language affects identitiy and family relationships

language value depends on who is speaking it

english only polices can affect students

testing bias makes bilingual kids seem behind

shows benefit even for low income kids

switching between languages improves brain control skills

a nuertoscientist is a credible reliable source

bilingualism improves brain fuctions for alll kids.

Synthèse sur l'Optimisation des Plaques de Gélatine DIY

Résumé Exécutif

Ce document de synthèse détaille les problèmes, solutions et innovations présentés dans le contexte de la fabrication et de l'utilisation de plaques d'impression à la gélatine faites maison (DIY).

L'analyse révèle trois problèmes majeurs avec les recettes traditionnelles :

un séchage prématuré de la peinture, des risques significatifs liés à l'utilisation d'alcool, et des réactions chimiques indésirables avec la peinture acrylique.

La solution centrale est l'adoption d'une nouvelle recette "sobre", qui élimine complètement l'alcool et le remplace par du propylène glycol.

Ce changement résout non seulement le risque d'incendie et les problèmes d'irritation, mais améliore également de manière significative la rétention d'eau de la plaque, prévenant ainsi le séchage de la peinture.

Parallèlement, de nouveaux protocoles de maintenance sont introduits, notamment une "routine de soins" en deux étapes (nettoyage et hydratation) pour préserver la surface de la plaque et inhiber la croissance microbienne.

Les recommandations de stockage ont été révisées pour préconiser un contenant hermétique, en conjonction avec cette nouvelle routine.

Enfin, des outils et méthodologies de précision sont proposés, comme le passage à des mesures en grammes et le lancement d'un "Calculateur de Recette 2.0".

Cet outil en ligne permet de personnaliser les recettes en fonction de la taille de la plaque et de la force (valeur de Bloom) de la gélatine.

Le document aborde également la cause du "caillage" de la peinture acrylique—un environnement acide—et fournit une solution de neutralisation à base de bicarbonate de soude.

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

1. Problèmes Identifiés avec la Recette Originale

L'analyse de la recette originale de la plaque de gélatine DIY a mis en évidence plusieurs problèmes récurrents rencontrés par les utilisateurs, transformant parfois l'expérience d'impression en un processus frustrant.

1.1. Le Problème de la "Plaque Assoiffée"

Le problème le plus courant est celui d'une plaque qui sèche trop rapidement, rendant la peinture quasi impossible à retirer.

• Cause principale : Un surdosage de gélatine ou l'utilisation d'une gélatine à haute valeur de Bloom. Une telle plaque n'est pas entièrement saturée en eau et devient "très, très assoiffée".

• Mécanisme : La plaque de gélatine sèche aspire instantanément l'eau contenue dans la peinture acrylique. Les pigments adhèrent alors de manière presque irréversible à la surface.

• Conséquence : La plaque se comporte comme un "aimant suceur de peinture acrylique" plutôt que comme une surface de transfert antiadhésive.

• Analogies : L'auteure compare ce phénomène aux premières crêpes que l'on jette, expliquant que la plaque a besoin de "s'échauffer", c'est-à-dire de se saturer en eau, avant de fonctionner correctement.

1.2. Les Risques et Inconvénients de l'Alcool

L'alcool, ingrédient clé de l'ancienne recette pour réduire l'aspect collant et améliorer la conservation, présente deux inconvénients majeurs.

• Risque d'incendie : L'utilisation d'alcool (isopropylique, dénaturé, ou à haute teneur) lors du chauffage du mélange présente un risque réel d'incendie.

Une utilisatrice nommée Rita a d'ailleurs connu un tel incident, ce qui a été un catalyseur pour le changement de recette.

• Irritation : Les vapeurs d'alcool peuvent irriter les yeux et les voies respiratoires des utilisateurs.

• Déshydratation de la plaque : L'alcool contribue significativement à la déshydratation de la plaque sur le long terme.

L'auteure fait une analogie avec la "sensation de Sahara dans la bouche" après une soirée arrosée pour illustrer cet effet.

1.3. Comportement Anormal de la Peinture Acrylique

Certains utilisateurs ont rapporté un comportement "super étrange" de la peinture acrylique, qui se met à cailler ou à se décomposer sur la plaque.

• Cause : Un environnement acide (pH bas).

• Origine du problème : L'ajout d'acides comme le jus de citron ou l'acide citrique dans le mélange, souvent dans le but d'agir comme conservateur.

• Effet : Dans un milieu fortement acide, le système liant de la peinture acrylique peut se rompre, provoquant son caillage.

La peinture adhère alors davantage au rouleau qu'à la plaque elle-même.

2. La Nouvelle Recette "Sobre" : La Solution Centrale

Pour remédier à ces problèmes, la recette a été entièrement reformulée, la modification la plus importante étant le retrait de l'alcool, qualifiant la nouvelle plaque de "sobre".

2.1. Le Remplacement de l'Alcool par le Propylène Glycol

L'alcool est remplacé par le propylène glycol, décrit comme le "partenaire parfait" de la glycérine.

• Propriétés : Le propylène glycol appartient chimiquement à la famille des alcools, mais il est beaucoup moins volatil, ne s'évapore quasiment pas et présente un risque d'incendie significativement plus faible dans des conditions de cuisine normales.

• Bénéfices dans la recette :

◦ Stabilité : Il aide à rendre la plaque plus ferme et stable sans lui "voler toute son eau".  

◦ Rétention d'humidité : Il aide la plaque à rester flexible, à moins rétrécir et à conserver son humidité, ce qui garantit de belles impressions.   

◦ Conservation : Il contribue à ralentir la croissance des bactéries et des moisissures, agissant comme un agent de conservation.

