Juan studies from January 20 to May 4.

¿Un lenguaje se vuelve grande solo con ser bueno? Considero firmemente que necesita de una comunidad que lo respalde.

Esto es acertado. En mi experiencia con diferentes lenguajes de programación los bugs más graves nacen en lo que parece algo muy simple.

Es algo difícil, pero cuando se logra aunque sea en parte se vuelve un lenguaje muy llamativo.

Esto es bastante real, ya que la mayoría de lenguas de programación nacen de una necesidad.

El trabajo colaborativo del código abierto es lo que permite que evolucione contantemente, si algo a ellos los creadores no se les ocurre, puede que a otro se le ocurra algo mejor. y ese constante cambio puede llegar a mejores cosas.

Ellos lo saben, con tantos desarrollos en proceso y la tecnología corriendo a grandes pasos, su proyecto no tendrá final, siempre estará en constante evolución, e impactando en su proceso.

Bueno, siento que como todo lo novedoso siempre tiene un declive, entre los aparatos tecnológicos o lenguajes de programación o simplemente en este mundo de tendencias, todo tiene un cierto declive si no se innova, aunque también puede ser momento de una nueva revolución si se desea. Bueno si ven que se puede mejorar o dar un nuevo salto en su necesidad.

Es curioso como se dio el comienzo de este lenguaje, sin tanto tramite o burocracia, solo una charla entre colegas con correos y pasaron manos a la obra, es gratificante ver como un proyecto así nace sin tanto papeleo, solo personas, una idea y ganas de desarrollar algo.

Las grandes invenciones son desde una necesidad, para estas personas que en su practica de uso de estos lenguajes y querer combinar y crear algún modelo mucho mejor y que fuera fácil, es como todo desde una idea o un pensamiento pequeño puede llegar a cambiar algo en gran medida.

Anyone should be able to become a member of the House of Representatives and should not be discriminated against on the basis is class, religion, or origin.

Madison believes that term limits and voting will help keep the House of Representatives from having corrupt individuals.

La gestión de estas áreas en conjunto, permiten que la información sea accesible, confiable y útil para la sociedad.

This emphasizes how bringing the world together is more about connection and not conquest.

To me this talks about how more robotic we sound the more we use devices and I personally agree. It always feels more connected talking face to face then texting.

Carr is expressing what many people have been expressing for a little while now and that's a low attention span and a struggle to focus on tasks.

Women in their 30s and they are not coming back to their jobs in the music industry.

Freelancing is prominent in the music industry.

Vick Bain’s 2019 study of the music industry found that just over 14% of those currently signed to 106 music publishers and just under 20% of those signed to 219 record labels were female.88 The BPI told us:

We are already seeing the changes in relation to the signing of women artists as a result of more women working in these crucial roles and targeted entry-level programmes, some specifically for women.

The lack of women in positions of authority sets the culture and influences decisions for the rest of the profession and can have a direct impact on women’s career opportunities and progression.

A lot of these organizations do not have many funds and are ran by volunteers.

Support systems for women in the music industry.

^^ Continuation from above

Women who are disabled or in the LGBTQ+ community are harassed and bullied more than other groups of women.

Women have lost work and opportunities because they had children, took family leave

Women are experiencing harassment both in real life and online on social media--not for their talent but their attractiveness and physical looks, worth is judged based on appeal and sexual ability.

QUESTION -> METHODOLOGY -> what was using focus groups like?

Research question: Why have crises like the Eurozone NOT triggered MORE EU citizen mobilisation? (like that reading back in the day that said citizens in EU-bailout crises did NOT treat EP elections any less like SONEs than non-bailout countries, despite being the direct recipients of EU policies)

Suggests / argues that: - BECAUSE EU citizens have limited / restricted knowledge of EU - "limited knowledge of the EU" - Discourse that frames EU as group of / consortium BETWEEN MEMBER STATES as opposed to integrated democracy OF those states - Do not understand that EU policies are in part responsible for crises -> attribute responsibility entiorely (or mostly) to national govts.

MEANS THAT politicisation of EU issues is more likely to / has already led to INCREASED "RE-NATIONALISATION" of these issues as opposed to making these issues SUPRANATIONAL in scope among the electorate -> implicitly less of a pan-EU identity then, too.

ARE SPECIFICALLY ASKING WHY THE EUROZONE CRISIS DID NOT LEAD TO FURTHER INTEGRATION VIA INCREASED ELECTORATE PARTICIPATION

"This article examines to what extent the Eurozone crisis has indeed contributed to such a shift towards European citizenship."

this is a good way to describe the situation as one side gives difficultly and more satisfaction when done vs the other where there is no difficulty at all.

this gives the author good support to her question of asking if AI is making us dumber.

logos

logos

Défis et controverses de l'éducation des parents : Analyse et perspectives

Résumé exécutif

Le présent document synthétise l'analyse du sociologue Claude Martin concernant l'évolution des pratiques et des politiques d'éducation des parents en France.

Le constat initial révèle un « effet de ciseaux » alarmant : une explosion de la souffrance psychique chez les jeunes coïncidant avec un affaissement de l'offre de soins et de soutien humain.

L'analyse souligne un basculement paradigmatique majeur : le passage d'un déterminisme social (collectif et structurel) à un déterminisme parental (individuel et comportemental).

Cette évolution a favorisé l'émergence d'un marché du conseil aux parents et d'une « parentalité positive » qui, bien que prônant la bienveillance, impose de nouvelles injonctions de performance et de bonheur.

Le document explore également les usages politiques des neurosciences et les controverses actuelles entourant les méthodes éducatives, concluant sur le paradoxe du « double bind » (double contrainte) auquel les parents modernes sont confrontés.

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1. L'état des lieux : Une jeunesse en souffrance

La situation actuelle de l'enfance en France est marquée par une dégradation notable de la santé mentale, un phénomène antérieur à la pandémie de COVID-19 mais accentué par celle-ci.

L'effet de ciseaux

Le Haut Conseil de l'enfance, de la famille et de l'âge (HCFEA) alerte sur deux phénomènes concomitants :

• Explosion de la demande : Une hausse massive des manifestations de souffrance psychique chez les enfants et adolescents.

• Affaissement de l'offre : Une réduction drastique des moyens de prise en charge (thérapies de parole, lieux d'accueil) et une crise du secteur de la pédopsychiatrie.

La réponse médicamenteuse

Faute de structures d'accompagnement suffisantes, la réponse s'est déplacée vers la prescription de psychotropes, avec des augmentations spectaculaires entre 2014 et 2021 :

| Type de médicament | Augmentation de la prescription (2014-2021) | | --- | --- | | Antidépresseurs | \+ 63 % | | Psychostimulants | \+ 78 % | | Hypnotiques et sédatifs | \+ 155 % | | Antipsychotiques | \+ 50 % |

Le phénomène du retrait social

Le document identifie l'émergence en France du phénomène de retrait social (type Hikikomori), touchant principalement des garçons lycéens (15-17 ans).

Ce refus d'entrer dans la course à la réussite scolaire est parfois analysé, de manière controversée, à travers le prisme de l'influence parentale (notamment des mères jugées excessives ou intrusives).

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2. Le basculement des déterminismes

L'approche sociologique a radicalement changé de nature entre les années 1960 et aujourd'hui.

Du collectif à l'individuel

• Le déterminisme social (Années 60-70) : La réussite ou l'échec d'un enfant était perçu comme le résultat de l'appartenance à une classe sociale et de la reproduction des inégalités. C'était un enjeu de lutte collective et politique.

• Le déterminisme parental (Actuel) : La focale s'est déplacée vers le comportement individuel des parents.

Les difficultés de l'enfant (santé mentale, échec scolaire, comportement antisocial) sont désormais imputées à un déficit de « compétences parentales ».

La psychologisation des problèmes publics

Cette vision individualiste conduit à une responsabilisation accrue des parents, générant souvent un sentiment de culpabilité.

Des auteurs comme Frank Furedi (Paranoid Parenting) ou d'autres parlent de « parentalité narcissique », soulignant un manque de confiance des adultes dans le futur qui compromettrait leur capacité à éduquer.

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3. Évolution historique du contrôle de la fonction parentale

L'éducation des parents n'est pas un concept nouveau, mais elle a traversé plusieurs phases distinctes :

1. Fin XIXe - Début XXe siècle (Hygiénisme et Protection) : Lutte contre la mortalité infantile et protection contre les « classes dangereuses ».

Il s'agissait alors d'enseigner aux mères les soins de base et de limiter la puissance paternelle absolue.

2. L'après-guerre (Le marché du conseil) : Émergence de manuels à succès (Benjamin Spock, Laurence Pernoud, Françoise Dolto).

Ce secteur économique puissant prospère sur l'inquiétude des parents : plus ils consomment de conseils, plus ils se sentent déroutés, alimentant une consommation accrue.

3. Années 1990 (L'invention de la « Parentalité ») : Le terme parenting (centré sur l'acte et le comportement plutôt que sur le statut) est traduit par « parentalité ».

Cela devient un segment à part entière de l'action publique, visant à « soutenir » les parents, mais les prenant en réalité comme cibles d'intervention.

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4. Neurosciences et "Neuro-parenting"

L'usage des neurosciences dans l'éducation des parents fait l'objet de critiques importantes, notamment concernant la surinterprétation de données scientifiques.

• Le mythe des trois premières années : Une fascination scientiste pour l'imagerie cérébrale a conduit à l'idée d'une « fenêtre d'opportunité » unique durant les trois premières années de vie.

Cette vision déterministe présente le bébé comme un « petit ordinateur » dont le câblage dépendrait entièrement des stimuli parentaux.

• L'adolescent stigmatisé : À l'inverse de la vision « mine d'or » du cerveau du nourrisson, le cerveau de l'adolescent est souvent présenté par les politiques publiques comme « mal foutu » ou intrinsèquement problématique, justifiant des interventions urgentes.

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5. La parentalité positive : Entre bienveillance et injonction

La « parentalité positive » est devenue un courant dominant, porté par un lobbying actif auprès des pouvoirs publics.

La controverse du "Time Out"

Une polémique oppose actuellement deux visions :

• Les partisans du cadre : Préconisent des méthodes simples comme le « Time Out » (envoyer l'enfant dans sa chambre) pour gérer les crises.

• Les radicaux de la bienveillance : Assimilent le « Time Out » à une « violence éducative ordinaire », créant une continuité entre ces pratiques et des dérives graves comme l'infanticide.

L'injonction au bonheur

La parentalité moderne impose une « norme sous la peau » : les mères ne doivent pas seulement bien agir, elles doivent être « authentiquement heureuses ». Un faux sourire est perçu comme dangereux pour l'enfant, créant une pression psychologique insoutenable pour les parents.

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6. Conclusion : Le paradoxe de la mission parentale

Le document conclut sur l'impasse du « double bind » parental actuel :

• D'un côté : Les parents qui « n'en font pas assez » sont désignés comme irresponsables ou absents.

• De l'autre : Les « parents hélicoptères » (parentalité intensive) sont critiqués pour générer une dépendance problématique chez l'enfant.

L'analyse de Claude Martin suggère que la politique de parentalité devrait redevenir un soutien collectif et générationnel plutôt qu'une focalisation sur les comportements individuels.

L'éducation est une improvisation située historiquement ; les modèles parentaux ne peuvent être des invariants déconnectés du contexte social et des limites de chaque génération.

I don't know if that's standard practice -- these are 2 EA-linked or adjacent groups -- moderate

I think that it is important to remember that we can only learn so much in a day and to manage what we want to teach students over time.

I think that it is important to have more than one way to deliver lectures and not just listening to the teacher talk about a subject in class.

I did not realize what cognitive load meant and I think that this is really interesting.

This connects to my own experience as a learner. I often become overwhelmed when too much information is presented to me at one time. I think it is super important to be intentional about what information to share and when to share it.

This article reminded me of how important it is to remember that everyone has different learning needs. Using both written and visual materials can help students who learn in different ways, making the classroom more inclusive.

Doing this makes learning more accessible and flexible. It ensures that all students have access to the material. An example of when this would be helpful is when a student misses a class or when someone needs to review what was covered in class.

Hanssen, Geir Kihle. 2026. The Typewriter Enthusiast’s Handbook: An Introduction to Analogue Typewriters in a Digital World. https://www.amazon.com/Typewriter-Enthusiasts-Handbook-Introduction-Typewriters/dp/B0GMYYG367 (February 16, 2026).

https://typewriterdatabase.com/typewriters.php?hunter_search=5641&collection_search=My+Collection

A second pair of eyes is usually beneficial. We do this at work and it allows us to help each other out.

This shows us that we are all human's and human errors are likely to happen. It has nothing to do with our intelligence, it has to do with mistakes that happen to all of us. And it's okay!

https://www.poetryfoundation.org/poets/james-whitcomb-riley

less ppl want to buy my expensive stuff, so i make less sales overall despite having more revenue per sale

ChatGPT, next to adding ads, is now also inviting users to upload their contact list according to this web article. The purpose is unclear, is OpenAI building a social media platform? It is a EU wide GDPR violation though. Even if I were a user and opt-out others who don't might upload my personal contact details.

I think it's crazy to think that even this long ago Native Americans were seen more as a resource then as people, as their value in the fur trade was seen as the main reason that they were not exterminated or enslaved like other native people.

It is very impressive to me that China has been such a world power for how long they have been. They were one of the first real world powers to ever exist and even to this day they are seen as a top world power along with the United States.

I never knew that the Philippines were named after the Spanish King Philip II and that it was made up of thousands of islands, I thought it was a lot smaller than that. However I did know of its importance to the Spanish as it was one of the territories fought over during the Spanish American War.

