly from my family, teachers, and listening to other people. I think using certain words and communicating is folkway because it's an everyday expectation. If someone uses inappropriate or disrespectful language people might judge the, or think differently of them.

    2. Lorena.monroy 05 Oct 2026
      Throughout history, and in societies across the world, leaders have used religious narratives, symbols, and traditions in an attempt to give more meaning to life and understand the universe. Some form of religion is found in every known culture, and it is usually practiced in a public way by a group. The practice of religion can include feasts and festivals, intercession with God or gods, marriage and funeral services, music and art, meditation or initiation, sacrifice or service, and other aspects of culture.

      Religion can be a big part of how people connect with each other. In my life, I learned certain religious traditions from my family and by seeing them practice them. its an expectation but not a strict rule, and if someone didn't participate they were questioned or seen as disrespectful.

    3. jasmine.ventura8 05 Oct 2026
      They help people know when to shake hands, where to sit, how to converse, and even when to laugh. We relate to others through a shared set of cultural norms, and ordinarily, we take them for granted.

      This part of the textbook helped me remember one of the norms that are in my own life. Whenever we arrive at an event, for example, like a birthday party or just a family gathering, you always have to say hi to everyone, whether that is a handshake or sometimes even a big hug(usually a hug). Even the guests that you don't know, if you don't, it can be seen as rude or impolite. If you just arrived, they are expecting you to come up to them and greet them, since they're already at the party; they just wait for you to approach them first. This is just a cultural norm for us. My parents were the ones who told me this is how it is practiced, so they'd always say, "Go say hi to your aunt," and if I didn't, my aunt would come up to me and say, "Where's my greeting??"

    4. jasmine.ventura8 05 Oct 2026
      In the U.S., by contrast, individuals are expected to leave home and live independently for a period before forming a family unit that consists of parents and their offspring.

      Another thing I can think of is that in some cultures, whenever parents get older, sometimes people don't have enough time to take care of their loved ones because either they have their own lives now that they are busy and too focused, or it can take a toll on their mental health. In my culture, who cares what you have going on with your life, but you WILL take care of your loved one. Some don't practice it, but many want you to because, since your parent took care of you their whole life, now it's your turn. While it is perfectly acceptable to have your parents live in a retirement home so they can be taken care of, for my family it is viewed as cruel or heartless.

    5. Lorena.monroy 05 Oct 2026
      As norms vary across cultures and time, it also makes sense that notions of deviance change. Sixty years ago, public schools in the United States had dress codes that often banned women from wearing pants to class. Today, it’s socially acceptable for women to wear pants, but less so for men to wear skirts.

      People have different expectations about what men and women should wear, in my life I've learned that certain clothes are seen as more appropriate for girls or boys by watching my family and other people around me. I think this is a folkway because it's an everyday expectation. If someone goes against it they might get judged, made fun of, or get weird looks from others.

    6. jasmine.ventura8 05 Oct 2026
      In many Asian cultures, for example, family members from all generations commonly live together in one household.

      This reminds me of another norm we practice that my family will always give us like an understanding that they are above anyone else in your life. They are more important than your friends, your partner, and anyone and everyone. Which, in my opinion, makes sense; however, this can sometimes be used against someone as an excuse for their wrong behavior. Kind of like "I am like this because I care about you"; however, that is sometimes not the case. If, for some reason, god forbid, you end up hanging out with friends at a family event, and they find out, they will scold you a bit. Trust me, I love my family, but they can be a lot sometimes, so a break is sometimes needed!

    7. nancy.luna 05 Oct 2026
      Norms are behaviors that reflect compliance with what cultures and societies have defined as good, right, and important.

      A norm in my family that was taught to us and something thats expected in our culture is that if you're visiting someone as a guest to clean up after yourself- wash your dish, clean any mess etc. One things I learned as I got older was people of different backgrounds didn't always do the same things. In my culture, not doing this as a guest was seen as 'uneducated' or 'rude'. While in other cultures it may be considered rude to have a guest to clean up after themselves.

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    openstax.org/books/introduction-sociology-3e/pages/1-3-theoretical-perspectives-in-sociology
  26. github.bmc.com github.bmc.com
    Authenticating Datasource Ingestion
    1
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  27. www.thegospelcoalition.org www.thegospelcoalition.org
    We Who Work with Words: Towards a Theology of Writing
    1
    1. apoorv.katoch 05 Oct 2026
      This divine, perpetual exchange of love and glory is what I call communion behavior, which fosters unbroken unity, and this behavior is what I have in mind when using the word “language.”

      Language is what enables the communicative behaviour (the exchange of love and glory) between the three persons of the trinity.

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    thegospelcoalition.org/themelios/article/we-who-work-with-words-towards-a-theology-of-writing/
  28. bookshelf.vitalsource.com bookshelf.vitalsource.com
    Embodied Cognition
    18
    1. abalaj 05 Oct 2026
      The hope is that a carefully controlled laboratory setting makes possible the discovery of which features of the world are encoded for use in a particular cognitive task, and what effect these features have on cognitive performance.

      Again.. how is this ecologically valid. Every person's software is different. What part of the world is embedded in the cognitive process varies significantly across participants. Moreover, every person has a unique set of unconscious top-down rules which is not very accessible to analysis.

    2. abalaj 05 Oct 2026
      They tend to draw the boundaries of cognition at the same place that a computer scientist might draw the boundaries of computation – at the points of interface with the world.

      Very True. I wonder why the solipsistic view of cognitive science fails? Mind is still a computer, but a computer that extends beyond the anatomical brain into the physical world. Why should embodied cognition resist this solipsistic view? Don't we just need solipsistic view to be redefined?

    3. abalaj 05 Oct 2026
      On the other hand, one might wonder how useful GPS might be as a guide to all of cognition, given the very constrained nature of problems to which means-end analyses are suited

      I am inclined to think that all of cognition is means-end analyses. It is just that the steps from the starting point to the goal remains out of our conscious awareness. This reminds me of the Libet's EEG study where the spike was seen way ahead of even the conscious will to press a button. So, we have no idea of the workings of the mind beyond conscious awareness. GPS can model only those tasks that are operable within our conscious awareness.

    4. abalaj 05 Oct 2026
      As we shall see in the next chapter, some critics of standard cognitive science have sought to undermine the idea that cognition relies on linguistic structures.

      I wonder how cognition cannot fit into a linguistic framework. A linguistic model might still serve as a good metaphor to understand cognitive processes. I am reminded of an example from Psych 20: an image of an apple provides the raw sensory data - lines, edges, contrast- which are meaningfully combined (by rules) to create a geometric shape. This is further manipulated using top-down knowledge to realize that the image is an apple. Mind thus combines pieces of raw information (letters/words) into a concept(sentence).

    5. abalaj 05 Oct 2026
      The conditions for being a symbol do not, apparently, mandate that symbols be made of any particular stuff, but instead focus on functional criteria, on criteria about what symbols must do. Among the relevant conditions seem to be these. First, symbols can “stand for” or represent things.

      True, symbols have little to do with what they are made of. The focus is primarily on function. However, when it comes to the mind machine, I believe function and anatomy/biochemistry are interlinked. It is the sheer biochemistry of the brain that drives the symbol/neuronal firing pattern.

    6. abalaj 05 Oct 2026
      Subjects merely had to identify the rules that would transform one syntactical object into another and then apply them.