• Conclusion de l'auteure : "Si je devais choisir entre 'Brûle bien' et 'Imprime bien'... je suis assez sûre que vous choisirez la plaque qui imprime parfaitement plutôt que le feu d'artifice dans la cuisine."

2.2. Expérimentations avec des Plaques "Fusion"

Des tests ont été menés sur des plaques "fusion" combinant les propriétés de la gélatine et d'agents gélifiants à base de plantes. Ces versions semblent résoudre nativement le problème de séchage de la peinture.

• Ingrédients testés :

◦ Gomme de xanthane   

◦ Konjac (ou glucomannane) : L'agent actif de la farine de konjac, connu pour son pouvoir gélifiant et épaississant extrême.

• Résultats préliminaires : Les plaques fusion semblent libérer plus de peinture sur le papier, laissant moins de résidus sur la surface. Les tests sont jugés "très prometteurs".

• Note : Une exploration plus approfondie de ces hydrogels est prévue dans une future vidéo.

3. Nouveaux Protocoles de Maintenance, de Stockage et de Réparation

La nouvelle approche s'accompagne de protocoles mis à jour pour entretenir, stocker et même réparer les plaques.

3.1. Réhydratation d'une Plaque Sèche

Une plaque devenue trop sèche peut être "ramenée à la vie" sans être refondue.

• Méthode : Un "bain" d'eau. La plaque est immergée dans l'eau pendant une durée allant de 3 à 48 heures, voire plus, jusqu'à ce qu'elle absorbe l'eau nécessaire et augmente de volume.

• Alternative : Si un contenant adapté n'est pas disponible, la surface peut être vaporisée d'eau, recouverte de papier essuie-tout humide et enveloppée dans un film plastique.

3.2. Nettoyage des Anciennes Couches de Peinture

Une découverte notable a été faite pour enlever les couches de peinture tenaces : La colle artisanale simple à base d'eau (colle blanche universelle) s'est avérée extrêmement efficace pour décoller les anciennes couches de peinture séchée de la surface de la plaque.

3.3. Nouvelle "Routine de Soins"

Un protocole de soins post-impression, comparé à une routine de soins pour la peau, est désormais recommandé pour préserver la plaque.

1. Nettoyage Doux : Vaporiser un spray nettoyant sur la plaque, essuyer avec un chiffon doux pour enlever les résidus de peinture.

2. Rinçage : Repasser sur la surface avec de l'eau claire pour éliminer tout tensioactif résiduel.

3. Hydratation et Protection : Masser une petite quantité d'un spray de soin sur la surface.

Les recettes pour ces sprays sont les suivantes :

Spray

Ingrédients (en grammes)

Instructions

Spray Nettoyant

- 500g Eau<br>- 2g Savon neutre<br>- 1g Alcool (pour dissoudre)<br>- 1g Huile essentielle (Arbre à thé ou Clou de girofle, optionnel)

Dissoudre l'huile essentielle dans l'alcool, ou directement dans le savon. Mélanger tous les ingrédients et verser dans un flacon pulvérisateur.

Spray de Soin

- 200g Eau<br>- 2g Huile pour bébé (huile minérale)<br>- 1g Huile essentielle d'arbre à thé<br>- 1g Huile essentielle de clou de girofle

Mélanger tous les ingrédients. Agiter vigoureusement avant chaque utilisation car le mélange est biphasique (l'huile se sépare de l'eau).

Le spray de soin laisse un "film protecteur huileux très fin" qui protège contre le dessèchement et rend la surface moins accueillante pour les microbes grâce aux propriétés des huiles essentielles.

3.4. Recommandations de Stockage Mises à Jour

• Ancienne recommandation (pour les plaques avec alcool) : Ne pas stocker dans un contenant hermétique les premiers jours pour permettre à l'humidité de s'échapper et éviter un "microclimat tropical" propice aux moisissures.

• Nouvelle recommandation (pour les plaques "sobres" avec routine de soin) : Stocker dans un contenant hermétique dès le début.

Cette approche est jugée optimiste pour minimiser la perte d'eau, les précautions étant prises par la routine de soin antimicrobienne.

4. Outils et Méthodologies de Précision

Pour améliorer la fiabilité et la reproductibilité des résultats, de nouvelles méthodologies ont été introduites.

4.1. Passage aux Mesures en Grammes

Toutes les nouvelles recettes sont désormais formulées en grammes plutôt qu'en unités de volume.

• Raison : La précision est cruciale, en particulier avec les agents gélifiants végétaux où "un demi-gramme de plus ou de moins peut déjà faire une énorme différence".

• Avantage pratique : Il devient très simple de calculer la perte d'eau lors de la refonte d'une plaque.

Il suffit de peser la plaque usagée, de comparer son poids au poids total initial des ingrédients, et d'ajouter la différence en eau lors de la refonte pour la restaurer à son état optimal.

4.2. Le Calculateur de Recette 2.0

https://ashrey.com/diy-gel-plate/

Un nouvel outil en ligne, le "Calculateur de Recette 2.0", a été développé.

• Fonctionnalités :

◦ Fonctionne entièrement en grammes.  

◦ Prend en compte la force de la gélatine (valeur de Bloom).   

◦ Permet de dimensionner les recettes précisément à la taille de plaque souhaitée.   

◦ Offre le choix entre différents types de plaques : standard, plus souple, ou la version expérimentale "fusion" avec hydrogel.

• Disponibilité : L'outil est accessible sur le site web de l'auteure. Le calculateur classique (en unités métriques et impériales) reste également disponible.

5. Diagnostic et Solution pour le Caillage de la Peinture

Le mystère du comportement anormal de la peinture acrylique a été résolu.

• Diagnostic : La peinture acrylique n'aime pas les environnements acides. Un pH bas provoque son caillage et la rupture de son système liant.