Looking at this now, I can't help but think of how it could've been very inefficient to rely on goods from trade that would take this long to obtain. Getting to the Mediterranean Sea from the Indian Ocean at this time would have taken an extremely long time as it would require going all the way around the African continent, and it would take a very long time to get to the bottom of the continent from both sides.

https://www.reddit.com/r/typewriters/comments/1r6bjkm/require_help_to_identify_typeriter/

This is my thought

https://old.reddit.com/r/typewriters/comments/1lw38tv/please_help_me_identify_this_typewriter/

The french took a more hieracical route to promote assimilation. Th French divided colonies into cantons that did not correspind to pre colonial govenemnte units, with them sometimes intetionally breaking up old poltical units. - chiefes were not selected according to customaryprocedure, but were selected based on their education, lineage or loyalty to the french. - their function was basically to be a mouth piece to the locals, maintained no judicial authrity, and they eventualluy removed

british - indrect rule. Done through chiefes by british political officer with their main purpose being ti intefere in the domestic affairs when neccessary. However in cases where authority f the chief in a given area was not as strong, political officers influence intensened. - had a system of judicial authoriuty

french

Video on running an ollama coding assistant on Hetzner. Does not mention the fact it runs on a GEX44 at Hetzner, which starts at 200 Euro/month

Example of person working with Obsidian and Claude Code. Note that it does not use the Obsidian CLI access, but its API.

Same author some months back mentioned running Claude Code on a small Hetzner VPS (but just Claude Code, no models) so he could access it from anywhere (except offline obviously).

Laying out the overarching questions to be explored in the article as the research results are discussed. Main ideas of the whole article are expressed here; preceding text is priming the audience and providing background.

This is essentially a traffic light problem.

mentions different ways of installing a local set-up for AI. Also mentions much cheeper options than Anthropic, specifically OpenRouter.ai. Does not mention running your own local install on a cloud VPS.

??

Comparison video of Claude Code using Anthropics cloud models vs local models on a M4 128GB. Still a heavy lift, fans spinning, memory usage almost at full capacity. But it works. Means that for my M1 16GB a smaller model is all that works, and you need to leave room for context loading too. For one-offs like code generation and for interactive in moving contexts there's different needs.

Decline in Golden Eagle prey habitat = decline in Golden Eagle population and therefore an overall disturbance to the ecosystem.

Warm-dry sagebrush steppe is a critically important habitat for Golden Eagles, serving as a nesting habitat, hunting ground (and primary habitat for prey). The health of this ecosystem is a key indicator of the Golden Eagle population's survival.

test 2123

Must look into informetrics; it looks very interesting.

test annotation

mean proportion of unburned shrubland decreased from 0.73 to 0.22 inches in 2014

MinBzk zoekt bmw op digitale autonomie.

de specifieke ordening, samenstelling of inrichting van onderdelen binnen een systeem, apparaat of structuur. Het heeft betrekking op hoe componenten (hardware, software, of atomen) ten opzichte van elkaar zijn geplaatst en ingesteld om een gewenst geheel te vormen.

How do districts ensure mentors are the best of the best and can properly support new teachers?

Creating the partnership between the university and school districts would be beneficial. The issue arises if there is not a program like this in areas without a university close.

How to school districts ensure this occurs throughout? Is it only in some buildings but not other?

Why is this? Are we examining the right things in education?

This study provided various data points which is helpful to find the commonalities. While there are commonalities there is a range.

This to me means university and district level. Programs that work together provide the necessary tools for success.

providing a mentor and counties offering PD is just the surface. For new teachers to see teaching in action from skills professionals is valuable. This is especially true in special education.

Does the level of support look the same for every teacher? This is where building administrators need to know their staff. Walk throughs and regular informal chats provide the insight needed.

This is where self-reflection needs to occur on all levels of the school system. Providing relevant professional development is necessary. Teachers do not want to feel like they are wasting time when there is so much they could be doing.

This is information school districts need to look at. Specifically within the individual schools.

Dossier de Synthèse : L'Implication des Usagers dans les Structures d'Exercice Coordonné

Synthèse

Ce document synthétise les enseignements du webinaire régional concernant l'indicateur « Implication des usagers » pour les Maisons de Santé Pluriprofessionnelles (MSP) et les Centres de Santé (CdS).

Initialement centré sur la satisfaction des patients, cet indicateur a évolué pour devenir un levier global de transformation du système de santé, incitant les structures à passer d'une logique de soin « pour » le patient à une logique de soin « avec » le patient.

Bien qu'optionnel, cet indicateur est considéré comme un objectif structurant pour l'exercice coordonné, conditionnant une partie du financement par l'Assurance Maladie via l'Accord Cadre Interprofessionnel (ACI).

En 2024, plus de 70 % des structures ont atteint le niveau 1 de cet indicateur, démontrant une maturité croissante.

Le passage au niveau 2, qui implique une co-décision et un partenariat pérenne, reste le défi majeur pour les équipes de soins primaires.

1. Cadre Stratégique et Enjeux de l'Indicateur

L'implication des usagers n'est plus perçue comme un objectif isolé, mais comme une démarche transversale visant à améliorer l'efficacité des soins et l'adéquation de l'offre de santé aux besoins réels des territoires.

Objectifs de la démarche

• Améliorer la qualité des soins : En intégrant l'expertise de vie du patient (maladie, handicap).

• Renforcer la démocratie en santé : Donner une voix légitime aux usagers dans la co-construction des actions de santé.

• Évolution du projet de santé : Utiliser les retours des usagers pour faire évoluer de manière vivante le projet de la structure.

• Qualité de vie au travail (QVT) : Le partenariat est identifié comme un levier d'amélioration du quotidien des professionnels.

Financement et Justification

Le financement par l'Assurance Maladie est conditionné par la fourniture de justificatifs probants.

Cette exigence est présentée non pas comme une suspicion, mais comme une garantie de transparence dans la gestion des fonds publics.

• Nouveauté : Les négociations en cours suggèrent une évolution du modèle pour supprimer les niveaux de complexité, tout en maintenant l'évaluation de la satisfaction et la co-décision.

• Dynamisme : Pour être rémunérée, une structure doit démontrer une progression ou une révision de ses outils d'une année sur l'autre.

2. La Philosophie du Partenariat en Santé

Le passage au partenariat repose sur un changement de paradigme, souvent appelé le « modèle de Montréal ».

| Modèle | Approche | Position de l'usager | | --- | --- | --- | | Paternaliste | Pour le patient | Objet de soin, passif. | | Centré sur le patient | Pour le patient | Au centre des préoccupations, mais exclu des décisions d'équipe. | | Partenariat | Avec le patient | Membre de l'équipe, reconnaissance de ses savoirs expérientiels. |

Le Continuum de l'Engagement

L'implication se décline en quatre étapes progressives :

1. Information : Diffusion de données de santé publique ou de fonctionnement de la structure.

2. Consultation : Recueil d'avis (questionnaires de satisfaction, boîtes à idées).

3. Collaboration : Travail conjoint sur des projets ponctuels (création d'une affiche, soirée thématique).

4. Partenariat : Co-construction, co-décision et co-réalisation sur le long terme.

3. Niveaux d'Atteinte et Justificatifs Requis

L'indicateur se structure en deux niveaux cumulatifs pour l'octroi de la rémunération.

Niveau 1 : Information et Consultation

• Actions : Mise en place d'outils pour évaluer la satisfaction et recueillir les besoins.

• Justificatifs : Exemplaires des questionnaires, synthèse des résultats, plan d'action découlant des retours usagers.

• Évolution annuelle : Si la structure reste au niveau 1, elle doit prouver que l'outil a été révisé ou analysé à nouveau.

Niveau 2 : Collaboration et Partenariat

• Actions : Intégration pérenne des usagers dans la gouvernance ou les groupes de travail.

• Justificatifs : Désignation d'un référent usager, compte-rendu de réunions de co-construction, description de l'apport réel de l'usager dans les décisions.

• Exemple de dynamique : « Si l'année suivante la structure reste au niveau 2, elle doit évaluer ce qui a été fait l'année précédente dans le cadre de la collaboration. »

4. Les Acteurs du Partenariat

La diversité des profils permet d'adapter l'implication selon les besoins du projet de santé.

• L'Usager : Patient, personne accompagnée ou proche-aidant.

• Le Patient Partenaire / Expert : Individu ayant développé des compétences suite à sa maladie et pouvant intervenir en Éducation Thérapeutique du Patient (ETP) ou en recherche.

• Le Représentant des Usagers (RU) : Membre d'une association agréée, formé au système de santé et siégeant dans des instances officielles.

• Le Citoyen Engagé : Habitant du quartier souhaitant contribuer à la vie de la structure de proximité.

• Le Médiateur en Santé : Facilite le lien dans les salles d'attente ou lors de l'accueil.

Donnée clé (Enquête BVA 2021) : 80 % des habitants d'Occitanie souhaitent le développement des regroupements de professionnels et 47 % se disent prêts à s'impliquer auprès de ces équipes.

5. Exemples Concrets et Ressources

Le webinaire a mis en avant des initiatives réussies illustrant la mise en œuvre de l'indicateur :

• Éducation Thérapeutique (ETP) : Une MSP a intégré un patient expert pour reconstruire totalement son programme diabète, augmentant significativement la satisfaction de la patientèle.

• Groupes de parole : En Haute-Garonne, une patiente partenaire et une psychologue co-animent mensuellement un groupe de parole sur le cancer.

• Gouvernance : Bien que les SISA (Sociétés Interprofessionnelles de Soins Ambulatoires) soient juridiquement limitées aux professionnels, des comités d'usagers peuvent être créés pour influencer les décisions stratégiques.

• Communication : Utilisation de lettres d'information, de panneaux en salle d'attente ou de vidéos "ambassadeurs" où des patients expliquent l'offre de soins de la structure à leurs pairs.

Ressources Disponibles

• COPS (Centre Opérationnel du Partenariat en Santé) : Dispositif financé par l'ARS Occitanie offrant des fiches pratiques, un répertoire de patients partenaires et des compagnonnages.

• France Assos Santé : Propose des formations gratuites pour les usagers souhaitant s'impliquer.

• Haute Autorité de Santé (HAS) : Guide sur l'engagement des usagers dans les structures de soins primaires.

6. Points de Vigilance et Obstacles

• Statut juridique et financier : Il n'existe pas encore de statut de « métier » pour le patient partenaire. La rémunération reste complexe (micro-entreprise ou bénévolat avec défrayage).

• Recrutement : Il est conseillé de recruter un patient partenaire « comme un collaborateur », sur la base de ses compétences, de son savoir-être et de valeurs partagées avec l'équipe.

• Représentativité : Il est illusoire de chercher une représentativité statistique parfaite. L'objectif est de combiner une diversité de visions et de compétences.

• Accompagnement : Compte tenu de l'absence de cadre légal rigide, les structures sont encouragées à se faire accompagner par des tiers facilitateurs pour sécuriser leurs projets.

Wow, AI is coming for the CPUs as well now?

Partindo da Taxonomia Digital de Bloom, parece-me realçar que o desenho de e-atividades tem evoluído com a integração de tecnologias emergentes, sobretudo IA generativa, learning analytics e ambientes imersivos. Se antes a operacionalização das categorias passava sobretudo por fóruns, wikis ou vídeos anotados, hoje já conseguimos desenhar e-atividades adaptativas e co-criativas. Por exemplo, ao nível de Criar e Avaliar, os estudantes podem desenvolver artefactos com apoio de IA, prototipar soluções, testar cenários e até validar resultados com dados reais. Ao nível de Analisar, ferramentas de analytics permitem explorar padrões de interação ou datasets educativos, aproximando a atividade de contextos autênticos. Isto desloca a Taxonomia de uma lógica apenas cognitiva para uma lógica também ecossistémica e aplicada, onde aprender implica produzir, iterar, validar e partilhar em rede.

writing becomes more lyrical as esther "gets better"? am i grasping at straws here? i'm noticing a pttern

girl i wish

The author is challenging the concept that time heals all wounds, and trauma can disrupt the normal timeline of a fading memory and can even intensify that memory.

может вообще убрать абзац?

beautiful wording

ander lettertpe

A,dere kleur

web-trail://💻/thinkpad/🧊/me/📓/2026/02/15/do/search/delejtű+ember

practical-exploriment-custom protocol for web-tails on the indyweb

• one that ties together concepts going back to 2002 when I learned about Universal Resource Identifiers

core of the core concepts for the World Wide Web, that like many others were clearly misguided, incoherent.

I did not understand at the time what;s going on

Never the less, clearly we need an alternative that does deliver on the original intent and promiss

So I started thinking about Universal Resource Names that are amenable to the PUN of being used to actually reTRIEve them. clearly Prefix TRIEs are a true inspiration for this work

This is just a quick note (to self and others I hare this annotation with) on the margins that me2we can come back to when WE can

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El autor menciona que la escritura propone colocar de forma directa sus palabras, lo que verdaderamente se quiere decir en escrito no lo que se piensa.

Mencionan a un poeta que escribía sus escritos imaginándose declamando el texto. Esto lo puedo asociar a la cuentera o narración oral, donde la forma de escribir puede estar relacionada con la forma en que se va a contar el cuento.

Este autor Albans mencionaba que al escribir tenía el poder de dictarse a si mismo

Se comienza a desarrollar el oficio de escribir que eran personas contratadas para ofrecer este servicio

La escritura es definida como. "Cualquier marca visible o sensoria con un significado determinado, la integran en la conducta meramente biológica"

La grafía es la representación de un enunciado que alguien dice o supone que se dice.

Porque lo que hace la tecnología es mejorar la vida humana. Si pensamos en una orquesta es una tecnologia compleja que tiene diversas herramientas como los instrumentos por ejemplo el órgano o el violín.

Una reflexión propia: Es que mejora la vida humana porque permite especializarse en los conocimientos especificos, por ejemplo, el uso del piano donde la persona va desarrollando habilidades que le permiten tocar mejor el instrumento y volverse un experto con esa tecnología, llegando a convertirse en un medio o método de trabajo. Lo que no pasaría sino existieran estas tecnologías porque tendriamos que hacer los sonidos directamente con nuestros cuerpos y eso nos podría dar esa especie de libertad creativa infinita porque el ser humano tiene la capacidad de crear cosas que no están establecidas, mientras que lo que nos aporta la tecnologia puede llegar a tener un limite en nuestro proceso creativo, pero que nos va a llevar a convertirnos en expertos.

La escritura como tecnología no deberiamos condenarla menciona el autor sino elogiarla, porque esta nos permite tener un valor inestimable y esencial para la realización de las aptitudes humanas. No sólo son recursos externos sino transformaciones interiores de la conciencia, y mucho más cuando afectan la palabra.