      This reminds me of the Chinese Room experiment which also involved symbol manipulation and means-end analyses. Though the methodology was essentially the same, I think the Chinese Room experiment inferred the opposite of GPS that mere symbol manipulation is mindless. Nonetheless, I still favor the idea of mind being computational.

    7. Niepa 05 Oct 2026
      The computational processes in this description of events take place in the temporal space between the receipt of inputs and the display of outputs. All the “action,” so to speak, begins and ends where the computational processes touch the world.

      Initially the “brain in a vat” idea seemed impossible to me, the idea of separating cognition from the real world was a foreign concept until I understood the computer analogy. Artificial electrical signals could definitely produce a real cognitive process, which made me wonder how much of our cognition depends on interacting with the real world. Our syllabus describes sight as the most informative sense we have but none of that would be possible without the brain and its use of symbols.

    8. Niepa 05 Oct 2026
      algorithms

      Terms like algorithms, and the idea of thinking about the mind like a computer made cognition feel a lot more concrete to me; whereas before it was a lot more difficult to visualize the same way you can for other concepts. I think this is also why the Hoffman argument from lecture helped understand these ideas better, some of the more abstract arguments started to make more sense to me.

    9. Niepa 05 Oct 2026
      Newell and Simon created a computer program they called General Problem Solver (GPS),3

      I was very interested by the GPS because it reminds me of what we’re seeing with AI today. Newell and Simon were working on a completely different problem and obviously the AI today has nothing to do with what they were even able to fathom back then, but it Is interesting that they were already trying to get a computer to imitate human problem solving and act like one. It was also refreshing to see an early example of trying to connect computers with human cognition that doesn’t feel so “end of the world”

    10. cccccccccccccccccccc 05 Oct 2026
      The words “lamb” and “αρνι´” are both symbols, and both happen to represent the same wooly object.

      Sure, but the latter phrase means nothing to me (I don't speak the language) and thus I wouldn't be able to recognize it as a symbol. This makes me wonder about how arbitrary symbols are. Who's to say a pile of dirt isn't a symbol for, say, a horse? To me, "αρνι" is as much a symbol for "lamb" as a pile of dirt is a symbol for a horse. Does it require that at least one observer somewhere in the universe understands dirt -> horse, just as αρνι meaning lamb just requires a subset of people to understand it as "lamb"?

    11. cccccccccccccccccccc 05 Oct 2026
      while perhaps there may be no certain answer to the coffee mug question in all cases,

      i wonder if this is true. to what extent is the symbolic representation of a coffee mug in my brain identical to the symbolic representation of a coffee mug in your brain? does knowing one make the other easier to identify, or is it a product of personal development and unique between people?

    12. cccccccccccccccccccc 05 Oct 2026
      Thus, Newell and Simon asked human subjects to “think out loud” while solving logic problems

      seems like this is just a narrow subset of the "internal thought processes" used in problem solving, i.e. leaving out subconscious thought and intermediary thoughts... i wonder if adding non-introspective problem solving observations would have improved this model (was fmri/eeg/etc around then to help?)

    13. tyorozum 05 Oct 2026
      They are, in a sense, passive recipients of stimulation that has been especially designed to elicit information about cognitive algorithms located exclusively in their brains. Embodied cognition, we shall see, resists the idea that cognition is solipsistic, and so rejects the idea that subjects are passive receivers of stimulation.

      If you only study cognition while people are cut off from their environment, it's reasonable to conclude that it only happens in the brain. Sternberg's tests in a lab function differently than say the grocery list example.

    14. tyorozum 05 Oct 2026
      On the other hand, one might wonder how useful GPS might be as a guide to all of cognition, given the very constrained nature of problems to which means-end analyses are suited.

      Some tasks do not fit means-end analysis such as recognizing a face, riding a bike, understanding a joke. This would be a limitation to GPS theory.

    15. tyorozum 05 Oct 2026
      The hypothesis that motivates GPS

      Hypothesis: Thought is symbol manipulation. Examples being detecting, storing, copying, and comparing symbols like a computer would.

    16. emelym1 05 Oct 2026
      Rather than roaming a noisy and distraction-laden environment, subjects are thus often seated in front of a computer monitor, focusing their attention on a very deliberately constructed set of stimuli, performing a task for which they may have received special training or instruction. They are, in a sense, passive recipients of stimulation that has been especially designed to elicit information about cognitive algorithms located exclusively in their brains

      How do researchers design these studies so that the data is as accurate as possible, will participants act almost the same way as they would out in their everyday environment?

    17. emelym1 05 Oct 2026
      The question that vision theory addresses is this: how does stimulation on the retina become a perception of the world? Cognitive scientists also emphasize the apparent fact that the patterns of stimulation on the retina do not carry enough information on their own to determine a unique visual perception. The visual system must therefore embellish visual inputs, make assumptions, and draw inferences in order to yield a correct solution—an accurate description of the visual scene

      It's fascinating to learn how our visual system is a really complex process.

    18. emelym1 05 Oct 2026
      The conditions for being a symbol do not, apparently, mandate that symbols be made of any particular stuff, but instead focus on functional criteria, on criteria about what symbols must do. Among the relevant conditions seem to be these. First, symbols can “stand for” or represent things.

      It is interesting how many things in our everyday lives are symbolic, and with it, its functional criteria. How there can be numerous symbols for one individual thing but at times represent the same thing.

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    bookshelf.vitalsource.com/reader/books/9781351719162
  29. thewasteland.info thewasteland.info
    Current - T. S. ELIOT
    1
    1. jjackson27 05 Oct 2026
      Consider Phlebas, who was once handsome and tall as you.

      Phlebas, the drowned sailor, loses his physical stature, beauty, and status as a man. In the third movement of "SECTION III—DREAM-FUGUE," the soldier is said to lament over men lost before sunrise: "I sat, and wept in secret the tears that men have ever given to the memory of those that died before the dawn, and by the treachery of earth, our mother." This excerpt follows the fated death of the girl in section III; unable to save her, the sailor watches "the treacherous sands gathering above her head..[soon enough] Already her person was buried; only the fair young head and the diadem of white roses around it were still visible to the pitying heavens; and last of all, was visible one white marble arm." Without consulting the sources, the image seems to establish a royal antiquity to the (similarly) drowned/suffocated girl. Wearing a crown of white rose, she is preserved in sand. The only lasting remitment of her body is a marble arm—an object that connotes traditional roman and greek drowned statues. The reference of "the drowned sailor" as a product of Madame Sosostris's fortune further develops the sailor and girl to doomed, irremediable figures.

      Line 320's reference to the "wheel" continues a recurrent refrain and phrase: most references to the "wheel" have been in reference to time, its movement, and destiny. Its use in the pulled passage is no difference: the term "wheel" is an explicit connection to the sailor's non-linear movement through time. Not only does he continue to be plagued and haunted by his mortal, tortured body in death, but he passes "the stages of his age and youth/ Entering the whirlpool" as might a lost soul in the River Styx or the River Acheron.

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    thewasteland.info/new-2/
  30. drive.google.com drive.google.com
    activites-9557.pdf
    3
    1. nataliamurrutiasanchez 05 Oct 2026

      Los autores argumentan que los diseñadores defienden una identidad basada en saber navegar la incertidumbre y abordar "problemas complejos", pero al mismo tiempo se ven obligados a condensar su metodología en esquemas visuales claros y vendibles para encajar en contextos corporativos o institucionales. Este argumento evidencia que la diagramación visual no es neutra, sino un espacio de negociación estratégica entre la intuición creativa y la demanda externa de predictibilidad.