• Action à éviter : Ne pas ajouter d'acides (jus de citron, acide citrique) comme conservateurs dans le mélange de la plaque de gélatine.

• Solution de Réparation ("Fix d'Urgence") : Pour une plaque déjà acide, il est possible de neutraliser sa surface.

1. Préparer une solution alcaline douce : Dissoudre 2 à 3 grammes de bicarbonate de soude (disponible sous des noms comme "Kaisernatron" en Allemagne) dans 1 litre d'eau.  

2. Appliquer : Verser ou vaporiser la solution sur la surface de la plaque.  

3. Attendre : Laisser agir pendant 30 à 60 secondes. 

4. Essuyer : Sécher la plaque, puis la nettoyer à nouveau avec de l'eau propre ou une lingette pour bébé.  

5. Répéter si nécessaire jusqu'à ce que la plaque fonctionne correctement.

People from all social levels, including everyday colonists, soldiers, and organizers, contributed to a major war for independence in the American colonies during the late 18th century, which later inspired movements for equality and rights across the following centuries.

group of people who came of age during the American Revolution and were actively involved in or deeply influenced by the fight for independence. This generation shaped the political, social, and cultural foundations of the new United States.

women in the early United States who were expected to raise their children especially sons to be virtuous, informed, and patriotic citizens. Their role was seen as essential for sustaining the republic, teaching civic values, and supporting the principles of liberty and self-government in the next generation.

Not all colonists supported the revolution—many stayed loyal to Britain or tried to remain neutral. As resistance grew in 1774–1775, divisions appeared, especially among elites like merchants, clergy, and royal officials who relied on ties to Britain. At first they tried to moderate the protests, but later some feared the resistance was becoming too radical and still hoped for a peaceful reconciliation.

describes the Boston Tea Party, where colonists boldly destroyed 342 chests of tea to protest British taxes. I like how it emphasizes their courage and carefulness they made a huge statement without damaging the ships. It really shows how direct action became a key part of colonial resistance.

This passage is really interesting because it shows how ordinary colonists and leaders worked together to resist Britain. The Boston Massacre, especially with Paul Revere’s engraving, turned a local incident into a story that united the colonies. It’s fascinating to see how communication and shared outrage helped build a sense of a bigger political community.

New resistance efforts united elites, middle-class, and working-class colonists through coordinated economic action. Merchants and common colonists agreed to boycott British goods, often signing public lists to pledge compliance. Publishing these lists in newspapers both recognized participants and pressured others to join the boycott.

Resistance to the Stamp Act took different forms based on class elites used legislative action, merchants used economic pressure, and common colonists engaged in popular protest.

Some colonists feared that British policies were part of a growing trend of higher taxes and limited freedoms. This perception of government overreach helped fuel revolutionary sentiment.

The American Revolution emerged largely from British attempts to reform and control its empire after the costly Seven Years’ War. Britain gained vast territories in North America and India but faced enormous debts and expenses to defend its empire. Efforts in the 1760s to consolidate control over the colonies sparked colonial resistance, setting the stage for revolution.

Colonial political culture differed from Britain’s because land was easier to obtain, allowing more men to participate in politics. Colonists embraced republicanism, influenced by Britain’s “country” party, emphasizing virtue, public good, and vigilance against corruption and centralized power. While these ideas were fringe in Britain, they became widespread in the colonies, shaping colonial political identity.

ritain struggled to define its relationship with the colonies between 1688 and the mid-18th century, failing to implement coherent imperial reforms. Continuous wars and competing visions of empire authoritarian Old Whigs/Tories versus trade-focused Patriot Whigs divided officials and distracted from effective governance. As a result, occasional reform attempts failed, leaving colonial management inconsistent and setting the stage for future tens

his highlights the lasting impact of the American Revolution beyond just military victory. The revolution created political institutions (like Congress) and a shared national identity, embedding ideas such as liberty, civic virtue, and rights into the cultural and political fabric of the U.S.

This demonstrates the deep emotional and symbolic attachment many colonists had to Britain, highlighting how loyalty and admiration for the monarchy made the eventual turn toward revolution appear improbable. It sets the stage for understanding the dramatic nature of the Revolution as a shift in identity and allegiance.

bilingual shows identity

language reflects power and it is not just for communication.

personal experience caused harm and it was forced english immersion

The debate over bilingual education is about america's identiity and if multilingualism should be valued

Tom’s presence disrupts the carefree atmosphere of Gatsby’s parties and bring tension.

Gatsby learns early on how charm and ambition can help him reshape his identity and move closer to his dreams.

yess even gastby sees daisy inside , i don;t get it why tom ignore her

even nick sees his emotion,he saw the interaction between daisy and gastby

yess they are finally interacting,i can tell gastby pretend to be calm

Gatsby rejects his real background and creates a new identity based on an idealized version of himself rather than reality.

This sentence reveals how unstable Gatsby’s self-narrative has become. His words no longer sound deliberate but defensive and automatic. It suggests that the persona he performs is slipping beyond his control.

インストール可能なPythonの一覧を出力する。が正しいです

List the available Python installations

This one seems to be the successor project: https://github.com/OHF-Voice/piper1-gpl/

Ways that you can draw your reader in. This could make your break the beginning of your essay.

Keep in mind

This was not used in this module nor was it mentioned as part of the leanring outcomes.

Shoudl the colour of this "correct" box be green like the other quizzes?

Is there a reaosn for the capital A?

is this a question? This is a sub-heading so remove the quesiton mark.

Tattoo ink enters the lymph nodes, where it can remain for life and cause chronic inflammation.