No necesitamos la proximidad sino la distancia, y esto es lo que nos aporta la escritura de la conciencia, comprendo que: - La proximidad es el ejercicio de acercarnos más al conocimiento, profundizando en las temáticas y llegando a conclusiones más complejas. Y tener un número ilimitado de pensamientos. - La distancia es el ejercicio de reflexionar sobre un pensamiento concreto que se lleva a la escritura y que nos permita comprender totalmente un pensamiento concreto.

Yo como lector reflexiono que: "La escritura es el monumento de la memoria", porque genera un registro de lo que vamos viviendo, construyendo o pensando, pero que al final de cuentas es un producto industrializado porque esta es una representa idealista sobre nuestro pensamiento que no tiene un contexto y no se puede complejizar tanto como nuestros pensamientos. Porque para mi, la escritura es el resultado escrito de nuestras reflexiones, pero para poder llegar a concretar esas ideas especificas que me parecieron valiosas para comunicarlas de forma escrita, tuve que haber tenido un proceso de pensamiento de dias, semanas o meses que no fue registrado. La escritura es el único lugar donde pueden existir las palabras habladas.

La escritura es una tecnología externa y ajena a nosotros mismos. La escritura constituye una tecnologia que necesita herramientas como el papel, plumas, tablas de madera, tintas o pinturas. esta tuvo como objetivo la reducción del sonido dinámico al espacio inmóvil - lo que también hacian la imprenta y la computadora.

Aunque el autor muera, el mensaje va a seguir vivo en la mente de los lectores. Es decir que: "El cuento no existe cuando alguien lo cuenta sino cuando alguien lo imagina"

El texto menciona una crítica a la invención de la imprenta porque consideraba que una abundancia de libros hace menos estudiosos a los hombres, como sino profundizarán en el conocimiento. Porque la crítica está en que ahorita se considera sabio a quien tiene o ha leído una mayor cantidad de libros, pero eso no quiere decir que profundice en el conocimiento.

Menciona que las ideas son visuales y que vienen arraigadas del mismo principio de video, ver, visión, visible. Entonces, se podría inferir que las ideas también pueden convertirse en una tecnología al poder crear una imagen mental de nuestros pensamientos.

Aquí mencionan algo importante, que Platon aunque estaba de en desacuerdo con la tecnología de la escritura, igual utilizo escritos para plasmas sus opiniones y pensamientos. O la otra realidad de los que inventaron las computadoras que criticaban el uso del papel tradicional y gracias a eso, lograron crear las cintas procesadas que inventaron la computadora.

Para el autor es simple, la escritura, la imprenta y la computadora son formas de tecnologizar la palabra, una vez tecnologizada no puede criticarse porque carece de ese contexto o de no tener al creador de frente.

Aquí se da el desarrollo de lo argumentos por la frase de platon donde menciona que: "es inhumana al pretender establecer fuera del pensamiento lo que en realidad solo puede existir dentro de él". Argumentos que sostienen esta frase: 1. Es un objeto, un producto manufacturado. Lo mismo se dice de las computadoras 2. La escritura destruye la memoria. Coloca el ejemplo de que la invención de las calculadoras es un recurso externo que produjo que las tablas aprendidas de memoria que son un recurso interno sean olvidadas. 3. Un texto escrito no produce respuestas, si se le pregunta a una persona que explica sus palabras presencialmente este podría hacerlo, pero en un texto escrito no se podría porque no tenemos al autor en frente. 4. El hecho de que la palabra escrita no puede defender como es capaz de hacerlo la palabra hablada natural. Menciona que el habla y pensamiento real está medida por un contexto de ida y vuelta, pero en la palabra escrita es pasiva, es decir, fuera de dicho contexto, irreal y artificial al igual que las computadoras.

Esta es una cita importante de Platon

Menciona que uno de estos ejemplos del lenguaje autonomo son las formulas rituales como las frases adivinatorias o las profecias

Menciona que esta clase de lenguaje está libre de contextos o es un discurso autonomo porque no se puede poner en duda ni cuestionarse directamente porque el discurso escrito está separado del autor

El texto menciona que la escritura es la tecnología que llego a estructurar directa o indirectamente el pensamiento y que por ende, la escritura ha transformado la comunicación oral como escrita porque cambia la manera en la que articulamos las palabras, y esta naturalmente favorece a los individuos que están funcionalmente escolarizados

Esta es una idea clave que menciona que no se puede refutar a un autor de un libro porque no lo tenemos en frente, es una forma de decirnos que lo que se escribe en el libro: "es cierto", aunque no lo sea para nosotros sino que es una verdad para el autor de ese libro

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Update the PDF version without an ''e'' at the end of général. and no comma at the bottom of the table 1

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Cette augmentation de 143.2 millions de dollars est principalement attribuable aux éléments suivants :

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core guys of medievalism in England also worked in Wales too Wales was a particular place of interest due to its castle's the most spectacular remoddling being that of Lord Bute's residences Cardiff Castle and Castle Coch, both designed by William Burges.

Lady guest reinforced the place of arthur as a welshman through her translation. however, did such a translation into english, and her setting into the 'broader context of medieval European romance', reduce the extent that arthur belonged to the welsh?

ModuleNotFoundError Traceback (most recent call last) Cell In[1], line 1 ----> 1 from phasic.utils import draw_coalescent_tree 2 import matplotlib.pyplot as plt 4 draw_coalescent_tree('GGTTTGGGA')

ModuleNotFoundError: No module named 'phasic'

What causes the differences in reactions when stimulating one spine versus multiple? What about the relationships between the spines is the reason behind this?

Is there a way in which we can subject a neuron to whole-cell dialysis without causing washout?

Why is this? What would happen if LTP was still induced in the spines that did not show enlargement? Would they just not have any synaptic potentiation occurring?

Enlargement was greater and longer-lasting in the smaller spines, and I’m wondering if this would change their response to stimulation at a later time (when their size is much larger than it was originally)? However, long-lasting enlargement was much higher in small spines than in large spines. This is super interesting. I would have thought that large spines would be easier to induce enlargement in than small ones. The fact that short-term enlargement occurred in all of the small spines and a large majority of the large spines indicates that the methods used were very effective, but is there a more impactful way that we could achieve higher levels of long-term enlargement, especially in larger spines? Would this improve the function of long-term memory?

Would different thicknesses of hippocampal slices cause variations in results?

It would be amazing if there was a way to treat the cause of depression directly in the brain, either by increasing/decreasing potentiation.

What would occur if a different concentration of CaCl was added to the extracellular solution? Would it, for example, speed up or slow down the enlargement of spines if the concentration was increased/decreased?

This is crazy to think about, but it makes sense! Smaller spines can become larger spines, and these larger spines are involved in long-term memory which is much harder to be altered once it’s formed. It seems that short-term memory is much more prone to influence from outside factors, before it becomes a permanent, long-term memory or is cast out by the brain as unimportant information.

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L’Attention aux Vulnérabilités : Une Priorité Éthique et Pédagogique

Résumé Exécutif

Ce document de synthèse examine le rôle critique de l'attention aux vulnérabilités dans le milieu scolaire, positionnant cette approche non seulement comme une obligation éthique, mais aussi comme un facteur déterminant de l'efficacité pédagogique.

L'analyse souligne que la relation enseignant-élève est intrinsèquement asymétrique, plaçant l'élève dans une position d'exposition aux risques — de la blessure émotionnelle au décrochage scolaire.

Les points clés abordés incluent :

• La redéfinition de la vulnérabilité : Elle n'est plus perçue comme un état permanent de la personne, mais comme une situation (momentanée ou durable) affectant jusqu'à la moitié des effectifs scolaires sur une année.

• L'impact des besoins fondamentaux : La satisfaction des besoins de compétence, d'autonomie et d'affiliation est essentielle à la sécurité relationnelle.

• La lutte contre la « Violence Pédagogique Ordinaire » : L'identification et l'élimination des micro-violences (verbales, comportementales) sont impératives.

• Le passage à la bienveillance active : L'adoption de gestes professionnels ciblés, tels que le feedback positif et l'exigence bienveillante, corrèle directement avec la réussite des élèves.

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1. La Nature de la Relation Pédagogique : Une Asymétrie Fondamentale

La relation éducative est définie par une asymétrie structurelle. L'enseignant détient la maîtrise des compétences, du statut, des objectifs pédagogiques, de l'espace et du temps, tandis que l'élève évolue dans une position de dépendance et de moindre conscience des enjeux.

La Vulnérabilité comme Situation

Le terme vulnérabilité (du latin vulnus, la blessure) désigne une fragilité qui expose l'élève à des risques de blessures concernant ses droits, sa dignité ou, plus fréquemment, ses besoins fondamentaux.

• Évolution conceptuelle : La recherche actuelle privilégie la notion de « situations de vulnérabilité » plutôt que de « personnes vulnérables ».

• Typologie des situations :

◦ Durables : Élèves en situation de handicap ou à besoins éducatifs particuliers (environ 470 000 à 800 000 élèves incluant les profils neurodéveloppementaux, haut potentiel et allophones).   

◦ Momentanées : Élèves traversant des crises familiales (séparation), économiques (perte d'emploi des parents), affectives ou liées au parcours migratoire.

On estime que près de 50 % des élèves vivent de telles phases chaque année.

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2. Cartographie des Besoins Fondamentaux en Milieu Scolaire

Pour garantir une relation éthique, l'enseignant doit répondre à une nomenclature de besoins multidimensionnels.

| Catégorie de Besoin | Composantes Clés | | --- | --- | | Besoins de base (Deci & Ryan) | Compétence, Autonomie, Affiliation. | | Sécurité et Confiance | Sécurité relationnelle, confiance en soi, confiance en l'adulte et en l'institution. | | Socialisation et Équité | Appartenance au groupe, besoin de justice, respect et considération. | | Accompagnement | Besoin d'aide, besoin de temps, besoin de dialogue avec l'adulte. |

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3. Gestes Professionnels et Leviers de Réussite

La recherche, notamment les méta-analyses de John Hattie, démontre que les facteurs relationnels ont un impact supérieur à la moyenne sur la réussite scolaire (coefficients de corrélation supérieurs à 0,7, là où le seuil de significativité est à 0,4).

Levier Majeur : Le Feedback

Le feedback positif agit comme un levier fondamental pour nourrir le besoin d'estime et de sécurité de l'élève. Il doit être intégré dans les moments pédagogiques critiques :

• L'accueil des élèves.

• La mise en activité.

• Les phases d'évaluation (annonce, correction, exploitation).

• La gestion des obstacles et des erreurs (dédramatisation).

Communication et Posture

La communication se divise en trois dimensions :

1. Verbale : Les mots utilisés.

2. Non-verbale : Gestes, mimiques, posture spatiale.

3. Paraverbale : Ton, volume et débit de la voix (cruciaux pour la perception de la satisfaction de l'enseignant par l'élève).

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4. La Violence Pédagogique Ordinaire (VPO)

La VPO regroupe des micro-violences souvent inconscientes mais délétères, désormais interdites par la loi du 10 juillet 2019.

• Manifestations : Cris, moqueries, intimidations, stigmatisations, discriminations sociales, comparaisons excessives ou injonctions paradoxales.

• Conséquences : Stress, mal-être, conduites antisociales et agressivité. Ces comportements ajoutent une vulnérabilité supplémentaire à celle déjà présente, créant un cercle vicieux de l'échec.

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5. Vers une Éthique de la Bienveillance Active

L'éthique est ici définie comme une disposition psychique visant à rechercher le comportement le plus juste pour l'élève.

Distinction entre Bienveillances

Le passage d'une posture passive à une posture active est nécessaire :

• Bienveillance Passive (ou minimale) : Se limiter à ne pas blesser l'élève et le laisser affronter seul ses difficultés par manque de temps ou de ressources.

• Bienveillance Active : Caractérisée par une qualité de présence, un soutien de proximité, des exigences adaptées et un intérêt réel pour la personne de l'élève au-delà de ses résultats.

Les 5 Modes d'Expression (selon Gwénola Reto)

1. S'intéresser à l'élève : Encourager sa pensée et accepter ses divergences.

2. Prendre en compte les besoins : Identifier les besoins cognitifs et fondamentaux.

3. Se soucier de son bien-être : Veiller à son intérêt et sa motivation.

4. Valoriser la personne : Distinguer l'individu de ses résultats normatifs lors des évaluations.

5. Manifester de la compassion : Montrer une sensibilité face aux difficultés rencontrées par l'élève.

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Conclusion

L'attention aux vulnérabilités ne doit pas être perçue comme une baisse d'exigence, mais comme une exigence bienveillante.

En sécurisant le cadre relationnel et en répondant aux besoins psycho-affectifs, l'enseignant rend l'exigence scolaire acceptable et fructueuse, garantissant ainsi que l'élève reste « dans le jeu de la réussite ».

This is his rebuttal against my argument that we arent original we have experienced this world and AI observes our observations. I don't disagree with him that feelings in our writing is the thing that matters the most. 'saying that there is a time and a place for AI and not to completely demonize it. For example art not every business has the money to spend hundreds of dollars to splurge on a logo that has every small detail they want so if they use AI for a logo is that completely bad?

I think that's an example of being too reliant on AI forgetting the actual point of the things that were doing day to day. Then again there are always people in this world that will go to those extreme path

Im split into two ways;. One side is saying if your a young person that is lazy and always want stuff the easy way you'll become intellectually crippled and become so reliant on AI that you won't be able to form thoughts without AI. The other side is saying that it can be a good starting point if your trying to learn about new subjects. It can tell you the beginning guide and then direct you into the sources you can delve into to further expand your knowledge

I think it's a bit of an exaggeration demonizing AI but I think we already know it's not alive and it says its happy to see us. It's like dora the explora saying how's your day going its just a cartoon and behind it is a voice actor that will never know you and your struggles in life.