    2. nataliamurrutiasanchez 05 Oct 2026

      Me pareció muy revelador como la investigación utiliza el célebre esquema del Double Diamond como punto de partida en las entrevistas con los diseñadores. Aunque se reconoce ampliamente como un estándar comunicativo útil para explicarse hacia afuera, los profesionales coinciden en que resulta demasiado rígido para reflejar su práctica real, la cual se organiza en procesos más sinuosos, narrativos o en espiral.

    3. nataliamurrutiasanchez 05 Oct 2026

      Los autores plantean que la cultura visual en el diseño no funciona únicamente como un canal de comunicación o entregable final para los clientes, sino como una herramienta cognoscitiva fundamental para el acto mismo de diseñar. La representación gráfica es el medio mediante el cual el diseñador procesa problemas complejos, coordina la colaboración interdisciplinaria y legitima su práctica profesional.

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    drive.google.com/file/d/1zuMYSeoW4L7ccmCLR-kENAadO9D1VP1I/view
  31. socialsci.libretexts.org socialsci.libretexts.org
    4.6: Chapter Activities
    10
    1. jullien 05 Oct 2026
      __________________________________________________________________

      review syllabus finish assignments before class think about what you learn in class

    3. jullien 05 Oct 2026
      __________________________________________________________________

      Yes because your attention keeps switching between different things this can make it harder to remember the information

    4. jullien 05 Oct 2026
      __________________________________________________________________

      connect information to things you already know relate information to real life situations to group similar information together

    5. jullien 05 Oct 2026
      __________________________________________________________________

      short term holds information that you need for a short period of time while long term memory stores information that you can remember and use later

    6. jullien 05 Oct 2026
      __________________________________________________________________

      it keeps your notes organized and makes them easier to study and review later

    7. jullien 05 Oct 2026
      __________________________________________________________________

      look over your notes, fix anything you missed, and add important information

    8. jullien 05 Oct 2026
      __________________________________________________________________

      Somewhere you won't get distracted easily lets you see and hear the instructor clearly.

    9. jullien 05 Oct 2026
      __________________________________________________________________

      paying attention to what someone is saying, understanding it and thinking about the information

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    socialsci.libretexts.org/Bookshelves/Counseling_and_Guidance/College_Success/04:_Listening_Taking_Notes_and_Remembering/4.06:_Chapter_Activities
  32. drive.google.com drive.google.com
    ART+9_ÑAWI_VOL10_N2.pdf
    3
    1. nataliamurrutiasanchez 05 Oct 2026

      La autora plantea la resignificación visual como una estrategia pedagógica en la que los estudiantes desplazan un objeto cotidiano de su uso utilitario para reinterpretarlo y dotarlo de nuevos sentidos simbólicos y narrativos. Apoyándose en el concepto de postproducción de Nicolas Bourriaud, el texto señala que en la cultura visual contemporánea la originalidad pura pierde protagonismo frente a la remezcla y edición inteligente de materiales preexistentes.

    2. nataliamurrutiasanchez 05 Oct 2026

      Un 95,7 % de los estudiantes encuestados afirmó que el ejercicio les permitió reflexionar críticamente sobre su rol profesional. El argumento central que surge de las respuestas abiertas es que la IA debe asumirse como un "banco de ideas" o un recurso de inspiración para superar bloqueos creativos, pero nunca como un sustituto del juicio, la selección ni la dirección conceptual del diseñador.

    3. nataliamurrutiasanchez 05 Oct 2026

      Me parece muy revelador el análisis comparativo de las obras en Padlet. Mientras que las propuestas iniciales hechas por los estudiantes mantenían decisiones compositivas más contenidas, sutiles y conceptualmente precisas, la intervención con inteligencia artificial tendió a intensificar el color, dramatizar el ambiente o volver demasiado explícita la metáfora.

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    drive.google.com/file/d/12TjZH_plM098cx1A_EDlHvVa9nAzGD3K/view
  33. rooseveltinstitute.org rooseveltinstitute.org
    AI and Government Workers: Use Cases in Public Administration - Roosevelt Institute
    10
    1. KennyJam123 05 Oct 2026
      The programs convert speech into text in the first language, then translatethe written text into a second language. Like in the slumber party game of“telephone,” each step in the process increases the potential for error

      This ties in with my earlier question about accuracy

    2. KennyJam123 05 Oct 2026
      Certainly, AI can facilitate basic translation work forcommon languages in government offices. But effective public administration stillrequires the labor of multilingual people

      As for translation, Ai can be a big helper, but not a solution

    3. KennyJam123 05 Oct 2026
      The Miami court system’s chatbot “SANDI” offers a moredynamic conversational example. SANDI uses AI to help residents find andcomplete forms for “non-complex family court matters” on its websites—and caninteract with users in both English and Spanish.

      What personal information does this bot have access to when helping someone complete a form? Earlier in the article, reference to chatbots being used for court matters and how privacy becomes a concern led me to think about the ethics of this particular chatbot.

    5. KennyJam123 05 Oct 2026
      . These conversations may include sensitiveinformation, financial data, and follow-up visits to an applicant’s home.

      This raises a big ethical concern if the chatbot isn't super secure.

    7. KennyJam123 05 Oct 2026
      An October 2023 survey from the Bloomberg and Johns Hopkins initiative AI CityConnect found that according to mayors, only 2 percent of cities were using AI. ASeptember 2024 Ernst and Young survey of federal, state, and local governmentworkers at the senior level, however, found that more than 50 percent of workerswere regularly using AI applications.

      This statistic shows how the presence of Ai use is emerging

    8. KennyJam123 05 Oct 2026
      Basic chatbots provide consistent andaccurate information because their responses are prewritten by content experts,but they can be frustratingly limited.

      Chatbots make communication easier for the reciever, but more difficult for the sender.

    9. KennyJam123 05 Oct 2026
      More than 75 percent of workers in a recentsurvey reported that, amid expectations of increased productivity from theirbosses, AI had made aspects of their job more difficult and “added to theirworkload in at least one way.”

      Ai is adding to problems

    10. KennyJam123 05 Oct 2026
      . Some good government jobs —which have been a sourceof middle-class stability in communities across the country for decades—arealready facing elimination.

      Ai is taking away jobs

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    rooseveltinstitute.org/wp-content/uploads/2025/07/RI_AI-and-Government-Workers_Report_202507.pdf
  34. www.tandfonline.com www.tandfonline.com
    Environmental sustainability in restaurants. A systematic review and future research agenda on restaurant adoption of green practices
    3
    1. Santiago_Grageda 05 Oct 2026
      Anothercase of emissions that has received some attention is the emission due to food delivery

      Siento que no nos ponemos a pensar suficiente en todo el impacto ambiental del delivery, osea es un canal enorme para que los restaurantes vendan sus platillos. Y no solo es el packaging que no sea reciclable en muchos casos sino por las emisiones que se generan durante el transporte al consumidor

    2. Santiago_Grageda 05 Oct 2026
      Scholars have suggested thatsince the top management is aware that green practices increase positive perceptionsamong customers, they may try to engage in window dressing or superficial greening

      Esto lo relaciono mucho con lo que vimos en clase sobre el green washing ya que las personas ven el lado bonito o "sustentable" del restaurante pero al final realmente no sabemos si estas acciones sustentables realmente ocurren o o el impacto que estas tienen, o si solo es publicidad para quedar bien

    3. Santiago_Grageda 05 Oct 2026
      Nudging can be done using a variety of visual cues. For instance, the information mayjust be displayed in simple text (for example, CO2 emission in the production of this dish isxx kg/portion) or more creative visual cues (for example, traffic light colors to representthe intensity of the impact) (

      ¿Si se implementara este tipo de menús en México, realmente la gente se fijaría en ellos e impactaría en las decisiones de los consumidores? Siento que es mas una estrategia de marketing para que el lugar se vea más sustentable aparte si solo se habla de los insumos y no de todo lo que se utiliza para operar un restaurante. (Gas, luz, agua, etc).