In mice, inflammatory markers were 5 times higher even 2 months after tattooing, and their bodies' response was weaker to mRNA vaccines and stronger to protein vaccines (e.g., influenza).

Macrophages overloaded with ink absorb fewer vaccine components. Studies of human lymph nodes have confirmed that pigment remains in them long after tattooing. Chronic inflammation can weaken the immune system, increasing the risk of infection and cancer.

In Europe, more than 60 million people have tattoos, and the ink may contain high levels of heavy metals that exceed acceptable standards.

this isuse edin one other module - the rest just have "Key takeaway" as the heading.

the rest of the module is all about teamwork. That appears to be the focus, not feedback.

I think the introduction could be more focused, particularly by shortening the background information on risk factors.

The unit of the age-standardized incidence rates (e.g., 26.7 vs 13.4) should be specified (for example, cases per how much population per year).

To Prof. Kassouf

Is this the email you want to be published in the paper?

only one period is correct.

Maybe we should add a phrase or an additional sentence here so readers do not jump to the conclusion that we have lost our way with regard to public health. not sure really

of BC in the (though obvious)

Michael Herrick specifically acknowledges the commonplace book as the source of inspiration for his Linkbook.

https://www.youtube.com/watch?v=rQ8U3yqKILM&list=PLWRUKnXAM0aJ4hM__GyzGrA1QZYxUJ7tc&index=10

Antithesis

simile

You'll notice that the links here point to much older, smaller numbers. 1013 3667. These are ideas from thousands of entries ago. ideas I'm still grappling with today. That's what I really love about my link book. The way old and new ideas collide on paper. That's where the real creative sparks happen. And that's why I say the linkbook is an innovation engine. It doesn't just store ideas. It helps them grow, interact, and evolve.<br /> —Michael Herrick [2:45](https://youtu.be/30_v2FHJ9e4?si=HclrmkAMnd6LVca_&t=165

Michael Herrick noticing what others have seen in the past. He doesn't give the idea a new name like he's done with "Linkbook" for commonplacing or various other iterations.

Honestly, I believe here the author is over-reacting.

Make sure to include all of these components ion your FIRST paragraph

A hook may be one of the most important parts of these kinds of papers. You do not want to bore your audience and deter them from reading your article.

Make sure to answer these questions when it comes to writing up your paper.

Definition of an informative research report. This is to relay results of a research question. Almost like an experiment but for text.

The article linked, talks about how many people do not have access to information. Information can be behind a paywall, or maybe the person lives in a place where they do not physically or financially have access to the internet.

a is agoraphobia but doesn't have to be in panic disorder c is seperation anxiety disorder and d is part of the ocd

Alcohol use Smoking and tobacco use having sufficient nutrition Exercising and sport *Attitude toward medical regiments(taking your meds and following the treatment etc.)

remember the movie we watched

When the person doesn't acknowledge the truth rather hide it and the y use defense mechanisms such as regression denial dipsplacement etc.

Free association The psychosexual stages Psychoanalysis Defense mechanisms *Id, ego, superego

He is an Austrain person who developed psychoanalysis. To him our unconscious mind also takes part in our daily life. Our dreams are reality we supress. We can make them conscious through free association. Also we have three parts : the id, the ego and superego. The conflicts betweeen them result in anxiety.Which we use defense mechanisms to cope wtih

You'll need to re-enable USB debugging at this point:

Enable USB/ADB debugging on the phone by typing ##DEBUG## (##33284##).

This seems to be something supported by the model provider. Something like saving the processing of previously sent input tokens. Like saving a context document to answer questions about it that a user may sent in different API requests.

This is what is submitted to the model apart from the user message.

the problem with beurocracy

URL form for a POST call to my Mastodon endpoints. Form submission with just status is enough. Repurpose jsonclassic.php for it.

Caspar has a EU focus.

use passion and facts and don't create unanswered questions

linking verb?

steer clear from jokes as not everyone shares a sense of humor

question, prove, revise, repeat

speak/tell your story with confidence

A thesis statement should make your argument and purpose clear

A thesis statement is like a condensed preview from the back of the book. It tells readers what to expect and portrays the message the author is trying to share

Fn-Control-F: Expand the active window to fill the desktop.

Fn-Control-F: Expand the active window to fill the desktop.

should state that bulk delete does not throw exception, it returns a list of errors.

only transformer that needs the ctx

wrong. use: .gdpr(gdprSpec -> gdprSpec .uouAndUserSupport(AppDefId, contextBuilder.secrets().appSecret()) ) before the change it is: .gdpr(gdprSpec -> gdprSpec .userSupportOnly(AppDefId, contextBuilder.secrets().appSecret()) )

fix to change in assert function not in the call

add note to put in a new trnaslators file, because when in class mappers the transformers aren't yet build so refrence to the transformer is unknown.

dont need to erase the existing, and also Void.class is unnecessary

need to add the step to run the bazel generate model command (in scala exists)

for - public good - being eroded

• SRG comment - public good being eroded - reference
• to - the Volcker Shock - Anna Bocca - How the Rich took over the Economy
  • https://via.hypothes.is/https://www.youtube.com/watch?v=gAV0bkTHui8
  • Anna explains how all this privatization began in the video

for

• SRG comment - this is also explained in Anna Bocca's video
• to - the Volcker Shock - Anna Bocca - How the Rich took over the Economy
  • https://via.hypothes.is/https://www.youtube.com/watch?v=gAV0bkTHui8

for - Our World in Data - discredited website - mix legitimate with illegitimate research to advance a biased ideology

for - progress champion - Steven Pinker - discredited - one example of many - outright lie - he says violence committed BY indigenous Uruguay people but it was colonialist violence done TO THEM!

for - AI - ponzi scheme - keep the progress narrative alive - while everything else is falling apart

for - history - progress - Francis Fukuyama - liberalism won - calling the bluff

for - history - progress - secular golden age promise - can procastinate by pushing it forward to the next generation

for - history - progress - religious golden age - is post death

for - history - progress - promised land - contemporary mythical promises - nothing new

for - observation - anti-science blowback

• SRG comment- anti- science blowback - competing narratives now out in the open, gaining momentum

for - history - Trump - back into age of religosity. He claims, like former kings, he was anointed by god!

for - trivia - philosopher John Locke - investor in slave trade and native american dispossesion!