I've heard that they got there data to train the AI illegally, but I don't think that's our problem I think that's between them and the person they stole from. As long as you inspect sources they use and credit them then that's not your issue. I think sometimes we dig deep into stuff thats none of our buisness. There are a lot of sketchy things going in the world and if we focus too much on its orgin we would die from boycotting everything. It's like if your boss does something illegally to get his money but your just working a normal job as long you don't use that money in a bad way then its none of your buisness what he does because your not directly assisting his act.

I guess there are details that you can find is profound that you won't find AI doing since it's mostly taking whats common but whose to say that It wont in the future make the small decisions we do. At the end of the day when we draw its from our in expereince in life and Ai just observing our observations

I'd have to disagree on this one I've seen what images Ai can generate. Based on the prompt you give it I've seen some good looking art bettter then most artist... does that go back to what he thinks about AI being bland? If AI takes whats bland only then why is its images so artistic? wouldnt they be bland and repetitive as well.

I've never really thought about that. Since Ai is mostly data it will take what's been repeated it's not gonna reveal to you the hidden gems that went through its system

this is the Main point,

"Inflectional gives information to the word" This will be a good way to help me remember the differences between derivational and inflectional morphology! I & I = Information.

eLife Assessment

This fundamental manuscript describes how the posterolateral cortical amygdala (plCoA) generates appetitive or aversive behaviors in response to odors. By combining optogenetic stimulation, single-cell RNA sequencing, and spatial analysis, the authors identify a topographically organized circuit within plCoA that governs these behaviors. The manuscript shows convincingly that multiple features (spatial, genetic, and projection) contribute to overall population encoding of valence. Overall, the authors conduct many challenging experiments, each of which contains the relevant controls, and the results are interpreted within the framework of their experiments.

Reviewer #1 (Public review):

Summary:

This study by Howe and colleagues investigates the role of the posterolateral cortical amygdala (plCoA) in mediating innate responses to odors, specifically attraction and aversion. By combining optogenetic stimulation, single-cell RNA sequencing, and spatial analysis, the authors identify a topographically organized circuit within plCoA that governs these behaviors. They show that specific glutamatergic neurons in the anterior and posterior regions of plCoA are responsible for driving attraction and avoidance, respectively, and that these neurons project to distinct downstream regions, including the medial amygdala and nucleus accumbens, to control these responses.

Strengths:

The major strength of the study is the thoroughness of the experimental approach, which combines advanced techniques in neural manipulation and mapping with high-resolution molecular profiling. The identification of a topographically organized circuit in plCoA and the connection between molecularly defined populations and distinct behaviors is a notable contribution to understanding the neural basis of innate motivational responses. Additionally, the use of fucntional manipulations adds depth to the findings, offering valuable insights into the functionality of specific neuronal populations.

Weaknesses:

Previously described weaknesses in the study's methods and interpretation were fully addressed during revision. Locomotor behavior of the mice during head-fixed imaging experiments was added and analysis of the correlation of locomotion with neural activity was also added.

This work provides significant insights into the neural circuits underlying innate behaviors and opens new avenues for further research. The findings are particularly relevant for understanding the neural basis of motivational behaviors in response to sensory stimuli, and the methods used could be valuable for researchers studying similar circuits in other brain regions. If the authors address the methodological issues raised, this work could have a substantial impact on the field, contributing to both basic neuroscience and translational research on the neural control of behavior.

Reviewer #2 (Public review):

Summary:

The manuscript by the Root laboratory and colleagues describes how the posterolateral cortical amygdala (plCoA) generates valenced behaviors. Using a suite of methods, the authors demonstrate that valence encoding is mediated by several factors, including spatial localization of neurons within the plCoA, glutamatergic markers, and projection. The manuscript shows convincingly that multiple features (spatial, genetic, and projection) contribute to overall population encoding of valence. Overall, the authors conduct many challenging experiments, each of which contains the relevant controls, and the results are interpreted within the framework of their experiments.

Strengths:

- The manuscript is well constructed, containing lots of data sets and clearly presented, in spite of the abundance of experimental results.

- The authors should be commended for their rigorous anatomical characterizations and post-hoc analysis. In the field of circuit neuroscience, this is rarely done so carefully, and when it is, often new insights are gleaned as is the case in the current manuscript.

- The combination of molecular markers, behavioral readouts and projection mapping together substantially strengthens the results.

- The focus on this relatively understudied brain region in the context is valence is well appreciated, exciting and novel.

Weaknesses:

The weaknesses noted in the primary review have all been addressed adequately.

Reviewer #3 (Public review):

Summary:

Combining electrophysiological recording, circuit tracing, single cell RNAseq, and optogenetic and chemogenetic manipulation, Howe and colleagues have identified a graded division between anterior and posterior plCoA and determined the molecular characteristics that distinguish the neurons in this part of the amygdala. They demonstrate that the expression of slc17a6 is mostly restricted to the anterior plCoA whereas slc17a7 is more broadly expressed. Through both anterograde and retrograde tracing experiments, they demonstrate that the anterior plCoA neurons preferentially projected to the MEA whereas those in the posterior plCoA preferentially innervated the nucleus accumbens. Interestingly, optogenetic activation of the aplCoA drives avoidance in a spatial preference assay whereas activating the pplCoA leads to preference. The data support a model that spatially segregated and molecularly defined populations of neurons and their projection targets carry valence specific information for the odors. Moreover, the intermingling of neurons in the plCoA is consistent with prior observations. The presence of a gradient rather than a distinct separation of the cells fits the model being proposed. The discoveries represent a conceptual advance in understanding plCoA function and innate valence coding in the olfactory system.

Strengths:

The strongest evidence supporting the model comes from single-cell RNASeq, genetically facilitated anterograde and retrograde circuit tracing, and optogenetic stimulation. The evidence clear demonstrates two molecularly defined cell populations with differential projection targets. Stimulating the two populations produced opposite behavioral responses.

Weaknesses:

The weaknesses noted in primary review have all been addressed adequately.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study by Howe and colleagues investigates the role of the posterolateral cortical amygdala (plCoA) in mediating innate responses to odors, specifically attraction and aversion. By combining optogenetic stimulation, single-cell RNA sequencing, and spatial analysis, the authors identify a topographically organized circuit within plCoA that governs these behaviors. They show that specific glutamatergic neurons in the anterior and posterior regions of plCoA are responsible for driving attraction and avoidance, respectively, and that these neurons project to distinct downstream regions, including the medial amygdala and nucleus accumbens, to control these responses.

Strengths:

The major strength of the study is the thoroughness of the experimental approach, which combines advanced techniques in neural manipulation and mapping with high-resolution molecular profiling. The identification of a topographically organized circuit in plCoA and the connection between molecularly defined populations and distinct behaviors is a notable contribution to understanding the neural basis of innate motivational responses. Additionally, the use of functional manipulations adds depth to the findings, offering valuable insights into the functionality of specific neuronal populations.

Weaknesses:

There are some weaknesses in the study's methods and interpretation. The lack of clarity regarding the behavior of the mice during head-fixed imaging experiments raises the possibility that restricted behavior could explain the absence of valence encoding at the population level.

We agree with idea that head-fixation may alter the state of the animal and the neural encoding of odor. To address this, we have provided further analysis of walking behavior during the imaging sessions, which is provided in Figure S2. Overall, we could not identify any clear patterns in locomotor behavior that are odor-specific. Moreover, when neural activity was sorted depending on the behavioral state (walking, pausing or fleeing) we didn’t observe any apparent patterns in odor-evoked neural activity. This is now discussed in the Results and Limitations sections of the manuscript.

Furthermore, while the authors employ chemogenetic inhibition of specific pathways, the rationale for this choice over optogenetic inhibition is not fully addressed, and this could potentially affect the interpretation of the results.

The rationale was logistical. First, inhibition of over a timescale of minutes is problematic with heat generation during prolonged optical stimulation. Second, our behavioral apparatus has a narrow height between the ceiling and floor, making tethering difficult. This is now explained the results section. The trade-off of using chemogenetics is that we are silencing neurons and not specific projections. However, because we find that NAc- and MeA- projecting neurons have little shared collateralization, we believe the conclusion of divergent pathways still stands. This is now discussed in the Limitations section.

Additionally, the choice of the mplCoA for manipulation, rather than the more directly implicated anterior and posterior subregions, is not well-explained, which could undermine the conclusions drawn about the topographic organization of plCoA.

We targeted the middle region of plCoA because it contains a mixture of cell types found in both the anterior and posterior plCoA, allowing us to test the hypothesis that cell types, not intra plCoA location, elicit different responses. Had we targeted the anterior or posterior regions, we would expect to simply recapitulate the result from activation of random cells in each region. As a result, we think stimulation in the middle plCoA is a better test for the contribution of cell types. We have now clarified this in the text.

Despite these concerns, the work provides significant insights into the neural circuits underlying innate behaviors and opens new avenues for further research. The findings are particularly relevant for understanding the neural basis of motivational behaviors in response to sensory stimuli, and the methods used could be valuable for researchers studying similar circuits in other brain regions. If the authors address the methodological issues raised, this work could have a substantial impact on the field, contributing to both basic neuroscience and translational research on the neural control of behavior.

Reviewer #2 (Public review):

Summary:

The manuscript by the Root laboratory and colleagues describes how the posterolateral cortical amygdala (plCoA) generates valenced behaviors. Using a suite of methods, the authors demonstrate that valence encoding is mediated by several factors, including spatial localization of neurons within the plCoA, glutamatergic markers, and projection. The manuscript shows convincingly that multiple features (spatial, genetic, and projection) contribute to overall population encoding of valence. Overall, the authors conduct many challenging experiments, each of which contains the relevant controls, and the results are interpreted within the framework of their experiments.

Strengths:

- For a first submission the manuscript is well constructed, containing lots of data sets and clearly presented, in spite of the abundance of experimental results.

- The authors should be commended for their rigorous anatomical characterizations and posthoc analysis. In the field of circuit neuroscience, this is rarely done so carefully, and when it is, often new insights are gleaned as is the case in the current manuscript.

- The combination of molecular markers, behavioral readouts and projection mapping together substantially strengthen the results.

- The focus on this relatively understudied brain region in the context is valence is well appreciated, exciting and novel.

Weaknesses:

- Interpretation of calcium imaging data is very limited and requires additional analysis and behavioral responses specific to odors should be considered. If there are neural responses behavioral epochs and responses to those neuronal responses should be displayed and analyzed.

We have now considered this, see response above.

- The effect of odor habituation is not considered.

We considered this, but we did not find any apparent differences in valence encoding as measured by the proportion of neurons with significant valence scores across trials (see Figure 1J).

- Optogenetic data in the two subregions relies on very careful viral spread and fiber placement. The current anatomy results provided should be clear about the spread of virus in A-P, and D-V axis, providing coordinates for this, to ensure readers the specificity of each sub-zone is real.

We were careful to exclude animals for improper targeting. The spread of virus is detailed in Figures S3, S8 & S9.

- The choice of behavioral assays across the two regions doesn't seem balanced and would benefit from more congruency.

The choice of the 4-quadrant assay was used because this study builds off of our prior experiments that demonstrate a role for the plCoA in innate behavior. It is noteworthy that the responses to odor seen in this assay are generally in agreement with other olfactory behavioral assays, so one wouldn’t predict a different result. Moreover, the approach and avoidance responses measured in this assay are precisely the behaviors we wish to understand. We did examine other non-olfactory behavioral readouts (Figures S3, S8), and didn’t observe any effect of manipulation of these pathways.

- Rationale for some of the choices of photo-stimulation experiment parameters isn't well defined.

The parameters for photo-stimulation were based on those used in our past work (Root et al., 2014). We used a gradient of frequency from 1-10 Hz based on the idea that odor likely exists in a gradient and this was meant to mimic a potential gradient, though we don’t know if it exists. The range in stimulation frequencies appears to align with the actual rate of firing of plCoA neurons (Iurilli et al., 2017).

Reviewer #3 (Public review):

Summary:

Combining electrophysiological recording, circuit tracing, single cell RNAseq, and optogenetic and chemogenetic manipulation, Howe and colleagues have identified a graded division between anterior and posterior plCoA and determined the molecular characteristics that distinguish the neurons in this part of the amygdala. They demonstrate that the expression of slc17a6 is mostly restricted to the anterior plCoA whereas slc17a7 is more broadly expressed. Through both anterograde and retrograde tracing experiments, they demonstrate that the anterior plCoA neurons preferentially projected to the MEA whereas those in the posterior plCoA preferentially innervated the nucleus accumbens. Interestingly, optogenetic activation of the aplCoA drives avoidance in a spatial preference assay whereas activating the pplCoA leads to preference. The data support a model that spatially segregated and molecularly defined populations of neurons and their projection targets carry valence specific information for the odors. The discoveries represent a conceptual advance in understanding plCoA function and innate valence coding in the olfactory system.

Strengths:

The strongest evidence supporting the model comes from single cell RNASeq, genetically facilitated anterograde and retrograde circuit tracing, and optogenetic stimulation. The evidence clear demonstrates two molecularly defined cell populations with differential projection targets. Stimulating the two populations produced opposite behavioral responses.

Weaknesses:

There are a couple of inconsistencies that may be addressed by additional experiments and careful interpretation of the data.

Stimulating aplCoA or slc17a6 neurons results in spatial avoidance, and stimulating pplCoA or slc17a7 neurons drives approach behaviors. On the other hand, the authors and others in the field also show that there is no apparent spatial bias in odor-driven responses associated with odor valence. This discrepancy may be addressed better. A possibility is that odor-evoked responses are recorded from populations outside of those defined by slc17a6/a7. This may be addressed by marking activated cells and identifying their molecular markers. A second possibility is that optogenetic stimulation activates a broad set of neurons that and does not recapitulate the sparseness of odor responses. It is not known whether sparsely activation by optogenetic stimulation can still drive approach of avoidance behaviors.

We agree that marking specific genetic or projection defined neurons could help to clarify if there are some neurons have more selective valence responses. However, we are not able to perform these experiments at the moment. We have included new data demonstrating that sparser optogenetic activation evokes behaviors similar in magnitude as the broader activation (see Figure S4).