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    tandfonline.com/doi/pdfdirect/10.1080/15022250.2022.2134203
  35. unshadowed.substack.com unshadowed.substack.com
    U.S. Harvest Under Water: Pre-Scripted Food Crisis Accelerates as Breadbaskets Fail [VIDEO]
    1
    1. milahu 05 Oct 2026

      just start depopulation here and now: give machine guns to the few worthy people, and let them eliminate the rest. natural selection. problem solved.

      then the only challenge is the disposal of all the human waste, millions of tons of rotting human flesh... but i guess they have already increased the number of waste incineration plants for this purpose

      overpopulation depopulation here and now
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  36. drive.google.com drive.google.com
    ART+6_ÑAWI_VOL10_N2.pdf
    3
    2. nataliamurrutiasanchez 05 Oct 2026

      Los autores argumentan que la revelación final reconfigura de forma retrospectiva toda la narrativa. Sustentándose en la teoría de Todorov sobre la vacilación de lo fantástico, defienden que este giro transforma la obsesión en una dinámica de control, manipulación y dependencia bidireccional. Sin justificar el acecho inicial, el argumento inicial demuestra que las categorías tradicionales de "víctima" y "victimario" resultan insuficientes para explicar la pérdida de autonomía de ambos personajes.

    3. nataliamurrutiasanchez 05 Oct 2026

      El autor define el horror obsesivo no como un nuevo subgénero cinefílico, sino como un descriptor analítico para comprender la fusión entre el thriller psicológico, la fijación romántica y el terror sensorial. A través de una focalización interna restrictiva, el lenguaje audiovisual fuerza al espectador a acompañar la subjetividad alterada del acosador.

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    drive.google.com/file/d/1CAedaw_vg9CW01EAmGpfhopLu2waJYRv/view
  37. rme-review.vercel.app rme-review.vercel.app
    Example board: pick the perfect example
    1
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    rme-review.vercel.app/example-board.html
  38. drive.google.com drive.google.com
    admin,+1_BOLD8_e015_Tesone.pdf
    3
    1. nataliamurrutiasanchez 05 Oct 2026

      El diseño no debe verse como una actividad meramente instrumental o técnica, sino como una profesión que articula de manera equilibrada lo funcional con lo estético, actuando como un constructor de sentido, cultura e identidad en constante diálogo con la sociedad.

    2. nataliamurrutiasanchez 05 Oct 2026

      Me parece un enfoque muy acertado, ya que la academia suele encasillar al diseño puramente en lo técnico u operativo, olvidando que las decisiones sobre el color, la forma, escala y el espacio tienen un profundo valor poético y expresivo que vincula directamente a ambas disciplinas.

    3. nataliamurrutiasanchez 05 Oct 2026

      El autor reflexiona sobre la falsa dicotomía entre el diseño y el arte, demostrando que el diseño no es ajeno solo por responder a un encargo o a la funcionalidad, sino que utiliza herramientas propias de este, compartiendo además los rasgos de lo contemporáneo como la serie, la reproducción y el anonimato.

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    drive.google.com/file/d/1Lfx2KG4AYwJN6FPR34bH9xipmTBEzk1a/view
  39. human.libretexts.org human.libretexts.org
    61: Cold, Hard Harvest: Making the Case for Frozen Produce (Pandolfi)
    2
    2. jiadan._ 05 Oct 2026
      the carbon footprint of a typical family meal was 5% smaller when made with frozen ingredients as opposed to fresh ones. Consider that for plant-based foods on the national market, transportation accounts for over 16% of total emissions and suddenly you are talking about substantial carbon savings when you choose local frozen produce as an alternative.

      logos

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    human.libretexts.org/Bookshelves/Literature_and_Literacy/Poetry_Plays_Essays_and_other_Genres/88_Open_Essays_-_A_Reader_for_Students_of_Composition_and_Rhetoric_(Wangler_and_Ulrich)/Open_Essays/61:_Cold_Hard_Harvest:_Making_the_Case_for_Frozen_Produce_(Pandolfi)
  40. drive.google.com drive.google.com
    recherchestravaux-12187.pdf
    3
    1. nataliamurrutiasanchez 05 Oct 2026

      El artículo muestra que una exposición no tiene porque limitarse solamente a mostrar textos o manuscritos. En casos como el de Tarkos, la escritura, el dibujo y otras formas artísticas se presentan como parte de una misma creación. Me parece importante porque cuestiona la idea de que la literatura y las artes visuales sean disciplinas completamente separadas.

    2. nataliamurrutiasanchez 05 Oct 2026

      El artículo plantea una dificultad interesante: la literatura puede ser más difícil de acercar al público general que las artes visuales, especialmente cuando se trata de autores que no son tan conocidos. Esto me hizo pensar en la importancia de buscar otras formas de presentar la literatura, como imágenes, sonidos o elementos interactivos, para que así más personas puedan acercarse a ella.

    3. nataliamurrutiasanchez 05 Oct 2026

      Me llamo la atención que organizar una exposición implica tomar muchas decisiones; como el que obras mostrar, como acomodarlas para que el público las pueda entender. En la exposición de Dotremont, por ejemplo, los organizadores tuvieron que encontrar un equilibrio entre la dimensión literaria y la artística. Esto demuestra que la exposición no solo presenta una obra, sino que también propone una manera de interpretarla.

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    drive.google.com/file/d/1lK8OrFWrq5HTeG8f-15bXBeCIS9VN43V/view
  41. www.biorxiv.org www.biorxiv.org
    Uncertainty-aware quantitative analysis of the structure and dynamics of T cell receptor repertoires
    4
    1. Public_Reviews 05 Oct 2026

      eLife Assessment

      This study presents a valuable tool for comparing immune receptor data across multiple samples while properly accounting for statistical uncertainty and receptor similarity. The evidence supporting the tool is solid overall. However, some key concerns regarding potential sequencing artifacts in one of the validation datasets and an unclear strategy for false discovery rate control in the proposed framework remain to be addressed.

      Summary
    2. Public_Reviews 05 Oct 2026

      Reviewer #1 (Public review):

      In this manuscript, the authors present ClustIRR, a computational tool that analyzes multiple TCR repertoires together, instead of one at a time. It builds one shared similarity graph across all the repertoires, then finds communities (CJs) on that graph that can be compared across samples. Building one shared graph across repertoires, rather than clustering each repertoire on its own, is a real improvement over existing tools. However, there are a few concerns that need to be addressed.

      (1) The Introduction motivates ClustIRR by contrasting it with beta-binomial regression, Fisher's exact test, and scCODA/tascCODA (lines 62-77), but none of these are actually run on the same data in the Results. Could the authors include a direct comparison, e.g., applying a standard beta-binomial or Fisher's exact test to the Dataset 1 CJ occupancy matrix, to show the Bayesian model gives a lower false-positive rate or better-calibrated intervals than the alternatives it's positioned against?