• SRG comment - John Locke - How "enlightened!"

for - history - progress - after Enlightenment - no long about converting savages to Christians - became about expanding markets

for - history - progress - European Enlightenment - broke secular off from religious progress very abruptly

for - history - progress - secular vs religious

for - history - progress - expansionism and gods - went hand-in-hand

for - history - progress - political and religious partnership narrative - top leader claimed to represent top god

for - history - progress - polytheistic gods - Roman and Greek - Jupiter & Zeus

for - history - progress - animism - gave way to polytheism in Mesopotamia - Marduk god

for - progress narrative - often accompanied by religious undertones

for - book - tracing history of progress / Growthist political economy narrative from Vikings to Mesopotamia to Judaism to Greeks to Islam to Enlightenment to US

for - youtube - Planet Critical interview - Samuel Miller MacDonald - The Myth of Progress

• SRG comment - interview - The Myth of Progress - Samuel Miller McDonald
  • Samuel summarizes key points from his research and book:
    • Progress: Humanity's Worst Idea
  • He discusses
    • what progress should be,
    • why it is so vital, and
    • how little time we really have to achieve

for - SRG comment - join us - MEconomy - forces us all into silos for survival

for - progress - myth of - 5000 years ago

The's is not an actual English word. In this case, it is probable that the singer means there are.

In this verse, as well as in the previous one, 2Pac establishes a relation of identity between blackness, restriction and stress: being Black inevitably entails a heightened sense of danger, an alert about one's surroundings that can never falter without risking life itself. If this theme, closely related to the body, interests you, I recommend reading Ta-Nehisi Coates' Between the World and Me, in which the author explores what being Black in 1990s Baltimore was like in a heartfelt letter to his son and, ultimately, to all members of the Black community.

Here, the condemnation towards the lack of equality and straight-up racism in America is explicit.

Given what has been already noted on the previous song, do you remember what is the war on drugs? Which President of the United States promoted it?

It means "What does a mother have to do?" This verse alludes to what has been examined in the "Setting the scene" section: the single-parent families.

What topic, already mentioned in the discussion, does this sentence point to?

What 2Pac considered impossible actually occurred in 2009, when Barack Obama was elected President of the United States and then again in 2012, when he was reelected. Nevertheless, his presidency was filled with conspiracy theories (especially revolving around his being born in the Unites States) and controversies that tried to undermine the figure of the President. Ultimately, this may suggest that the United States was not ready for a black President.

Setting the scene: the song was recorded in 1992 and released six years later in 2Pac's posthumous album Greatest Hits. It features Talent, an R&B trio formed by Ernest “Bishop” Dixon, Marlon “Castor Troy” Hatcher and Keith “Casino” Murrell. As mentioned earlier, the song samples Bruce Hornsby and the Range's The Way It is and addresses the same social themes. A dive into the historical context: what happened in the 1990s? * A new President, the Democratic Bill Clinton, was elected and stayed in charge from 1993 to 2001. in 1996, he promoted the Personal Responsibility and Work Opportunity Reconciliation Act which restricted governmental assistance to families in distress. * In the early 1990s, the country entered a severe recession, which was apparently overcome by the mid-1990s. * Black businesses bloomed and Black celebrities (such as Micheal Jackson, Michal Jordan and Oprah Winfrey) started to appear and constituted the first "Black bourgeoisie". However, this was not the reality of common, middle or lower-class African-Americans: in some inner cities, between 30% and 43% of the population in poor neighborhoods (such as East New York, the South Bronx, South Central Los Angeles, Chicago's South Side) was jobless. Many of those "who had once held stable blue-color jobs, low-wage service jobs, such as in the fast-food industry" relied on occasional and informal activities (braiding hair, childcare, car repair...). * The abuse of drugs that had exploded the previous decade went on, especially among poors. * Mass incarceration persisted: in 1990, prisoners reached one million. "By 2000, one-third of all black males in their twenties were under the control of the criminal justice system—either in prison or jail, on parole, probation, or awaiting trial. The major reason for this disproportion in incarceration is the stark racism that continues to pervade the criminal justice system. […] The socio-economic and political consequences of mass incarceration for the black community have been profound. Hundreds of thousands of households have been destroyed". (https://www.amistadresource.org/the_future_in_the_present/social_and_economic_issues.html). As a consequence, in many instances, there were one-parent families: mothers were left to raise their children alone. https://www.amistadresource.org/the_future_in_the_present/social_and_economic_issues.html https://www.ncpedia.org/anchor/united-states-1990s#:~:text=Labor%20unions%20believed%20it%20would,countries%20with%20weak%20pollution%20controls

Although in the previous verse, Huey Newton seems to be a source of inspiration for the singer, hope seems to be nowhere to be found in this verse. As any African-American, even Huey ends up being another victim of police brutality. Considering the bigger picture, it can be hypothesized that 2Pac was even questioning the actual impact and ideals that animated the Black Panther Party itself.

There is a missing word here: what is it? Clue: the following verb is in the present tense, so...

Do you notice anything strange in this line? For an answer, see the comment on the line "My mama didn't raise no fool".