The authors show that inhibiting slc17a7 neurons blocks approaching behaviors toward 2-PE. Consistent with this result, inhibiting NAc projection neurons also inhibits approach responses. However, inhibiting aplCOA or slc17a6 neurons does not reduce aversive response to TMT, but blocking MEA projection neurons does. The latter two pieces of evidence are not consistent with each other. One possibility is that the MEA projecting neurons may not be expressing slc17a6. It is not clear that the retrogradely labeling experiments what percentage of MEA- and NACprojecting neurons express slc17a6 and slc17a7. It is possible that neurons expressing neither VGluT1 nor VGluT2 could drive aversive or appetitive responses. This possibility may also explain that silencing slc17a6 neurons does not block avoidance.

We have now performed RNAscope staining on retrograde tracing to better define this relationship. Although the VGluT1 and VGluT2 neurons have biased projections to the MeA and NAc, respectively, there is some nuance detailed in Figure S10. Generally, MeA projecting neurons are predominately VGluT2+, whereas NAc projecting have about 20% that express both. Some (less than 35%) retrogradely labeled neurons were not detected as VGluT1 or VGluT2 positive, suggesting that other populations could also contribute. We agree that the discrepancy between MeA-projection and VGluT2 silencing is likely due to incomplete targeting of the MeA-projecting population with the VGluT2-cre line. This is included in the Discussion section.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Main:

(1) For the head-fixed imaging experiments, what is the behavior of the mice during odor exposure? Could the weak reliability of individual neurons be due to a lack of approach or avoidance behavior? Could restricted behavior also explain the lack of valence encoding at the population level?

We agree that this is a limitation of head-fixed recordings. In the revised manuscript we did attempt to characterize their behavioral response, and look for correlations in odor representation. Although we did find different patterns of odor-evoked walking behavior, these patterns were not reliable or specific to particular odors (Figure S2). For example, one might expect aversive odors to pause walking or elicit a fast fleeing-like response, but we did not observe any apparent differences for locomotion between odors as all odors evoked a mixture of responses (Figure S2A-D, text lines 208-232). We then examined responses to odor depending on the behavioral state (walking, pausing or fleeing) and didn’t observe any apparent patterns in odor responses (Figure S2E,F). Lastly, we acknowledge in the text that the lack of valence encoding may be an artifact of head-fixation (see lines 849-857).

(2) For the optogenetic manipulations of Vglut1 and Vglut2 neurons, why was the injection and fiber targeted to the medial portion of the plCoA, if the hypothesis was that these glutamatergic neuron populations in different regions (anterior or posterior) are responsible for approach and avoidance? 

We targeted the middle region of plCoA because it contains a mixture of cell types found in both the anterior and posterior plCoA, allowing us to test the hypothesis that cell types, not intraplCoA location, elicit different responses. Had we targeted the anterior or posterior regions, we would expect to simply recapitulate the result from activation of random cells in each region. As a result, we think stimulation in the middle plCoA is a better test for the contribution of cell types. We have clarified this in the text (Lines 417-419).

Could this explain the lack of necessity with the DREADD experiments? 

For the loss of function experiments, a larger volume of virus was injected to cover a larger area and we did confirm targeting of the appropriate areas. Though, it is always possible that the lack of necessity is due to incomplete silencing.

Further, why was an optogenetic inhibition approach not utilized? 

Although optogenetic inhibition could have plausibly been used instead, we chose chemogenetic inhibition for two reasons: First, for minutes-long periods of inhibition, optical illumination poses the risk of introducing heat related effects (Owen et al., 2019). In fact, we first tried optical inhibition but controls were exhibited unusually large variance. Second, it is more feasible in our assay as it has a narrow height between the floor and lid that complicates tethering to an optic fiber. Past experiments overcame this with a motorized fiber retraction system (Root et al., 2014), but this is highly variable with user-dependent effects, so we found chemogenetics to be a more practical strategy. We have added a sentence to explain the rationale (see lines 561-563).

(3) The specific subregion of the nucleus accumbens that was targeted should be named, as distinct parts of the nucleus accumbens can have very different functions. 

We attempted to define specific subregions of the nucleus accumbens and found that plCoA projection is not specific to the shell or core, anterior or posterior, rather it broadly innervates the entire structure. We have added a note about this in manuscript (see lines 470-471). Given that we did not find notable subregion-specific outputs within the NAc, targeting was directed to the middle region of NAc, with coordinates stated in the methods. 

(4) Why was an intersectional DREADD approach used to inhibit the projection pathways, as opposed to optogenetic inhibition? The DREADD approach could potentially affect all projection targets, and the authors might want to address how this could influence the interpretation of the results.

This is partly addressed above in point 2. As for interpretation, we acknowledge that the intersectional approach silences the neurons projecting to a given target and not the specific projection and we have been careful with the wording. Although this may complicate the conclusion, we did map the collaterals for NAc and MeA projecting neurons and find that neurons do not appreciably project to both targets and have minimal projections to other targets. We have now taken care to state that we silence the neurons projecting to a structure, not silencing the projection, and we acknowledge this caveat. However, since the MeA- and NAcprojecting neurons appear to be distinct from each other (largely not collateralizing to each other), the conclusion that these divergent pathways are required still stands. We have added discussion of this in the Limitations section (see lines 859-863).

Minor:

(1) Line 402 needs a reference.

We have added the missing reference (now line 441).

(2) The Supplemental Figure labeling in the main text should be checked carefully.

Thank you for pointing this out. We have fixed the prior errors.

(3) Panel letter D is missing from Figure 2.

This has been fixed.

Reviewer #2 (Recommendations for the authors):

Major Concerns, additional experiments:

- In the calcium imaging experiments mice were presented with the same odor many times. Overall responses to odor presentations were quite variable and appear to habituate dramatically (Figure S1F). The general conclusion from these experiments are a lack of consistent valence-specific responses of individual neurons, but I wonder if this conclusion is slightly premature. A few potential explanatory factors that may need additional attention are: -First, despite recording video of the mouse's face during experiments, no behavioral response to any odor is described. Is it possible these odors when presented in head-fixed conditions do not have the same valence?

Yes, we agree that this is a possibility. We have added a discussion in the Limitations section (see lines 849-857). We have also added additional behavioral analysis discussed below.

On trials with neural responses are there behavioral responses that could be quantified? 

We have now added data in which we attempt to characterize their behavioral response, to look for correlations in odor representation (see lines 208-228). Although we did observe different patterns of odor-evoked walking behavior, these patterns were not reliable or specific to particular odors (Figure S2). One might expect aversive odors to pause walking or elicit a fast fleeing-like response, but we did not observe any apparent differences for locomotion between odors (Figure S2A-D). Next, we examined responses to odor depending on the behavioral state (walking, pausing or fleeing) and didn’t observe any meaningful differences in odor responses (Figure S2E,F). Lastly, we acknowledge that the odor representation may be different in freely moving animals that exhibit dynamic responses to odor (see lines 859-857).

- Habituation seems to play a prominent role in the neural signals, is there a larger contribution of valence if you look only at the first delivery (or some subset of the 20 presentations) of an odor type for a given trial? 

Indeed, we considered this, but we did not find any apparent differences in valence encoding as measured by the proportion of neurons with significant valence scores across trials (see Figure 1J).

- Is it reasonable to exclude valence encoding as a possibility when largely neurons were unresponsive to the positive valence odors (2PE and peanut) chosen when looking at the average cluster response (Figure 1F)? 

It is true that we see fewer neurons responding to the appetitive odors (Figure 1H) and smaller average responses within the cluster, but some neurons do respond robustly. If these were valence responses, we would predict that neural responses should be similarly selective, but we do not observe any such selectivity. The sparseness of responses to appetitive odors does cause the average cluster analysis (Figure 1F) to show muted responses to these odors, consistent with the decreased responsivity to appetitive odors. Moreover, single neuron response analysis reveals that a given neuron is not more likely to respond to appetitive or aversive odors with any selectivity greater than chance. For these reasons, we think it is reasonable to conclude an absence of valence responses, which is consistent with the conclusion from another report (Iurilli et al., 2017).

- While the preference and aversion assay with 4 corners is an interesting set-up and provides a lot of data for this particular manuscript. It would be helpful to test additional behaviors to determine whether these circuits are more conserved. As it stands the current manuscript relies on very broad claims using a single behavioral readout. Some attempts to use head-fixed approaches with more defined odor delivery timelines and/or additional valenced behavioral readouts is warranted.

We appreciate the suggestion, but are not able to perform these experiments at the moment. The choice of the 4-quadrant assay was used because it built off of our prior experiments that demonstrate a role for the plCoA in innate behavior. It is noteworthy that the responses to odor seen in this assay are generally in agreement with other olfactory behavioral assays, so one wouldn’t predict a different result. The approach and avoidance responses measured in this assay are precisely the behaviors we wish to understand. Moreover, we did examine other nonolfactory behavioral readouts (Figures S3, S8), and didn’t observe any effect of manipulation of these pathways. Lastly, we have tried to define parameters for head-fixed behavior that would permit correlation of neural responses with behavior, including longer stimulations and closed loop locomotion control of odor concentration, but were unsuccessful at establishing parameters that generated reliable behavioral responses. We acknowledge that one limitation of the study is the limited behavioral tests with two odors and whether the circuits are more broadly necessary for other odors. 

Minor comments:

• Please define PID in the Results when it is first introduced.

Done (see line 154)

• Line 412 Figure S5C-N should be Figure S6C-N.

Fixed. Now Figure S8C-N due to additional figures (see line 451).

• Throughout the Discussion it would be helpful if the authors referred to specific Figure panels that support their statements (e.g. lines 654-656 "[...] which is supported by other findings presented here showing that both VGluT2+ and VGluT1+ neurons project to MeA, while the projection to NAc is almost entirely composed of VGluT1+ neurons".

Thank you for the suggestion. We have added figure references in the discussion.

• Line 778 "producing" should be "produce".

Corrected (see line 840)

• The figures are very busy, especially all the manipulations. The authors are commended for including each data point, but they might consider a more subtle design (translucent lines only for each animal, and one mean dot for the SEM), just to reduce the overall clutter of an already overwhelming figure set. But this is ultimately left to the authors to resolve and style to their liking. 

Thank you for the suggestion. We have tried some different styles but like the original best.

Reviewer #3 (Recommendations for the authors):

If within reach, I suggest that the author determine the percentage of retrogradely labeled neurons to NAc or MEA that expresses GluT1 and GluT2. 

We have done this for the middle region plCoA that has the greatest mixture of cell types (See Figure S10, lines 504-517). We find that the MeA projecting neurons are mostly VGluT2+ with a minority that express both VGluT1 and VGlut2. NAc-projecting neurons are primarily VGluT1+ with about 20% expressing VGlut2 as well.

It would also be nice to sparse label of aplCoA and pplCoA using ChR2 to see if sparse activation drives approach or avoidance. 

We agree that it would be useful to vary the sparseness of the ChR2 expression, to see if produces similar results. We examined this using sparsely labeled odor ensembles, as previously done (Root et al., 2014). Briefly, we used the Arc-CreER mouse to label TMT responsive neurons with a cre-dependent ChR2 AAV vector targeted to the anterior or posterior regions, while previously we had broadly targeted the entirety of plCoA. We had established that this labeling method captures about half of the active cells detected by Arc expression, which is on the order of hundreds of neurons rather than thousands by broad cre-independent expression. Remarkably, we get effects similar in magnitude that are not significantly different from that with broader activation of the anterior or posterior domains (see new Figure S4, lines 267-288). It still remains possible that there is a threshold number of neurons that are necessary to elicit behavior, but that is beyond the scope of the current study. However, these data indicate that the effect of activating anterior and posterior domains is not an artifact of broad stimulation.

This is still a little unclear to me. "Inflectional". I think I need to spend a little more time getting familiar with this word and defintion.

This is why language can be so difficult, because every language determines things differently! Every language identifies grammar differently.

eLife Assessment

This is an important study with direct implications for the rational selection of antimalarial drug combinations. The authors present data demonstrating antagonism between 4-aminoquinoline antimalarials and peroxide drugs under physiologically relevant conditions, including robust effects at the trophozoite stage and for chloroquine at the ring stage. While the conclusions are based on in vitro assays and further work will be needed to fully resolve the underlying mechanism, the findings are convincing and provide a strong rationale for evaluating drug combinations in relevant preclinical models prior to clinical testing.

Reviewer #1 (Public review):

Summary:

This study set out to investigate potential pharmacological drug-drug interactions between the two most common antimalarial classes, the artemisinins and quinolines. There is strong rationale for this aim, because drugs from these classes are already widely-used in Artemisinin Combination Therapies (ACTs) in the clinic, and drug combinations are an important consideration in the development of new medicines. Furthermore, whilst there is ample literature proposing many diverse mechanisms of action and resistance for the artemisinins and quinolines, it is generally accepted that the mechanisms for both classes involve heme metabolism in the parasite, and that artemisinin activity is dependent on activation by reduced heme. The study was designed to measure drug-drug interactions associated with a short pulse exposure (4 h) that is reminiscent of the short duration of artemisinin exposure obtained after in vivo dosing. Clear antagonism was observed between dihydroartemisinin (DHA) and chloroquine, which became even more extensive in chloroquine-resistant parasites. Antagonism was also observed in this assay for the more clinically-relevant ACT partner drugs piperaquine and amodiaquine, but not for other ACT partners mefloquine and lumefantrine, which don't share the 4-aminoquinoline structure or mode of action. Interestingly, chloroquine induced an artemisinin resistance phenotype in the standard in vitro Ring-stage Survival Assay, whereas this effect was not as extensive for piperaquine.

The authors also utilised a heme-reactive probe to demonstrate that the 4-aminoquinolines can inhibit heme-mediated activation of the probe within parasites, which suggests that the mechanism of antagonism involves the inactivation of heme, rendering it unable to activate the artemisinins. Measurement of protein ubiquitination showed reduced DHA-induced protein damage in the presence of chloroquine, which is also consistent with decreased heme-mediated activation, and/or with decreased DHA activity more generally.