      (2) Prior predictive and posterior predictive checks are both reported as "(data not shown)" (lines 644, 728). Since the manuscript's central claim is rigorous, uncertainty-aware inference, it would help to include these diagnostic plots, along with Rhat and ESS values, in the supplement rather than stating they were checked.

      (3) With 7,505 CJs tested simultaneously for differential occupancy in Dataset 1 alone (Figure 1 legend), what is the expected false discovery rate under the non-overlapping-HDI criterion used throughout? A short discussion of multiple-comparisons correction, or an argument for why it isn't needed under this framework, would strengthen the statistical claims.

      (4) Line 115 states the Dataset 1 joint graph produced 10,301 CJs, of which 3,038 were singletons, leaving 7,263 non-singleton CJs. The Figure 1 legend reports 7,505 CJs used for the β modeling. Could the authors clarify how these two numbers relate - whether some singletons were included in the model, or a filtering step was applied that isn't described in Methods?

      (5) The Methods section states that archival pretreatment tumor tissue was available for four patients (Pt4, Pt32, Pt36, Pt38; line 531), but the T+/T- DCJ analysis in Fig. 3B-C is only shown for Pt4. Was this analysis attempted in the other three patients? Extending it, even partially, would substantially strengthen the claim that contracting DCJs are enriched for tumor-infiltrating TCRs, which is currently based on a single patient.

      (6) Dataset 1 was generated by deliberately stimulating T cells with EBV or MART1 antigen, so recovering EBV/MART1-annotated CJs from VDJdb is closer to a positive control than a blinded validation. Do the authors have, or could they obtain, any independent confirmation (e.g., tetramer data or an orthogonal cohort) for the CJs with large β that lack VDJdb annotation (orange dots, Figures 1B-C)?

      Review 1
    3. Public_Reviews 05 Oct 2026

      Reviewer #2 (Public review):

      Summary:

      The study confronts a major obstacle in repertoire analysis. Given that individual TCR sequences are diverse and sparsely detected across repertoires, identifying sample-specific enrichment of TCRs based on sample-to-sample comparison of exact clonotype sequences can be intractable. This paper attempts to build on insights that sequence-similar TCRs can share antigen recognition, such that aggregating similar sequences derived from multi-sample joint-graph communities (i.e. clusters of tightly connected nodes) could reduce sparsity and boost signal.

      Strengths:

      The study is a well-motivated effort to address a need in the field. The paper takes a unique approach. The core method is modeling community occupancy with a hierarchical Dirichlet-Multinomial model that attempts to account for the high level of overdispersion present in repertoire sampling, a technique that has been previously applied to compositional microbiome data.

      The authors apply this framework to both single-cell paired-chain and bulk single-chain TCR data. They develop a set of examples from public and synthetic data, with the most promising real-world application shown in reanalysis of longitudinal data during treatment of cancer patients with checkpoint inhibitors.

      The manuscript is well structured and cogent. The authors are to be commended for contributing a well-documented, open-source R/Bioconductor package and for providing the underlying analysis datasets in a well-organized repository. In the joint graph construction step, the authors opt to use an existing implementation of the BLAST algorithm on CDR3 sequences, which is a slightly odd choice since it ignores potential contributions of other V-gene germline-encoded CDRs, but the authors also envision that their statistical package could be extended to include community graphs developed with other established TCR clustering tools. This will allow others to potentially explore the utility of Bayesian hierarchical Dirichlet-Multinomial models for differential occupancy analysis of immune receptors under varied clustering criteria.

      The methods described here were demonstrated on relatively small datasets from 2-5 samples, and future work is likely needed to extend the joint graph differential occupancy concept to larger datasets. The authors are transparent about this and some of the other limitations in their current tool, most notably the computational cost of graph construction based on an all-versus-all sequence alignment to construct a joint sequence graph and the challenge of Bayesian parameter estimation as the number of subgraph entities scales with input data size. Since efficient approximate methods exist to find edges between similar text strings, the underlying idea of applying uncertainty-aware statistical inference to subgraph communities is promising, and the paper advances its primary goal.

      Weaknesses:

      The paper proposes the utility of the joint-graph community occupancy framework through three examples. I discuss potential weaknesses apparent in each separate example in turn.

      (1) Weaknesses in Example 1

      A broad weakness of the first results section ("Detecting EBV- and MART1-antigen reactive T cell communities from single cell datasets") is its reliance on a single vendor-generated dataset generated by the company ParseBio with no published experimental protocols and limited, if any, prior peer review. For reasons I will explore in greater detail below, the EBV-sample data may be particularly prone to chimeric pairings that confound the authors' primary analysis goals, and, at the very least, may not reflect physiologically realistic conditions for identifying antigen-reactive TCRs in other contexts.

      Let us first consider Dataset 1 in more detail. The authors compare 2 antigen-stimulated repertoires with 3 unstimulated controls. To improve on single clonotype-level comparisons, the authors propose comparing the cell count aggregated across cells within joint graph communities constructed from paired CDR3 sequences across all the samples. Thus, one of the most relevant questions one hopes the authors answer in this section is whether the resolved graph communities are made up of many distinct clonotypes (i.e., are they polyclonal), allowing the method to function as intended by aggregating across multiple clones with putative shared antigen-reactivity.

      Supplementary Figure 1B shows the size of all the communities with callouts for the putative EBV-expanded communities. The authors listed communities strongly enriched in the EBV-stimulated sample as e1, e2, e3, and e5. Each contains {greater than or equal to}100 clonotypes, and the authors note they contain at least one clonotype with a CDR3 sequence matching an EBV-annotated clone in VDJdb - a database of TCRs with some experimental evidence of epitope-reactivity. Community "e2" is notable for its remarkable size, including 1,339 unique clonotypes. At first glance, this seems promising for a method attempting to boost signal through community detection. However, it is worth re-investigating the individual clone sizes and sequences within this extraordinary community.

      In the EBV-associated community "e2", a look at the data provided by the authors on the paper's GitHub repository suggests a single clonotype (clonotype_9; TRAV12-3 CATQGSNDYKLSF / TRBV9 CASSTGQVATNEKLFF) comprises 26,917 cells. As such, it makes up 29% of the sample with a total of 90,588 cells. If one clone supplies most of a graph community's cell counts, the community-level posterior estimate of β (Figure 1B) effectively tracks a single-clone estimate, and the premise of borrowing statistical power across a polyclonal expansion in this example is hard to assess.

      There is also considerable evidence to believe that the apparent mega-polyclonality of cluster e2 may be partially an illusion, stemming from an artifact of this hyper-expanded clone's massive size and the experimental method used to assign TCRα-TCRβ chain pairings. In fact, the same α-chain CDR3 (CATQGSNDYKLSF) appears in ~1,219 clonotypes paired to distinct β chains, generating much of e2's remarkable 1,339-clonotype count. I believe two features warrant caution here. First, a single clonotype making up ~29% of total T cells in the sample is highly unexpected in ex vivo repertoires, suggesting intense non-physiological expansion conditions unlikely to generalize to other settings. That is, one would almost never expect to see a signal this strong.