In this disheartening scenario, 2Pac seeks refuge in the pleasant memories of an idealized childhood, which is perceived as the only moment in space and time where he may find solace.

In the society in which the narrative voice lives, both poverty and blackness have a negative connotation. In other words, being Black adds up to being poor.

This is directly connected to what has been said about the pervasiveness of drugs (of crack, in particular) among poor families.

The verb blast can have multiple meanings; in this case, the most probable is shoot (sparare in Italian) https://www.merriam-webster.com/dictionary/blast#dictionary-entry-2

Note I skipped a large chunck of fairly unrelated history about sports, war, and measurement that predated videogames. Recently, there has been a large increase in data collection devices which is much more interesting.

The datafication from phones, car GPS, AI, and radar war vehicle detection is much more in-line with game interfaces and game positivist metrics, KPI, retention, etc. Some of these have been instrumentalised for gamification, or speedruns too, like deaths/coins per-level, or playtime/speed.

This is also inherited from inherited from Industrial school tests, IQ, Normality, Matrices, and the tabular accounting quantification of money, born in imperialist countries and urban gangs that tried to keep track of trading stocks.

Arguably, per Kropotkin, this could be a form of rationing and a way to promote worker-guild control, so as to avoid top-down impositions from cartel pricing... which goes to show how far we've branched from games, and how economics the Enlightment science, and quantification aren't the devil, rather they have been repurposed as such, mainly through the industrial education system pipeline, and illusion of explanatory depth specialisation (which is harder to disentangle with ellusive numeric objectivist abstractions).

There, you don't need to read those 15 pages.

Procedural generation games. Roguelikes, if you wish.

A similar thing happened with fishing, you can see the book "Animals in Video Games and Humanity".

As different from football or other popular sports this is meant as a "private" and "individual" passtime, initially only for the burgeoise (then gun culture spread to all America).

What follows is only partly new, but mainly no. Sports as at the work level are hardly games. People there don't play, they train, they work, they compete. Sports as competition is football, basketball, you may know more ancient forms of them... and they have rules, referees, time, and metrics like assists, goals, even dating very far back.

The olympics of wrestling, of lifting, or ball throwing. They had metrics in the past, in Greece, in Rome. This ain't new, come on.

The point being, there is exogenous, or what is forced from outside, and endogenous, or what is forced from inside. UK, France, Spain, etc. were and are colonialists countries that forced upon others their empire, and they still do, but in a more palatable less visibly violent fashion.

They opress, homogenise, silence, displace, much like the US, Mainland China, India, or Mexico. Their internal dissidents, varied ideologies, immigrants, subcultures, languages, natives, are squished, minoritised, colonised.

Yes, arguably that is the game's goal, but as I see it, most of the game's players are actually very far from being mindless colonialists. They are not warmongers either. Instead, I see historians, economists, people that may be white, sure, but that are also concerned with whiteness, that try to tackle it by explaining it. Strategy games demand systems thinking that is somewhat incompatible with reductionist us vs them narratives.

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Reply to the reviewers

Manuscript number: RC-2025-03091

Corresponding author(s): Chia-Tsen, Tsai, Liuh-Yow Chen

1. General Statements [optional]

We thank the reviewers for their valuable time and constructive feedback on our study, which ultimately improved our manuscript. Herein, we provide a detailed response to each of the reviewers' comments, supported by new data that have been integrated into both the main text and the supplementary figures.

2. Point-by-point description of the revisions

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

Summary This manuscript builds upon the authors' prior findings that targeting COUP-TF2 to TRF1 induces ALT-associated phenotypes and G2-mediated synthesis in telomerase-immortalised BJT human fibroblasts. In this study, the authors show that telomere-coupled COUP-TF2 promotes H3K9me3 enrichment in these cells, and that this effect is blocked by TRIM28 depletion. Furthermore, TRIM28 depletion also suppresses the formation of ALT phenotypes in VA13 ALT cells. Given that TRIM28 has been implicated in regulating H3K9me3 deposition via SETDB1, and has been reported to co-purify with TR2 and TR4 (though not previously in the context of ALT telomeres), these findings add mechanistic depth to how heterochromatin regulators contribute to ALT activity. Overall, the manuscript's conclusions are generally supported by the presented data, but several aspects require clarification or additional experimental validation.

The authors report a modest reduction in telomeric H3K9me3 following COUP-TF2 and TR4 depletion in U-2 OS and VA13 cells (Figure 1B). To strengthen the claim that these orphan receptors specifically regulate H3K9me3, the authors should 1) Assess additional heterochromatic histone marks (e.g., H4K20me3) at telomeres, 2) Normalize telomeric signals to both parental histone levels and input, and 3) Evaluate whether global H3K9me3 levels also decrease upon receptor depletion

Response: We appreciate the reviewer's suggestion. To address the concern regarding specificity, we assessed H3K27me3 and H4K20me3 levels upon COUP-TF2/TR4 depletion and found no significant changes (Supplementary Fig. 1C). Furthermore, we reprocessed the telomeric ChIP data, normalizing to both input DNA and parental histone levels (Figure 1B). This refined analysis reinforces our original conclusion. Finally, Western blot analysis showed no significant changes in global H3 or H3K9me3 levels upon COUP-TF2/TR4 depletion (Figure 1A). Altogether, these results further support the specificity of COUP-TF2/TR4 for H3K9me3 at telomeres. We have revised the main text (page 3) and updated Figure 1A, 1B, and Supplementary Figure 1C for these changes.

Most experiments explore chromatin changes in telomerase-positive BJT fibroblasts (Figure 2, Figure 4D). It remains unclear whether similar manipulations in ALT cells yield consistent effects, which would give a broader context for ALT phenotype induction. Are ALT phenotypes similarly induced in ALT cells? Does altered chromatin status affect telomere length or telomerase recruitment/activity? Can these pathways drive ALT phenotypes in non-immortalised cells?