Overall, the study clearly demonstrates a mechanistic antagonism between DHA and 4-aminoquinoline antimalarials in vitro. It is interesting that this combination is successfully used to treat millions of malaria cases every year, which may raise questions about the clinical relevance of this finding. However, the conclusions in this paper are supported by multiple lines of evidence and the data is clearly and transparently presented, leaving no doubt that DHA activity is compromised by the presence of chloroquine in vitro. It is perhaps fortunate the that the clinical dosing regimens of 4-aminoquinoline-based ACTs have been sufficient to maintain clinical efficacy despite the non-optimal combination. Nevertheless, optimisation of antimalarial combinations and dosing regimens is becoming more important in the current era of increasing resistance to artemisinins and 4-aminoquinolines. Therefore, these findings should be considered when proposing new treatment regimens (including Triple-ACTs) and the assays described in this study should be performed on new drug combinations that are proposed for new or existing antimalarial medicines.

Strengths:

This manuscript is clearly written and the data presented is clear and complete. The key conclusions are supported by multiple lines of evidence, and most findings are replicated with multiple drugs within a class, and across multiple parasite strains, thus providing more confidence in the generalisability of these findings across the 4-aminoquinoline and peroxide drug classes.

A key strength of this study was the focus on short pulse exposures to DHA (4 h in trophs and 3 h in rings), which is relevant to the in vivo exposure of artemisinins. Artemisinin resistance has had a significant impact on treatment outcomes in South-East Asia, and is now emerging in Africa, but is not detected using a 'standard' 48 or 72 h in vitro growth inhibition assay. It is only in the RSA (a short pulse of 3-6 h treatment of early ring stage parasites) that the resistance phenotype can be detected in vitro. Therefore, assays based on this short pulse exposure provide the most relevant approach to determine whether drug-drug interactions are likely to have a clinically-relevant impact on DHA activity. These assays clearly showed antagonism between DHA and 4-aminoquinolines (chloroquine, piperaquine, amodiaquine and ferroquine) in trophozoite stages. Interestingly, whilst chloroquine clearly induced an artemisinin-resistant phenotype in the RSA, piperaquine only had a minor impact on the early ring stage activity of DHA, which may be fortunate considering that piperaquine is a currently recommended DHA partner drug in ACTs, whereas chloroquine is not.

The evaluation of additional drug combinations at the end of this paper is a valuable addition, which increases the potential impact of this work. The finding of antagonism between piperaquine and OZ439 in trophozoites is consistent with the general interactions observed between peroxides and 4-aminoquinolines, and it may be interesting to see whether piperaquine impacts the ring-stage activity of OZ439.

The evaluation of reactive heme in parasites using a fluorescent sensor, combined with the measurement of K48-linked ubiquitin, further support the findings of this study, providing independent read-outs for the chloroquine-induced antagonism.<br /> The in-depth discussion of the interpretation and implications of the results are an additional strength of this manuscript. Whilst the discussion section is rather lengthy, there are important caveats to the interpretation of some of these results, and clear relevance to the future management of malaria that require these detailed explanations.

Overall, this is a high quality manuscript describing an important study that has implications for the selection of antimalarial combinations for new and existing malaria medicines.

Weaknesses:

This study is an in vitro study of parasite cultures, and therefore caution should be taken when applying these findings to decisions about clinical combinations. The drug concentrations and exposure durations in these assays are intended to represent clinically relevant exposures, although it is recognised that the in vitro system is somewhat simplified and there may be additional factors that influence in vivo activity. This limitation is reasonably well acknowledged in the manuscript.

It is also important to recognise that the majority of the key findings regarding antagonism are based on trophozoite-stage parasites, and one must show caution when generalising these findings to other stages or scenarios. For example, piperaquine showed clear antagonism in trophozoite stages, but minimal impact in ring stages under these assay conditions.

A key limitation is the interpretation of the mechanistic studies that implicate heme-mediated artemisinin activation as the mechanism underpinning antagonism by chloroquine. This study did not directly measure the activation of artemisinins. The data obtained from the activation of the fluorescent probe are generally supportive of chloroquine suppressing the heme-mediated activation of artemisinins, and I think this is the most likely explanation, but there are significant caveats to consider. Primarily, the inconsistency between the fluorescence profile in the chemical reactions and the cell-based assay raise questions about the accuracy of this readout. In the chemical reaction, mefloquine and chloroquine showed identical inhibition of fluorescence, whereas piperaquine had minimal impact. On the contrary, in the cell, chloroquine and piperaquine had similar impacts on fluorescence, but mefloquine had minimal impact. This inconsistency indicates that the cellular fluorescence based on this sensor does not give a simple direct readout of the reactivity of ferrous heme, and therefore, these results should be interpreted with caution. Indeed, the correlation between fluorescence and antagonism for the tested drugs is a correlation, not causation. There could be several reasons for the disconnect between the chemical and biological results, either via additional mechanisms that quench fluorescence, or the presence of biomolecules that alter the oxidation state or coordination chemistry of heme or other potential catalysts of this sensor. It is possible that another factor that influences the H-FluNox fluorescence in cells also influences the DHA activity in cells, leading to the correlation with activity. It should be noted that H-FluNox is not a chemical analogue of artemisinins. It's activation relies on Fenton-like chemistry, but with a N-O rather that O-O bond, and it possesses very different steric and electronic substituents around the reactive centre, which are known to alter reactivity to different iron sources. Despite these limitations, the authors have provided reasonable justification for the use of this probe to directly visualise heme reactivity in cells, and the results are still informative.

Another interesting finding that was not elaborated by the authors is the impact of chloroquine in the DHA dose-response curves from the ring stage assays. Detection of artemisinin resistance in the RSA generally focuses on the % survival at high DHA concentrations (700 nM) as there is minimal shift in the IC50 (see Fig 2), however, chloroquine clearly induces a shift in the IC50 (~5-fold), where the whole curve is shifted to the right, whereas the increase in % survival is relatively small. This different profile suggests that the mechanism of chloroquine-induced antagonism may be different to the mechanism of artemisinin resistance. Current evidence regarding the mechanism of artemisinin resistance generally points towards decreased heme-mediated drug activation due to a decrease in hemoglobin uptake, which should be analogous to the decrease in heme-mediated drug activation caused by chloroquine. However, these different dose response curves suggest different mechanisms are primarily responsible. Additional mechanisms have been proposed for artemisinin resistance, involving redox or heat stress responses, proteostatic responses, mitochondrial function, dormancy and PI3K signalling among others. Whilst the H-FluNox probe generally supports the idea that chloroquine suppresses heme-mediated DHA activation, it remains plausible that chloroquine could induce these, or other, cellular responses that suppress DHA activity.

Impact:

This study has important implications for the selection of drugs to form combinations for the treatment of malaria. The overall findings of antagonism between peroxide antimalarials and 4-aminoquinolines in the trophozoite stage are robust, and the this carries across to the ring stage for chloroquine.

The manuscript also provides a plausible mechanism to explain the antagonism, although future work will be required to further explore the details of this mechanism and to rule out alternative factors that may contribute.

Overall, this is an important contribution to the field and provides a clear justification for the evaluation of potential drug combinations in relevant in vitro assays before clinical testing.

Reviewer #2 (Public review):

Summary:

This manuscript by Rosenthal and Goldberg investigates interactions between artemisinins and its quinoline partner drugs currently used for treating uncomplicated Plasmodium falciparum malaria. The authors show that chloroquine (CQ), piperaquine, and amodiaquine antagonize dihydroartemisinin (DHA) activity, and in CQ-resistant parasites, the interaction is described as "superantagonism," linked to the pfcrt genotype. Mechanistically, application of the heme-reactive probe H-FluNox indicates that quinolines render cytosolic heme chemically inert, thereby reducing peroxide activation. The work is further extended to triple ACTs and ozonide-quinoline combinations, with implications for artemisinin-based combination therapy (ACT) design, including triple ACTs.

Strengths:

The manuscript is clearly written, methodologically careful, and addresses a clinically relevant question. The pulsing assay format more accurately models in vivo artemisinin exposure than conventional 72-hour assays, and the use of H-FluNox and Ac-H-FluNox probes provides mechanistic depth by distinguishing chemically active versus inert heme. These elements represent important refinements beyond prior studies, adding nuance to our understanding of artemisinin-quinoline interactions.

Weaknesses:

Several points warrant consideration. The novelty of the work is somewhat incremental, as antagonism between artemisinins and quinolines is well established. Multiple prior studies using standard fixed-ratio isobologram assays have shown that DHA exhibits indifferent or antagonistic interactions with chloroquine, piperaquine, and amodiaquine (e.g., Davis et al., 2006; Fivelman et al., 2007; Muangnoicharoen et al., 2009), with recent work highlighting the role of parasite genetic background, including pfcrt and pfmdr1, in modulating these interactions (Eastman et al., 2016). High-throughput drug screens likewise identify quinoline-artemisinin combinations as mostly antagonistic. The present manuscript adds refinement by applying pulsed-exposure assays and heme probes rather than establishing antagonism de novo.

The dataset focuses on several parasite lines assayed in vitro, so claims about broad clinical implications should be tempered, and the discussion could more clearly address how in vitro antagonism may or may not translate to clinical outcomes. The conclusion that artemisinins are predominantly activated in the cytoplasm is intriguing but relies heavily on Ac-H-FluNox data, which may have limitations in accessing the digestive vacuole and should be acknowledged explicitly. The term "superantagonism" is striking but may appear rhetorical; clarifying its reproducibility across replicates and providing a mechanistic definition would strengthen the framing. Finally, some discussion points, such as questioning the clinical utility of DHA-PPQ, should be moderated to better align conclusions with the presented data while acknowledging the complexity of in vivo pharmacology and clinical outcomes.

Despite these mild reservations, the data are interesting and of high quality and provide important new information for the field.

Editor's Review of the Revision: The authors have provided a well-reasoned rebuttal to the comments of the three reviewers. Most of the changes were incorporated in their revised Discussion. Their data with the active heme probe H-FluNox are novel and the authors reveal interesting interactions between peroxide and 4-aminoquinoline-based antimalarials that open new avenues of research especially when considering antimalarial combinations that combine these chemical scaffolds. This study will be of broad interest to investigators studying and developing antimalarial drugs and combinations and the impact of Plasmodium falciparum resistance mechanisms. A minor recommendation would be that the authors state H-FluNox when referring to their small molecule probe in the abstract, so that it is captured in PubMed searches.

Reviewer #3 (Public review):

Summary:

The authors present an in vitro evaluation of drug-drug interactions between artemisinins and quinoline antimalarials, as an important aspect for screening the current artemisinin-based combination therapies for Plasmodium falciparum. Using a revised pulsing assay, they report antagonism between dihydroartemisinin (DHA) and several quinolines, including chloroquine, piperaquine (PPQ), and amodiaquine. This antagonism is increased in CQ-resistant strains in isobologram analyses. Moreover, CQ co-treatment was found to induce artemisinin resistance even in parasites lacking K13 mutations during the ring-stage survival assay. This implies that drug-drug interactions, not just genetic mutations, can influence resistance phenotypes. By using a chemical probe for reactive heme, the authors demonstrate that quinolines inhibit artemisinin activation by rendering cytosolic heme chemically inert, thereby impairing the cytotoxic effects of DHA. The study also observed negative interactions in triple-drug regimens (e.g., DHA-PPQ-Mefloquine) and in combinations involving OZ439, a next-generation peroxide antimalarial. Taken together, these findings raise significant concerns regarding the compatibility of artemisinin and quinoline combinations, which may promote resistance or reduce efficacy.

With the additive profile as the comparison and a lack of synergistic effect in any of the comparisons, it is hard to contextualize the observed antagonism. Including a known synergistic pair (e.g., artemisinin + lumefantrine) would have provided a useful benchmark to assess the relative impact of the drug interactions described.

Strengths:

This study demonstrates the following strengths:

• The use of a pulsed in vitro assay that is more physiologically relevant over the traditional 48h or 72h assays

• Small molecule probes, H-FluNox, and Ac-H-FluNox to detect reactive cytosolic heme, demonstrating that quinolines render heme inert and thereby block DHA activation.

• Evaluates not only traditional combinations but also triple-drug combinations and next-generation artemisinins like OZ439. This broad scope increases the study's relevance to current treatment strategies and future drug development.

• By using the K13 wild-type parasites, the study suggests that resistance phenotypes can emerge from drug-drug interactions alone, without requiring genetic resistance markers.

Weaknesses:

• The study would benefit from a future characterization of the molecular basis for the observed heme inactivation by quinolines to support this hypothesis - while the probe experiments are valuable, they do not fully elucidate how quinolines specifically alter heme chemistry at the molecular level.

• Suggestion of alternative combinations that show synergy could have improved the significance of the work. The invitro study did not include pharmacokinetic/pharmacodynamic modeling, hence it leaves questions about how the observed antagonism would manifest under real-world dosing conditions, necessitating furture work based on these findings.

Author response:

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

eLife Assessment

We appreciate the positive assessment. We recognize that since all of the work in this manuscript was done in vitro, there are reasonable concerns about the translatability of these data to clinical settings. These results should not directly inform malaria policy, but we hope that these data bring new considerations to the approach for choosing strategic antimalarial combinations. We have modified the manuscript to clarify this distinction.

Public Reviews

Reviewer #1 (Public Review):

We thank the reviewer for their thoughtful summary of this manuscript. It is important to note that DHA-PPQ did show antagonism in RSAs. In this modified RSA, 200 nM PPQ alone inhibited growth of PPQ-sensitive parasites approximately 20%. If DHA and PPQ were additive, then we would expect that addition of 200 nM PPQ would shift the DHA dose response curve to the left and result in a lower DHA IC50. Please refer to Figure 4a and b as examples of additive relationships in dose-response assays. We observed no significant shift in IC50 values between DHA alone and DHA + PPQ. This suggests antagonism, albeit not to the extent seen with CQ. We have modified the manuscript to emphasize this point. As the reviewer pointed out, it is fortunate that despite being antagonistic, clinically used artemisinin-4-aminoquinoline combinations are effective, provided that parasites are sensitive to the 4-aminoquinoline. It is possible that superantagonism is required to observe a noticeable effect on treatment efficacy (Sutherland et al. 2003 and Kofoed et al. 2003), but that classical antagonism may still have silent consequences. For example, if PPQ blocks some DHA activation, this might result in DHA-PPQ acting more like a pseudo-monotherapy. However, as the reviewer pointed out, while our data suggest that DHA-PPQ and AS-ADQ are “non-optimal” combinations, the clinical consequences of these interactions are unclear. We have modified the manuscript to emphasize the later point.