      Second, one TCR-α chain paired to ~1,220 distinct and diverse β chains in one sample is also biologically unexpected given what we know of the best-characterized epitope-specific responses for EBV, including to the well-known HLA-A*02 EBV BMLF-1 epitope, which recruits a tetramer-stained repertoire with conserved CDR3 motifs in both chains and at least some constraint on favored V-gene/α-β pairing (See Extended Data Figure 5 in Dash et al., Nature 2017). This raises the concerning possibility of barcode collision and mispairing against a hyperexpanded clone in the ParseBio split-pool method, unlikely to be robust to a clone occupying a third of the sample. Most of the ~1,219 β chains paired to the dominant α have a cell count of 1, further raising the concern of artifactual pairing versus genuine convergence. The second largest community "e1" also seems to suffer from the same issue, with a TCRβ sequence from one super clone making up 7% of the sample potentially being artifactually over-paired to >500 rare single-cell-count TCR α chains.

      Taken together, these observations suggest that the authors' first positive control example passes but probably for the wrong reason since at least some of the "antigen-specific communities" are strongly anchored by a single hyper-clone. This could be remedied by repeating the same type of analysis on an ex vivo single-cell repertoire following more modest stimulation or natural infection (e.g., yellow-fever vaccine, influenza, or SARS-CoV-2 single-cell TCR datasets). I would advise future work using an alternative data source with better-documented experimental protocols, given the concerns above.

      (2) Weaknesses in Example 2

      Example 2 explores the application of Bayesian methods for identifying sample-enriched joint graph communities found in longitudinal data from many participants at two time points and longitudinal data from 1 person (Pt4) at 5 time points. A potential weakness of Case Study 2 is that it yields limited additional biological insight compared to what was previously shown by the authors of the underlying input data. Previously, Formenti et al. 2018 showed that the number of expanded clones after treatment strongly reflects responder status in this cohort, greater in patients with CR/PR versus SD or PD (See Figure 2b of the study). This 2018 primary analysis showed that tracking individual clonotypes was sufficient to reveal biological insight without the need for the computational demands of constructing a massive sequence similarity graph, finding communities on joint graphs, or Bayesian statistical inference. Thus, the impact of Case Study 2 in proving the unique utility of ClustIRR is somewhat diminished.

      It is not clear how this study's result is more "robust" than the original analysis. Perhaps the authors could further clarify what is learned from the uncertainty-aware approach that could not be learned from exact clone tracking in time series.

      Thus, example 2 shows that a complex method recapitulated the finding of a much simpler method for analyzing longitudinal TCR data where a strong signal of expansion was already present at the single-clonotype level. Since the Dirichlet method is sensitive to absolute counts, the large expanding clone in each community at 22 days may alone have carried most of the signal, which is not fully explored.

      In this section, the authors make an interesting observation that CDR3β detected in both PBMC and patient-matched tumor samples were enriched in contracting communities (7/14) versus expanded communities (1/41). The authors do not indicate whether a similar enrichment of tumor-infiltrating lymphocytes (TILs) matched Day 0-22 contracting communities in the other 4 patients with tumor-matched samples, which, if consistent, would strengthen their finding.

      (3) Weaknesses in Example 3

      The final example explores "convergent repertoire differences between species." This is intriguing in principle but less informative due to the use of synthetic data with somewhat predictable properties that some may reasonably consider baked in by the data-generating process. That is, some of the results might be guaranteed by the way the data is constructed using the OLGA/IGoR generative model. For instance, the authors observe a positive correlation between CJ community size and Pgen of constituent clonotypes, stating: "This indicates that CDR3 sequences with high Pgen are statistically more likely to be generated, leading to convergence of similar sequences into public CJs." I may be mistaken, but this conclusion is almost guaranteed by the way the data-generating OLGA model outputs more similar high-Pgen sequences and fewer lower-Pgen sequences.

      An interesting finding in this section is shown in Figure 4B, where community-level aggregation allowed for discrete clustering of human samples away from mouse samples that was not possible by comparing cosine similarity of a sparse clonotype occurrence matrix, a result that would be higher impact if it could be shown to separate real repertoire samples from humans with differential serology, vaccination status, or HLA backgrounds.

      (4) Weaknesses in General

      More generally, one aspect of the method that seems under-emphasized is the fact that many of the nodes in a multi-sample joint sequence similarity graph may have no edges. These zero-degree nodes would probably frequently occur in only one sample but be absent in other samples. It is not strongly emphasized in the paper how the model would infer whether such a singleton found in only one sample in the graph could be reliably inferred to be sample-specific enriched (see, for example, the large single node in Figure 3D, the orange node labeled "GQYF" in the far-right position of the lowest row in panel D). Presumably the number of cell counts represented in this single-sequence node is so great at sampled timepoint Day 22 as to yield a statistically strong signal in the multinomial model; however, the authors may wish to comment on how, for such singleton sequences, the power to detect sample-specific enrichment differs from prior single-clonotype-based methods.

      With any large effort to find statistically significant features from a large candidate set examined all at once, a reader might be concerned with the potential for false discovery. Throughout, the authors seem to assign statistical significance when the 95% high-density interval (HDI) of the posterior estimate excludes zero or when the 95% HDIs of two features do not overlap. There is little discussion of how this implicitly handles multiplicity adjustment via shrinkage, which the authors could address more directly and explain more clearly to a broad audience, including many non-statisticians, who will read this paper.

      Review 2
    4. Public_Reviews 05 Oct 2026

      Reviewer #3 (Public review):

      Summary:

      Analysis of immune receptor repertoires (IRR) needs to take into account the underlying diversity of the repertoires analysed, and the limitations inherent to the technologies used to measure IRRs: limited sampling depth relative to total number of cells and clonotypes, and experimental noise. In this work, Kitanovski and colleagues present ClustIRR. ClustIRR proposes to improve the analysis of immune receptor repertoires, specifically TCRs in the presented applications, by performing two steps: (1) consistent and comparable sequence clustering across repertoires, to account for sparsity of sampling, and (2) estimation of sequence clusters of interest using a Bayesian approach. They showcase ClustIRR performance in 3 scenarios: detection of antigen-specific T cells in a peptide-stimulation, detection of T cells responding to immunotherapy in the context of lung cancer, and analysis of mouse and human T cell repertoires.

      Strengths:

      (1) The cluster occupancy calculation presents an important conceptual framework which would be of interest and useful to the TCR repertoire field as it smoothly integrates information over a set of experimental conditions or time points. The application to longitudinal TCR sequencing datasets is particularly interesting, and could easily be extended to BCR sequencing datasets. Moreover, the calculation can be performed with any user-defined grouping of TCR clones, which allows for usage of other existing methods as the user wishes.

      (2) The results of the human and mouse repertoires provide a very insightful argument for the use of metaclones as opposed to single clones for analysis of repertoires compared to single clones.

      Weaknesses:

      (1) While ClustIRR provides an interesting framework to analyse TCR sequencing datasets, it is not clear whether ClustIRR can perform more informative sequence clustering than state-of-the-art methods. A comparison of obtained clusters with existing methods would provide a useful benchmark. Moreover, computation time scales quite fast with the number of sequences included. This is a major limitation, as the authors state that a time of 2.5 hours is required for clustering of ~100,000 sequences, a number of clones that can easily be reached when analysing multiple samples together.

      (2) The authors claim in the abstract that ClustIRR is integrated with gene expression data. However, in the results presented, the gene expression and TCR sequencing data are analysed separately, and the results are simply correlated. No real integration in the analysis exists for these two data types. The claim should be removed from the abstract. Moreover, the differential gene expression section, while it presents interesting results, lacks clarity and transparency. Presentation of the data in more transparent ways (such as showing violin plots or clustering on the UMAP) would increase the strength of the claims.