Response: We appreciate the reviewer's suggestion and have explored chromatin changes in telomerase-negative BJ and IMR90 primary fibroblasts (Supplementary Fig. 2C, D). Consistent to the result in BJ-telomerase cells, we found that VP64-TRF1 decreased telomeric H3, H4, and H3K9me3 levels, whereas KRAB-TRF1 increased these marks. Moreover, expression of either VP64-TRF1 or KRAB-TRF1 was sufficient to induce APB formation and ATDs in BJ and IMR90 cells. These results indicate that the chromatin changes at telomeres can drive ALT phenotypes in both primary and telomerase-immortalized fibroblast cells.

    Additionally, regarding whether chromatin alteration affects telomere length or telomere regulation, we have explored telomere length changes in BJT cells expressing vector, TRF1, KRAB-TRF1 or VP64-TRF1. The result of telomere restriction fragment (TRF) assay showed that the cells of all conditions maintained static telomere lengths through 30 days in culture (data shown below), suggesting that the chromatin alterations may not impact telomerase recruitment or activity. As this result is beyond the scope of current study, this data is only shown here in the rebuttal letter for a reference and is not included in the revised manuscript.

    Moreover, according to the reviewer's suggestion, we also carried out VP64-TRF1 or KRAB-TRF1 expression experiments in WI38-VA13/2RA cells that express high TERRA and have altered chromatin structures. Our data revealed that VP64-TRF1 suppresses telomere H3K9me3 and ALT activity, while KRAB-TRF1 increases both (Supplementary Figure 2E), suggesting an association of heterochromatin state with ALT activation in WI38-VA13/2RA cells.

    The observation that VP64-TRF1 reduces ALT activity in WI38-2RA/VA13 cells contrasts with findings in BJT cells. It is worth noting that studies from the Azzalian and Linger groups demonstrated that experimentally induced TERRA expression promotes ALT activity in ALT and non-ALT cells (PMID: 36122232, PMID: 40624280). Therefore, we propose that TERRA upregulation by VP64-TRF1 may contribute to the ALT induction observed in BJT cells (Supplementary Figure 2A, B), whereas the ability of VP64-TRF1 to suppress ALT activity in WI38-2RA/VA13 cells could be attributed to the reduction of telomere H3K9me3 and heterochromatin loss. Importantly, KRAB-TRF1 concurrently enhanced histone H3, H4, and H3K9me3 occupancy and ATL activity in both human fibroblasts and ALT cells. Altogether, these results support the notion that heterochromatin formation triggers ALT.

    We also examined TRIM28 recruitment to telomeres by telomere-ChIP and found that COUP-TF2LBD-TRF1 promotes TRIM28 telomere enrichment in BJ, IMR90 and U2OS, similar to BJT cells (Supplementary Fig. 5A-D).  Moreover, in ALT cell lines WI38-2RA/VA13, U2OS, and Saos-2, depletion of COUP-TF2 or TR4 reduced TRIM28 telomeric association (Figure 4A, B). Together, the data from human fibroblasts and ALT cells supports a role of orphan NRs in recruiting TRIM28 to ALT telomeres.

We acknowledge the reviewer's suggestions, which allow us to clarify and strengthen the conclusions. The corresponding data are presented in Figure 4A-B and Supplementary Figure 2B-D and 5E-F, and the main text has been modified on page 4-6 in the revised manuscript.

When referring to Figure 3G, the authors state that that telomeric H3K9me3 was abolished upon depleting TRIM28 from the U2OS and WI38-VA13/2RA cells. Abolished is a strong word for a 50% decrease, and this sentence should be revised. The reduction appears greater than that seen with COUP-TF2/TR4 depletion. Are the effects additive? If so, might TRIM28 act, at least in part, independently of COUP-TF2/TR4?

Response: We appreciate the reviewer's comments. We have revised the manuscript on page 5, replacing "abolished" with "significantly reduced" to better describe the effect of TRIM28 depletion on telomeric H3K9me3. To further investigate the interplay between TRIM28 and orphan NRs in regulating telomeric H3K9me3, we conducted single and combined knockdown experiments in U2OS and WI38-VA13/2RA cells, followed by telomere-ChIP analysis (Supplementary Figures 4D, E). Our results showed that single depletion of either orphan NRs or TRIM28 lead to a similar decrease in telomeric H3K9me3, and that combined knockdown do not result in any further reduction. These findings support an epistatic interaction between orphan NRs and TRIM28 in the regulation of telomeric H3K9me3. We have expanded on this interpretation in the main text (page 6) and included the relevant data in Supplementary Figures 4D, E.

VA13 cells consistently exhibit stronger effects than U-2 OS (e.g., Figures 1 and 3). This discrepancy could be linked to the high content of variant repeats in VA13 cells. The authors should assess whether variant repeat content underlies the differential response. Repeating key experiments in additional ALT lines with varied repeat compositions would be informative.

Response: We appreciate the reviewer's suggestion and have extended our analyses to two additional ALT osteosarcoma cell lines, SAOS-2 and G292. In both lines, depletion of orphan NRs resulted in a consistent decrease in telomeric H3K9me3 levels (Supplementary Figures 1A, B). We also examined the contribution of TRIM28 to telomeric H3K9me3 in these cells. siRNA-mediated depletion of TRIM28 in SAOS-2 and G292 cells similarly caused a significant reduction in telomeric H3K9me3 and ALT phenotypes (Supplementary Figure 4A-C). Together, these results from 4 ALT cell lines confirm that orphan NRs and TRIM28 promote telomeric H3K9me3 formation in ALT cells. We have modified the main text on page 3 and 5-6 for these results.