While the Ac-H-FluNox and ubiquitin data point to a likely mechanism for DHA-quinoline antagonism, we agree that there are other possible mechanisms to explain this interaction.  We have addressed this limitation in the discussion section. Though we tried to measure DHA activation in parasites directly, these attempts were unsuccessful. We acknowledge that the chemistry of DHA and Ac-H-FluNox activation is not identical and that caution should be taken when interpreting these data. Nevertheless, we believe that Ac-H-FluNox is the best currently available tool to measure “active heme” in live parasites and is the best available proxy to assess DHA activation in live parasites. These points are now addressed in the discussion section. Both in vitro and in parasite studies point to a roll for CQ in modulating heme, though an exact mechanism will require further examination. Similar to the reviewer, we were perplexed by the differences observed between in vitro and in parasite assays with PPQ and MFQ. We proposed possible hypotheses to explain these discrepancies in the discussion section. Interestingly, our data corelate well with hemozoin inhibition assays in which all three antimalarials inhibit hemozoin formation in solution, but only CQ and PPQ inhibit hemozoin formation in parasites. In both assays, in-parasite experiments are likely to be more informative for mechanistic assessment.

It remains unclear why K13 genotype influences RSA values, but not early ring DHA IC50 values. In K13^WT parasites, both RSA values and DHA IC50 values were increased 3-5 fold upon addition of CQ. This suggests that CQ-mediated resistance is more robust than that conferred by K13 genotype. However, this does not necessarily suggest a different resistance mechanism. We acknowledge that in addition to modulating heme, it is possible that CQ may enhance DHA survival by promoting parasite stress responses. Future studies will be needed to test this alternative hypothesis. This limitation has been acknowledged in the manuscript. We have also addressed the reviewer’s point that other factors, including poor pharmacokinetic exposure, contributed to OZ439-PPQ treatment failure.

Reviewer #2 (Public Review):

We appreciate the positive feedback. We agree that there have been previous studies, many of which we cited, assessing interactions of these antimalarials. We also acknowledge that previous work, including our own, has shown that parasite genetics can alter drug-drug interactions. We have included the author’s recommended citations to the list of references that we cited. Importantly, our work was unique not only for utilizing a pulsing format, but also for revealing a superantagonistic phenotype, assessing interactions in an RSA format, and investigating a mechanism to explain these interactions. We agree with the reviewer that implications from this in vitro work should be cautious, but hope that this work contributes another dimension to critical thinking about drug-drug interactions for future combination therapies. We have modified the manuscript to temper any unintended recommendations or implications.

The reviewer notes that we conclude “artemisinins are predominantly activated in the cytoplasm”. We recognize that the site of artemisinin activation is contentious. We were very clear to state that our data combined with others suggest that artemisinins can be activated in the parasite cytoplasm. We did not state that this is the primary site of activation. We were clear to point out that technical limitations may prevent Ac-H-FluNox signal in the digestive vacuole, but determined that low pH alone could not explain the absence of a digestive vacuole signal.

With regard to the “reproducibility” and “mechanistic definition” of superantagonism, we observed what we defined as a one-sided superantagonistic relationship for three different parasites (Dd2, Dd2 PfCRT^Dd2, and Dd2 K13^R539T) for a total of nine independent replicates. In the text, we define that these isoboles are unique in that they had mean ΣFIC50 values > 2.4 and peak ΣFIC50 values >4 with points extending upward instead of curving back to the axis. As further evidence of the reproducibility of this relationship, we show that CQ has a significant rescuing effect on parasite survival to DHA as assessed by RSAs and IC50 values in early rings.

Reviewer #3 (Public Review):

We thank the reviewer for their positive feedback. We acknowledge that no combinations tested in this manuscript were synergistic. However, two combinations, DHA-MFQ and DHA-LM, were additive, which provides context for contextualizing antagonistic relationships. We have previously reported synergistic and additive isobolograms for peroxide-proteasome inhibitor combinations using this same pulsing format (Rosenthal and Ng 2021). These published results are now cited in the manuscript.

We believe that these findings are specific to 4-aminoquinoline-peroxide combinations, and that these findings cannot be generalized to antimalarials with different mechanisms of action. Note that the aryl amino alcohols, MFQ and LM, were additive with DHA. Since the mechanism of action of MFQ and LM are poorly understood, it is difficult to speculate on a mechanism underlying these interactions.

We agree with the reviewer that while the heme probe may provide some mechanistic insight to explain DHA-quinoline interactions, there is much more to learn about CQ-heme chemistry, particularly within parasites.

The focus of this manuscript was to add a new dimension to considerations about pairings for combination therapies. It is outside the scope of this manuscript to suggest alternative combinations. However, we agree that synergistic combinations would likely be more strategic clinically.

An in vitro setup allows us to eliminate many confounding variables in order to directly assess the impact of partner drugs on DHA activity. However, we agree that in vivo conditions are incredibly more complex, and explicitly state this.

We agree that in the future, modeling studies could provide insight into how antagonism may contribute to real-world efficacy. This is outside the scope of our studies.

Recommendations for the Authors:

Reviewer #1 (Recommendations for the Authors):

The key weaknesses identified in this manuscript are described in the 'weaknesses' section of the public review. The major one is the inconsistency around the H-FluNox response in the chemical vs biological experiments. I can't think of a simple experiment to resolve this issue, but it is good that this data is openly provided in the manuscript. I believe there could be more discussion to clarify this limitation with the current study, and the conclusions, and particularly the title, should be softened regarding the mechanism of antagonism being based on heme reactivity.

We have softened the title and conclusions to take into account the limitations of our studies.

(1) Please double-check the definitions for isobologram interpretation. In most antimicrobial interaction studies, I see the threshold for antagonism at sumFIC50 of 1.5, or even 2. 1.25 is often interpreted as additive in many studies.

We acknowledge that different studies use various cutoff values. Our interpretations for additive versus antagonistic versus superantagonistic were based not only on mean ΣFIC50 values, but also isobologram shape. For example, the flat isoboles for MFQ-DHA were clearly distinct from the curved isoboles of PPQ-DHA. It is unclear what cutoff value(s) would be most clinically relevant.

(2) For the MFQ-PPQ interaction study, please make it clear that these drugs have very long half-lives (weeks), so the 4 h pulse assay isn't really relevant to their overall activity. It probably shows a slower onset of action, but there is plenty of drug remaining for many days in the clinical scenario, so perhaps the data from the traditional 48h assay is more relevant. The same consideration applies to OZ439, which may impact the interpretation of that data.

We have now included the half-lives of these compounds in the discussion section. Our intent was to use a pulsing format to make these isobolograms comparable with the other assays. It is important to note that pulses can reveal stronger phenotypes that might be missed with traditional methods. Thus, while 48 h assays may better mimic in vivo conditions, they could also mask important phenotypes.

Reviewer #3 (Recommendations for the Authors):

I have included most of my concerns in the public review. Below are some additional specific points for consideration:

(1) It is expected to include a synergistic combination as a control (e.g., artemisinin + lumefantrine) to contextualize the degree of antagonism observed. The experimental design should show some synergistic profiles in comparison. Adding a few experiments by including a synergistic control is needed.

Both MFQ-DHA and LM-DHA combinations were additive, which provides context for antagonistic combinations. This is now stated in the results section pertaining to Figure 1. We have also included a reference to our previous publication in which we demonstrated that proteasome inhibitor-peroxide combinations are synergistic to additive using this same pulsing format.

(2) Consider in vivo validation or pharmacokinetic/pharmacodynamic modeling to strengthen the translational relevance of the findings when it comes to doses and the IC50 correlations.

We agree that this would be useful to do in future, but it is outside the scope of the current study.

(3) It would be beneficial to include a discussion section on how the findings are generalizable to different Plasmodium falciparum genotypes (3D7, Dd2, MRA-1284) and their relevance.

Findings were consistent across three parasite backgrounds depending on PfCRT genotype. This point has been included in the discussion section. The background of these parasites is also provided in Table 1.

(4) Potential evaluation criteria to understand where certain combinations should be reconsidered can be included as a suggestion for the wider audience.

Our in vitro studies suggest that pulsing isobolograms would be a useful assay to include when evaluating combination therapies. While we believe that synergistic combinations would be more strategic than antagonistic combinations, we cannot provide evaluation criteria or make recommendations for reconsidering currently used combinations.

(5) Further elaborate on the mechanistic basis of heme inactivation by quinolines. If data are available, please include more data on the specificity of the process.

Despite our best efforts, we were unable to evaluate quinoline-heme interactions in parasites. Even in vitro, this interaction has remined elusive for decades. We agree that this would be an important future step towards supporting a specific mechanism for quinoline-DHA antagonism.

Hypothesis

eLife Assessment

In this study, the authors identify EOLA1 as a novel mitochondrial protein required for mitochondrial translation and normal cardiac function. The characterization of the molecular role of EOLA1 is still incomplete, and additional controls will be necessary. Nevertheless, the identification of a novel factor critical for mitochondrial gene expression and oxidative phosphorylation will be useful for cell biologists working on mitochondrial dysfunction.

Reviewer #1 (Public review):

Summary:

Mitochondria encode a small set of proteins that are made inside the organelle by specialized ribosomes. When this mitochondrial translation system fails, oxidative phosphorylation is impaired, an outcome that is particularly harmful to energy-demanding tissues such as the heart. In this manuscript, the authors use a targeted CRISPR/Cas9 screen in cultured cells grown on galactose (a condition that forces reliance on oxidative phosphorylation) to identify genes required for mitochondrial activity. They highlight EOLA1, previously studied mainly in inflammatory contexts, as a top candidate.

Strengths:

The authors present data suggesting that EOLA1 is imported into mitochondria via an N-terminal targeting sequence and resides in the mitochondrial matrix. Loss of EOLA1 reduces oxygen consumption and is associated with altered mitochondrial ultrastructure. Mechanistically, affinity purification suggests interaction with mitochondrial elongation factors TUFM (mtEF-Tu), and RNA immunoprecipitation experiments enrich 12S mt-rRNA, consistent with a relationship to the small ribosomal subunit. Multiple assays, including sucrose-gradient profiling, reduced abundance of selected mtDNA-encoded proteins, and a click-chemistry labeling approach, support the conclusion that mitochondrial protein synthesis is decreased in EOLA1-deficient cells. Finally, whole-body Eola1 knockout mice show echocardiographic findings consistent with dilated cardiomyopathy and reduced levels of representative mitochondrially encoded proteins in cardiac tissue.

How to interpret the work:

The data support a role for EOLA1 in maintaining mitochondrial gene expression and oxidative phosphorylation capacity, and they plausibly implicate mitochondrial translation.

Weaknesses:

The main caveat is that the study does not yet establish how EOLA1 acts, whether it directly modulates translation elongation through TUFM, whether it is primarily required for mitoribosome biogenesis/rRNA stability, or whether it influences translation indirectly through mitochondrial stress pathways. The in vivo phenotype is intriguing, but without tissue-specific deletion/rescue and deeper cardiac pathology/mitochondrial functional measurements, it remains uncertain how directly the heart phenotype reflects a cardiomyocyte-autonomous defect in mitochondrial translation.

Reviewer #2 (Public review):

Summary:

In this study, the authors identify a previously uncharacterised regulator of mitochondrial function using a genetic screen and propose a role for this protein in supporting mitochondrial protein production. They provide evidence that the protein localises to mitochondria, interacts with components of the mitochondrial translation machinery, and is required for normal heart function in an animal model.

Strengths:

A major strength of the work is the use of multiple independent approaches to assess mitochondrial activity and protein production, which together provide support for the central conclusions. The in vivo data linking loss of this factor to impaired heart function are particularly compelling and elevate the relevance of the study beyond a purely cell-based context.

Weaknesses:

Given prior reports placing this protein outside mitochondria, its mitochondrial localisation would benefit from more rigorous and quantitative validation, and the proposed mechanism of the interaction with the mitochondrial translation machinery remains only partially explored. In addition, the physiological analysis is largely limited to the heart, leaving open questions about how broadly this pathway operates across tissues.

Major comments:

(1) Evidence for mitochondrial localization of EOLA1<br /> EOLA1 has previously been reported as a nuclear and cytosolic protein and is not annotated in MitoCarta 3.0, making rigorous validation of its mitochondrial localization particularly important. Although the authors provide several lines of evidence, interpretation is complicated by the use of different cell lines across localization, interaction, and functional experiments. Greater consistency in the cellular models used would strengthen the conclusions. The immunofluorescence analysis of tagged EOLA1 would also benefit from quantification across more cells and the inclusion of an additional mitochondrial marker (e.g., an outer membrane marker such as TOM20), as HSP60 staining can vary with mitochondrial state.

(2) Normalization of OCR measurements<br /> Clarification of how Seahorse oxygen consumption rate measurements were normalized (e.g., cell number or protein content) would aid interpretation, particularly given potential effects of Eola1 loss on cell growth.

(3) Linking interaction data to functional phenotypes<br /> Loss-of-function analyses are performed in mouse cell lines, whereas localization and interactome studies are conducted in human HEK293T cells. The absence of a human EOLA1 knockout model makes it difficult to directly connect the interaction data to the observed functional phenotypes. Additional validation or discussion of species conservation would improve clarity.

(4) Mechanistic interpretation of the EOLA1-TUFM-12S rRNA interaction<br /> The identification of TUFM and 12S mt-rRNA as EOLA1 interactors is an interesting finding; however, the basis for prioritizing TUFM among the many mitochondrial proteins identified in the interactome is not fully explained. Providing enrichment statistics and functional categorization of mitochondrial interactors would increase transparency. In addition, the proposed role of the ASCH domain in RNA binding would be strengthened by structure-informed or mutational analysis of the conserved RNA-binding motif.