      (3) The score calculation does not seem to be normalized to take into account the underlying diversities of CDR3a and CDR3b. While still a useful metric for sequence clustering, I worry about the impact of the lack of normalisation on the conclusion that the "alpha chain drives functional convergence through germline bias". The observation that clustering is mostly driven by the J gene is known and expected (https://pmc.ncbi.nlm.nih.gov/articles/PMC5553937/), as the J gene has lower diversity and greater overlap with the definition of CDR3. Because CDR3a is lower diversity generally from CDR3b, it will likely dominate the sequence similarity signal. Thus, it will appear that Ja drives the signal. The authors do try to address this by looking at clusters driven by CDR3b similarity. However, a low number of CDR3b-driven clusters is consistent with the similarity definition. I wonder if instead the appropriate control for this analysis would be to run the same analysis disregarding CDR3a sequence altogether, and quantify whether similar species-specific DCJ are identified when only CDR3b similarity is used? Absence or reduction of species-specific clustering would confirm that the effect is driven by the CDR3a sequence.

      Review 3
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    biorxiv.org/content/10.64898/2026.06.28.735097v1
  42. www.pnas.org www.pnas.org
    Ecological consequences of human niche construction: Examining long-term anthropogenic shaping of global species distributions
    5
    1. sc4rlettw 05 Oct 2026
      If an archaeological perspective is key to conservation efforts, it also challenges elements of their foundation. If change is the only constant in human–ecology relationships, it remains unclear what “natural” targets ecological restoration should aim for (139). The wholesale appropriation of land and resources for environmental ends—“green grabbing”—at the expense of the needs and livelihoods of local and indigenous groups (often seen as destructive of pristine ecologies) is also further problematized (146). Appreciation of historical data shifts conservation ecology away from concern with a return to original ecological conditions, suggesting the need for pragmatic solutions that acknowledge the integral role humans have long played in shaping natural systems (36, 139). The impact of human agency on ecosystems is neither completely avoidable nor entirely undesirable. People have inhabited a growing range of environments at ever increasing densities only through the continual anthropogenic transformation of ecosystems. Rather than an impossible return to pristine conditions, what is needed is the historically informed management of emerging novel ecosystems to ensure the maintenance of ecological goods and services (139). Such efforts need to account for the needs of all stakeholders and balance local livelihoods against first world agendas.

      Further thought: One aspect of the reading that caught my attention is the authors’ statement regarding how “pristine” ecosystems never really existed due to the fact that human interference within ecosystems dates back several thousand years. The question I had from this is how archaeologists and conservationists will be able to establish what a “natural” ecosystem would be supposed to look like when humans have already interfered with the system for thousands of years.

    2. sc4rlettw 05 Oct 2026
      Third, there is widespread evidence for increasing rates of human-mediated species translocation, extinction, and ecosystem and biodiversity reshaping through time. This acceleration is not constant but is characterized by pulses and pauses that reflect cultural, ecological, and climatic transformations at local, regional, and global scales. These changes have increasingly concentrated biomass into particular sets of human-favored plants and animals (134).

      Question set 3 - #3 It seems there is a pattern of growing human impact over time, although not a totally steady process. According to the reading, the first human impacts started during the Late Pleistocene period and intensified when agriculture emerged and spread in the Holocene epoch. The impacts intensified further when islands were colonized, when urbanization occurred, and when trade routes extended, moving species around. The point made by the authors is that all these developments have proceeded in “pulses and pauses,” which means that the acceleration of human-induced impacts did not occur evenly across all regions. It is quite fascinating that this process depended on many cultural and environmental factors.

    3. sc4rlettw 05 Oct 2026
      One of the major outcomes of the Neolithic was the inexorable spread of agriculture from ∼14–20 centers of early domestication (56) to encompass large swaths of the Old and New Worlds. This expansion had unprecedented and enduring impacts on species distributions. Key among these transformations was the promotion and expansion of a range of human-favored taxa, including newly created species (and subspecies) of domesticated crops and animals. Cumulative archaeological data show that crops and animals saw significant prehistoric and historic range expansion (Fig. 3). The scale of agriculture and land use in some regions was significant; for example, expansion of land area used for livestock and rice (Oryza sativa) paddy agriculture was sufficient to increase atmospheric methane emissions between 4,000 and 1,000 y B.P. (57) whereas deforestation and tillage are suggested to have contributed to increasing CO2 over the past 8,000 y (58).

      Question set 3 - #1 The first important pattern is that plants and animals did not stay confined to their places of domestication, but they expanded into new geographical regions as people moved around. As stated in the reading, for example, sheep, goats, and cattle were domesticated in the Near East and in just a few thousands of years, they became widespread in Europe, Africa, and South Asia. Not only crops but weeds and other organisms were transported by people at the same time; thus, people transported entire ecosystems, not just specific domesticated organisms. It means that we have to view domestication as an expansion process and human-environment interaction process.

    4. sc4rlettw 05 Oct 2026
      Species translocations to islands were so common in the past that archaeologists often speak of “transported landscapes” (84). These new landscapes included a broad range of domesticated animals, commensals, crops, weeds, microbes, and other species carried by humans. For example, Neolithic colonizers who arrived on Cyprus brought domestic cereals, pulses, sheep, goat, cattle, pigs, domestic dogs, and cats (Felis catus), as well as mainland game animals such as fallow deer (Dama dama), fox (Vulpes vulpes), and wild boar beginning 10.6 ka (64, 85). Polynesian people, expanding across the Pacific after ∼3,500 y B.P. (84), introduced a broad range of domesticated species, including the crops taro (Colocasia esculenta), yam (Dioscorea spp.), and banana (Musa spp.), and such animals as the domestic pig, chicken, dog, and Pacific rat (Rattus exulans). In the Caribbean, Archaic and Ceramic period peoples introduced a variety of species, including wild avocado (Persea americana), manioc/cassava (Manihot esculenta), maize (Zea mays), tobacco (Nicotiana rustica), and various trees, as well as dogs, opossums (Didelphis sp.), guinea pigs (Cavia porcellus), and shrews (Nesophontes edithae) (86).

      Question set 2 - #2 A particularly fascinating case is that of Polynesians settling the Pacific. As people spread through the Pacific Islands around 3,500 years ago, they actively introduced crops like taro, yams, bananas, pigs, chickens, and dogs, as well as Pacific rats that may have been introduced unintentionally. These species allowed the formation of more habitable islands as many of the islands had very few sources of edible plants and animals. Therefore, colonization of the islands differs from migration to a region that was already fertile before people arrived there. People carried fragments of their old ecosystems with them to create new anthropogenic ecosystems.

    5. sc4rlettw 05 Oct 2026
      The human-mediated translocation of species now dates back to the Late Pleistocene. For example, the northern common cuscus (Phalanger orientalis), endemic to New Guinea, was transported to eastern Indonesia, the Solomon Islands, and the Bismarck Archipelago beginning ∼20–23 ka, becoming a key subsistence species (26, 27). Other taxa were also moved; together with a species of bandicoot (Echymipera kalubu) and the Admiralty cuscus (Spilocuscus kraemeri), the Canarium indicum tree was introduced to Manus by ∼13 ka, followed a few millennia later by the rat Rattus praetor (26). Translocation patterns mirror patterns of maritime obsidian exchange in Melanesia in the Late Pleistocene and Early Holocene (26).

      Question set 2 - #1 Species movements were made possible by the human migration into new environments as well as their purposeful or accidental movement of organisms as they traveled. This is demonstrated by the example provided in the reading where it states that the Northern Common Cuscus was moved from New Guinea to eastern Indonesia, the Solomon Islands, and the Bismarck Archipelago as early as 20,000-23,000 years ago. Mankind was not only migrating to new territories but also relocating plant and animal life which were beneficial to their survival. Advances in transportation and human mobility made this practice possible. I believe that human migration and other species migration were directly related due to the fact that human migrants were affecting the ecosystems they inhabited.

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    pnas.org/doi/10.1073/pnas.1525200113
  43. research.ebsco.com research.ebsco.com
    Closely Watched Films : An Introduction to the Art of Narrative Film Technique - EBSCO
    1
    1. AhmedW 05 Oct 2026
      Griffith’s powerful shot juxtapositions suggested to theSoviet filmmakers and theoreticians that editing was the foundation offilm art.
      1. The soviets took a lot of inspiration from the editing style of griffith in a brave new world which was considered the heart of the art form to Eisenstein. I think there exactly right. The ultimate advantage of film compared to theatre or the advantage of pre-made vs live production is that the director, artist, painter, etc. get to choose exactly what you see and don't, and thats what editing is all about
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    research.ebsco.com/c/aneu5t/ebook-viewer/pdf/xvk7iadnhf/page/pp_22
  44. research.ebsco.com research.ebsco.com
    Closely Watched Films : An Introduction to the Art of Narrative Film Technique - EBSCO
    1
    1. AhmedW 05 Oct 2026
      “What diabolical mechanism lies hidden inthis art that I serve!” he wrote. “It’s not merely a cheat and a swindle.It’s poison—a dreadful, terrifying poison. For, if you can get your en-joyment through fantasy, who is going to make the effort any more tofind in real experiences what can be had without moving from the the-atre seat?”7
      1. I think the critic differentiates " real" and "artistic" for the similar reasons Eisenstein dislikes theatre for the needs of the soviet union. Just as theatre is a poison that can break the revolutionary spirits of the people, purely "artistic" do not derive the same feeling as a "real" showing of the feeling the people have put on film
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  45. docdrop.org docdrop.org
    JUH317600_WEB.qxd
    2
    1. Stardlp 05 Oct 2026
      While the Crawford case would not be settled until the 1980s, re-zoningand busing became a threat as early as the 1960s, given the activities of inte-gration advocates in the courts, in city hall, and in the schools. An article inthe Pico Post in February of 1968 quoted Superintendent Jack Crowtherbeseeching citizens to maintain calm about desegregation and potential bus-ing: “We cannot allow these false rumors to continue, for their only effect isto create a feeling of panic and dissension. I plead to all responsible citizensand especially to the communications media that they do everything possibleto combat these rumors.”45A few months thereafter, the Los Angeles City Board of Education passeda motion in favor of small-scale busing. Before the vote was taken, Dr. RalphSchneider / WHITE FLIGHT FROM LOS ANGELES1003

      It is disheartening that simple efforts to integrate schools were met with huge panic and disguised racism. Dr. Ralph Richardson’s words where trying to make a point through polite excuses to expose the pure racism behind the fight against busing. Its so bad how people used money concerns and school funding as an excuse to hide their underlying racism. I belive this shows that school segregation in Los Angeles was so entrenched that even tiny steps toward integration met fierce backlash.

    2. Stardlp 05 Oct 2026
      For six days in August of 1965, the Watts riot consumed Los Angeles, andwould forever change the context of integration in the city. A total of thirty-four people were officially reported killed during the riot, 1,100 were injured,4,000 were arrested, and an estimated $35 million in damage was done. Watts,and by extension Los Angeles, joined New York and Philadelphia, which hadexperienced race riots in 1964, as an urban crisis zone. Coverage of the Wattsriot brought the concept of violent race-conflict into public discourse. Post-riot news coverage included “considerably more attention to interracial vio-lence . . . mainly due to discussions of the riot . . . [and] unfavorable [media]references rose from 15% before the riot to 34% afterward.”30A 1971 study by Johnson, Sears, and McConahay, led the authors to con-clude that race conflict in Los Angeles in the late 1960s, though it was pre-sented as a present and significant threat, “served as the justification forfurther inattention and inaction, and for added repression.”31It worked. In herSchneider / WHITE FLIGHT FROM LOS ANGELES1001

      It is sad to realize how quickly leaders can use a tragedy as an excuse to ignore problems instead of actually fixing them. Especially since public protests often cause the very problems they fought against, because the media uses the violence to justify even more harsh government control. Bad press shows that how we talk about a crisis usually spreads fear instead of encouraging societies to help. This passge progress stops when we meet inequality with surveillance and neglect instead of healing.

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  46. press.rebus.community press.rebus.community
    The BIG Terms | Interdisciplinary Studies: A Connected Learning Approach
    1
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  47. human.libretexts.org human.libretexts.org
    "Learning to Read and Write" by Frederick Douglass
    3
    1. llinton 05 Oct 2026
      The moral which I gained from the dialogue was the power of truth over the conscience of even a slaveholder.

      He could finally read, and it began to become hurtful to him

    2. llinton 05 Oct 2026
      I continued to do this until I could write a hand very similar to that of Master Thomas. Thus, after a long, tedious effort for years, I finally succeeded in learning how to write.

      The thesis is, showing the life that he went through as well as the struggles in order to have the ability read and write.

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    human.libretexts.org/Courses/Butler_Community_College/Idea_to_Essay:_Collected_Strategies_and_Readings_for_the_College_Writer_4th_ed/07:_The_Reader/7.01:_Reading_About_Identity_and_Education/7.1.04:_Learning_to_Read_and_Write_by_Frederick_Douglass
  48. human.libretexts.org human.libretexts.org
    "Everything Has a Name" by Helen Keller
    2
    1. llinton 05 Oct 2026
      I do not remember what they all were; but I do know that mother, father, sister, teacher were among them —words that were to make the world blossom for me, "like Aaron's rod, with flowers." It would have been difficult to find a happier child than I was as I lay in my crib at the close of that eventful day and lived over the joys it had brought me

      She showed the struggles she went through in life and how hard it was to overcome those struggles.

    2. llinton 05 Oct 2026
      Have you ever been at sea in a dense fog, when it seemed as if a tangible white darkness shut you in, and the great ship, tense and anxious, groped her way toward the shore with plummet and sounding-line, and you waited with beating heart for something to happen?

      This showed a good example of how she was feeling and worded it so that other people could understand it.

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  49. social-media-ethics-automation.github.io social-media-ethics-automation.github.io
    1.2. Kumail Nanjiani’s Reflections on Ethics in Tech
    1
    1. kadenyip 05 Oct 2026
      Why do you think the people who Kumail talked with didn’t have answers to his questions?

      I've personally watched the show Silicon Valley and I think the show answers this question quite easily. In the show Silicon Valley, the main characters, along with Kumail's character, develop software and technology primarily for the purpose of technological ambition and the pursuit of money, disregarding ethics or social consequences. The show Silicon Valley is a mock comedy show about a real city, certain aspects are exaggerated, but certainly not the reason these engineers build their technology. Lots of engineers in real life build their inventions because they want to see the world progress technologically as fast as possible and partially because they want to live a financially comfortable life. These are two of the many reasons that engineers don't consider ethics and never even realize that it could be an issue.

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    social-media-ethics-automation.github.io/book/bsky/ch01_intro/02_kumail.html