(5) Interpretation of mitochondrial translation and protein abundance data<br /> Several assays supporting impaired mitochondrial translation would benefit from additional controls and quantification. The de novo mitochondrial translation assay (Fig. 3h) is not quantified, making it difficult to assess the magnitude and reproducibility of the effect. In addition, western blots showing reduced levels of mitochondrially encoded OXPHOS subunits (Figure 3g) lack a mitochondrial loading control (e.g., TOM20 or VDAC). Since loss of EOLA1 may affect mitochondrial mass, normalization to a mitochondrial marker is necessary. Relatedly, it would be informative to assess whether steady-state levels of mitoribosomal proteins (e.g., MRPS15, MRPL37) and nuclear-encoded OXPHOS subunits are altered upon Eola1 loss, both in knockout cell lines and in the knockout mouse.

(6) Physiological scope of the in vivo analysis<br /> The cardiac phenotype observed in the whole-body Eola1 knockout mouse is compelling, but the focus on a single tissue limits interpretation of EOLA1's broader physiological role. Examination of additional high-energy-demand tissues would help clarify whether the observed effects are heart-specific or more general. In addition, the presence of residual EOLA1 protein bands in western blots (Figure 4a) and remaining Eola1 transcripts in qRT-PCR analyses (Extended Figure 4e) from knockout tissues should be addressed. The authors should clarify whether these signals reflect incomplete knockout, alternative isoforms, antibody cross-reactivity, or technical background.

(7) Relationship to previously reported MT2A interaction<br /> Given prior reports of EOLA1 interaction with MT2A, a brief comment on whether MT2A was detected in the authors' co-immunoprecipitation experiments and how this relates to the proposed mitochondrial role would be useful.

Reviewer #3 (Public review):

The authors identified EOLA1 in a CRISPR/Cas9 screen for essential mitochondrial genes in a mouse B16-F10 cell line; however, no information on the library used for this screen or the list of all identified essential genes is provided. What was the p-value for EOLA1 in Figure 1b?

The authors show that EOLA1 is indeed a mitochondrial protein (using both mouse and human cell lines). It is valuable that the authors use different cell lines to investigate the function of this protein; however, this also presents a challenge, as four different cell lines (two mouse and two human) are used across individual experiments, with no consistency between them. Knock-out (KO) experiments were performed in mouse cell lines only, and human cell lines were used in overexpression experiments, in which EOLA1 was tagged with FLAG-HA. It would be beneficial if a knock-out were also generated in a human cell line to confirm the effect on the expression of mitochondria-encoded proteins, along with a rescue experiment in which the EOLA1 protein is reintroduced into KO cells.

Functional analysis of EOLA1: The authors performed affinity immunoprecipitation of FLAG-HA-tagged EOLA1 from stably overexpressing cells, and identified 202 co-immunoprecipitating proteins, of which 71 were known mitochondrial proteins; however, no list of these proteins is provided. Why did the authors choose TUFM? Were any mitochondrial ribosomal proteins co-immunoprecipitated, if EOLA1 is suggested to regulate translation? Were levels of TUFM affected in EOLA1-KO cells?

The authors continued to analyze mitochondrial ribosomes using sucrose gradient fractionation and in-vitro mitochondrial translation. However, there are several technical problems with the presented data: It has been established that mitochondrial ribosomes do not form polysomes in mammalian cells but rather perform translation as monosomes. The authors indirectly confirm this: almost no 12S or 16S rRNA (Fig. 3f) or MRP proteins (Extended data 3c) are present in "polysome" fractions. Although indeed 12S and 16S rRNAs are decreased in monosome fractions, the levels of mRNAs are not different between KO and WT cells, and neither is the migration of mitochondrial ribosomal proteins. As there is no loading control provided for the sucrose gradients blots (such as SDHA, VDAC), it is not possible to assess the overall levels of mitochondrial ribosomes. The gel presented for mitochondrial translation is of poor quality, as it is impossible to identify any of the expected 13 polypeptides. Although the intensity of the signal is weaker for KO, so is the intensity in the portion of Coomassie stained gel. A better-quality gel and quantification need to be provided to support the claims.

What is the difference between endogenous and exogenous RIP-qPCR? EOLA1 pulled down 12S rRNA without cross-linking (Figure 3d) or with UV-crosslinking (Figure 3e), however, both 12S and 16S rRNAs were enriched in UV-crosslinked cells (Figure 3c) and by UV-RIP-seq (Extended data 3b; although no control is provided here). Is no discussion offered for this observation? Is it possible that EOLA1 plays a role in the maturation of the mito-ribosome, rather than translation? Does EOLA1 co-migrate with the mito-ribosome on sucrose gradients?

Altogether, there is insufficient evidence to support the conclusion that EOLA1 plays a role in mitochondrial translation.

To investigate EOLA1 biological function, the authors created a whole-body EOLA1-/- mouse that exhibited no overall developmental abnormalities; however presented with an abnormal cardiac function. This is an ideal model to confirm prior observations in cellular models; however, apart from one western-blot for three mitochondrial encoded subunits, no other experiments were provided (such as measurements of the levels of 12S, or 16S rRNA, TUFM levels, ribosomes profile, mitochondrial translation, OXPHOS assembly, respirometry).

In Figure 2 g-i: TEM images are presented, but the method is not described, nor is any information on the cells used provided, nor is it clear how the circularity was determined. KO cells certainly look abnormal; however, are the authors sure that the indicated structures are mitochondria? They rather resemble autophagosomes/lysosomes with lamellar inclusions.

This caution protects the concept of hope from collapsing into a celebration of endurance. It foregrounds the difference between coping and complicity, and invites attention to where hope supports reorientation and contestation.

This anchors creative labour in a time structure where present sacrifice is rationalized through prospective recognition. The concept helps articulate why project based cultural work often normalizes uneven reward schedules and prolonged uncertainty.

Document de Synthèse : Le Projet FUSÉ – Une Approche Structurelle pour la Réussite des Élèves Fragilisés

Résumé Exécutif

Le projet FUSÉ (Formation à l’utilisation de stratégies efficaces pour l’engagement) est une initiative novatrice mise en œuvre à l’école secondaire Carrefour (Centre de services scolaire des Draveurs) pour contrer le décrochage scolaire précoce.

Ce projet cible les élèves du premier cycle du secondaire en situation de grande vulnérabilité, particulièrement ceux ayant des acquis de 6e année mais se trouvant en échec dans plusieurs matières à sanction.

Partant du constat que le redoublement traditionnel ne produisait aucun résultat positif (33 % de taux de sortie sans diplôme), la direction a instauré une structure rigoureuse remplaçant la culture du redoublement par un accompagnement intensif basé sur l’autodétermination et la réussite immédiate.

Après une année d'application, les résultats sont probants : sur 39 élèves ciblés, 20 ont réussi leur passage en secondaire 3, évitant ainsi des trajectoires de formation moins qualifiantes.

Le projet repose sur une mobilisation des services complémentaires, une réorganisation des horaires et l'utilisation d'un quartier général dédié : le « Bistrado ».

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1. Contexte et Problématique

1.1 Un constat d'échec systémique

L'école secondaire Carrefour, située en milieu urbain défavorisé, accueille environ 2 000 élèves. Avant l'implantation de FUSÉ, l'école faisait face à des défis majeurs :

• Taux de décrochage élevé : 33 % de sorties sans diplôme au régulier, contre une moyenne québécoise de 24,6 % pour des milieux équivalents.

• Décrochage précoce : Le profil type du décrocheur se dessinait dès l'âge de 15 ans, souvent suite à une reprise de la première année du secondaire.

• Inefficacité du redoublement : Les données montraient que les élèves reprenant leur secondaire 1 obtenaient des résultats inférieurs à leur première tentative, tout en développant des problèmes de comportement et de motivation accrus.

1.2 L'urgence d'agir

En mars 2024, les prévisions indiquaient que 42 élèves sur 200 au régulier étaient en échec dans au moins trois matières à sanction.

Face à la pression du personnel pour un redoublement massif ou un transfert en adaptation scolaire (non justifié par les acquis académiques), la direction a choisi de rompre avec les pratiques établies.

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2. Fondements et Vision du Projet FUSÉ

Le projet s'appuie sur une philosophie de « création du possible » lorsque les méthodes traditionnelles échouent.

2.1 Objectifs centraux

• Maintenir la trajectoire scolaire : Éviter que les élèves ne soient dirigés prématurément vers des parcours comme la FMS (Formation menant à l'exercice d'un métier semi-spécialisé).

• Favoriser l'autodétermination : Baser l'intervention sur les besoins fondamentaux d'appartenance, de relation et de compétence.

• Inverser l'effort : Faire en sorte que l'élève devienne l'acteur principal de sa réussite, plutôt que de voir les adultes « travailler plus fort que l'élève ».

2.2 Cadre théorique et leviers

Le projet s'inspire de modèles existants tels que :

• L'approche Check & Connect (utilisée au 2e cycle sous le nom de « Boussole éducative »).

• Le Plan d'intervention autodéterminé, soutenu par une formation de la conseillère pédagogique du centre de services.

• L'utilisation de données probantes pour identifier les facteurs de risque et de protection.

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3. Structure Opérationnelle et Mise en Œuvre

La réussite de FUSÉ repose sur une structure « bétonnée » plutôt que sur un simple changement de culture imposé au personnel enseignant.

3.1 Le « Bistrado » : Le Quartier Général

Le bistro étudiant de l'école est transformé chaque matin en centre de services centralisé pour les élèves FUSÉ. C'est un lieu sécurisant, loin de l'agitation des classes, où s'effectue l'accueil quotidien.

3.2 L'Intervenant Pivot

Chaque élève est lié à un intervenant pivot (agent de réadaptation, orthopédagogue, enseignant ressource ou intervenant en toxicomanie). Ce dernier :

• Centralise les communications.

• Assure un accueil quotidien (les « Soleils FUSÉ »).

• Suit les objectifs personnels de l'élève.

3.3 Analyse des données et sous-groupes

Les élèves sont regroupés selon la nature de leurs besoins, tout en restant intégrés dans leur profil ou programme d'origine (pas de classes fermées) :

| Profil de sous-groupe | Nature des difficultés | | --- | --- | | Comportement | Manifestations comportementales perturbatrices. | | Motivation / Assiduité | Taux d'absentéisme élevé, désengagement. | | Apprentissage | Lacunes académiques graves en français ou mathématiques. |

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4. L'Expérience Élève et Engagement

4.1 Le contrat d'engagement

La participation est volontaire. L'élève doit signer un contrat d'engagement. Si l'engagement fait défaut, l'élève peut être retiré du projet, avec la possibilité d'y revenir lorsqu'il se sent prêt.

4.2 Le déroulement quotidien

• Période solée (8h40 - 9h00) : Accueil au Bistrado, petit-déjeuner pour les élèves en milieu défavorisé, et fixation d'objectifs quotidiens ou hebdomadaires (ex: arriver à l'heure, participer en classe).

• Suivi des objectifs : Les réussites sont soulignées par des « billets de tirage » et des certificats de reconnaissance, favorisant l'émulation.

• Horaire différencié : Pour certains élèves, des matières comme les arts, l'anglais ou le CCQ sont temporairement allégées pour permettre des périodes de rattrapage intensif en français et mathématiques avec des enseignants ressources.

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5. Résultats et Impact

5.1 Statistiques de la première cohorte (39 élèves)

Les résultats ont surpassé les attentes initiales de la direction :

• 20 élèves ont intégré le secondaire 3 régulier.

• 4 élèves ont été dirigés vers la FMS.

• 2 élèves vers le Pré-DEP.

• 1 élève vers la formation générale des adultes.

• 8 élèves ont repris leur secondaire 2 (mais avec un meilleur accompagnement).

• Seulement 4 abandons (dont 2 en cours d'année).

5.2 Gains qualitatifs

• Amélioration du lien école-famille : Les parents, souvent découragés, ont retrouvé de l'espoir grâce à une communication axée sur le positif.

• Cohérence organisationnelle : Le personnel partage désormais un langage commun autour de l'autodétermination.

• Épanouissement social : Participation à des activités d'émulation (ex: sorties au théâtre) et implication bénévole des élèves au sein de l'école.

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6. Évolution : FUSÉ 2.0 et Perspectives

Fort de son succès, le projet entame sa deuxième année avec des ajustements majeurs :

1. Enseignement multiniveaux : Création de groupes en français et mathématiques pour les élèves ayant des lacunes profondes (niveau 5e année primaire), tout en évitant le cloisonnement.

2. Expansion au secondaire 1 : Identification précoce des élèves fragiles dès la rentrée pour prévenir l'échec.

3. Intégration systémique : Fusion de l'approche FUSÉ dans la « Boussole éducative » globale de l'école pour assurer une transition fluide entre le premier et le deuxième cycle.

4. Adaptation scolaire : Réflexion sur l'application de l'approche fusée pour les élèves en adaptation afin de viser une progression constante plutôt que la simple réussite de fin d'année.

Le projet FUSÉ démontre qu'en réallouant les ressources existantes et en structurant rigoureusement l'accompagnement, il est possible de modifier radicalement la trajectoire d'élèves que le système considérait autrefois comme perdus.

I think this is the design that has been superseded by the notion of having a rack level power supply unit, Which is significant in more efficient, And which then also provides DC directly to all the machines. So rather than having batteries on the rack, you'd have batteries in a rack. So that serves multiple machines. I think.

so the batteries are part of the rack design now

Boek: Over het algemeen wordt een lichaam gezien als een entiteit die deelneemt aan het economisch verkeer; kapitaalvennootschappen, zoals een bv of nv, maar ook stichtingen en verenigingen, mits ze een onderneming drijven

Microsoft has a commitment to entirely remove fossil fuel backup generation by 2030?

I know it's implied by the 100/100/0 statement, but I hadn't realised they had been explicit

Even US firms are using Chinese models because they are so much cheaper. They do not use them for everything, but where they can get away with lower cost options they will.

This was 2020, so around five years ago they started to do this.

testing this

He talks of a "thing", meaning he does not know what it is or is not sure about it. This gives the storyline a bit of eeriness, making the reader expect this "thing" to be something that is scary. anonymous?

He replies with the speaker's name even though the speaker never revealed his name after being asked, "Who's there?" This gives an expectation that they know each other, and or they work together.

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