In line with the previous point, it would be useful to show whether TRIM28 telomeric enrichment is affected by COUP-TF2/TR4 depletion in U2OS cells (Figure 4C). To improve confidence in these findings, the authors should perform telomeric ChIP assays, especially with the COUP-TF2^LBDΔAF2-TRF1 mutant construct.

Response: Following the reviewer's suggestion, we performed telomere-ChIP assays to assess TRIM28 enrichment at telomeres upon expression of COUP-TF2LBD-TRF1 and its ΔAF2 mutant in U2OS cells. Consistent with our immunofluorescence results, telomere-ChIP revealed that COUP-TF2LBD-TRF1 expression promotes TRIM28 telomere enrichment, while the AF2 deletion mutant failed to recruit TRIM28 (Supplementary Figure 5D). We have modified the main text on page 6 for this result.

The immunoprecipitation experiments showing TRIM28 association with orphan receptors should include benzonase treatment to rule out DNA-mediated co-association (Figure 4F-G).

Response: We appreciate the reviewer's suggestion. To address the possibility of DNA-mediated interactions, we pre-incubated cell lysates with benzonase prior to Co-IP (Page 7). This treatment did not disrupt the association between TRIM28 and COUP-TF2 or TR4 in WI38-VA13/2RA and BJT cells (Supplementary Figures 5E-G), indicating a DNA-independent interaction. We have modified the main text on page 7 for this result.

The study would benefit from a direct assessment of whether COUP-TF2LBDΔAF2-TRF1 fails to induce ALT phenotypes in BJTfibroblasts.

Response: We thank the reviewer for this suggestion. As the role of the COUP-TF2 AF2 domain in ALT induction in BJT fibroblasts has recently been thoroughly investigated and published by our group (PMID: 38752489), we have directed the current study towards a more detailed mechanistic question. Specifically, we have carried out experiments to further demonstrate that COUP-TF2 recruits TRIM28 to telomeres via its AF2 domain in both human fibroblasts and ALT cells (Supplementary Figures 5A-D). On Page 6, we have modified the main text for these results and included a citation to our previous publication to provide the necessary background.

The experiments performed in Figure 5E-H lack a vector-only + siCtrl control.• In Figure 5E, the observation that APB formation is restored in siTRIM28 + Vector-treated cells is unexpected. The authors should address this finding and clarify whether this reflects biological noise or a compensatory effect.

Response: We thank the reviewer for this suggestion. We have repeated the experiments with a revised design, ensuring a consistent vector background across all groups (Vector + siCtrl, Vector + siTRIM28, TRIM28 WT + siTRIM28, and TRIM28 ΔRBCC + siTRIM28) (Figure 5E-H). This improved design confirms that expression of wild-type TRIM28, but not TRIM28 ΔRBCC, restores APB formation, ATDS, ssTeloC, and telomeric H3K9me3 levels in TRIM28-depleted cells. The updated dataset also resolves the previous unexpected increase in APB formation in the siTRIM28 + Vector condition, which is now excluded. We have modified the main text accordingly on page 8.

Reviewer #1 (Significance (Required)):

This work offers valuable mechanistic insight into how COUP-TF2 and TRIM28 coordinate to regulate heterochromatin deposition and ALT phenotype formation. It adds to the growing understanding of chromatin-mediated telomere regulation. What remains unclear is how important this interaction is for ALT maintenance, as H3K9me3 is only moderately altered upon TRIM28 depletion in ALT cells. Depletion of TRIM28 has been shown previously to induce APB formation and telomere elongation in U-2 OS ALT cells (Wang et al., 2021), the opposite to what the authors observed here in VA13 cells (Figure 5E-H). Clarifying whether these differences are variant repeat-dependent, or reflect intrinsic features of specific ALT cell lines, would substantially elevate the study's impact.

Response: We appreciate the reviewer's recognition of the significance of our work in elucidating the molecular basis of ALT regulation through COUP-TF2-TRIM28-mediated heterochromatin formation. In response to the reviewer's insightful comment regarding the importance of this interaction for ALT maintenance, we have expanded our study. We now include data from three additional primary human fibroblasts and a total of four ALT cancer cell lines (Figure 4, Supplementary Figure 4). These new data further strengthen the conclusion that TRIM28 promotes telomeric H3K9me3 and ALT-associated features. Furthermore, our rescue experiments support the model that the ALT-promoting function of TRIM28 in both fibroblasts and ALT cell lines is mediated through its physical interaction with COUP-TF2 (Supplementary Figure 5). We believe these results provide a solid foundation for demonstrating a cooperative role of COUP-TF2 and TRIM28 in ALT maintenance, and address the reviewer's concern regarding the generalizability of our findings.

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

Summary This manuscript investigates the role of orphan nuclear receptors (ORs), specifically COUP-TF2 and TR4, in promoting H3K9me3 enrichment at ALT telomeres via recruitment of TRIM28 (KAP1). The authors propose that the AF2 domain of COUP-TF2, located in its ligand-binding domain (LBD), is sufficient to recruit TRIM28 to telomeres. This, in turn, promotes heterochromatinization and induces hallmarks of the Alternative Lengthening of Telomeres (ALT) pathway, including APB formation and telomeric DNA synthesis outside of S-phase. This study addresses one important and unresolved question in the field: by what mechanism is the heterochromatic state established at ALT telomeres? Another timely question, not addressed here is: how is heterochromatin (specifically H3K9me3) functionally linked to ALT? The findings are potentially novel and mechanistically insightful. However, key elements of the study, particularly the central tethering experiments, require stronger quantification and clarity. Additional mechanistic tests and literature adjustments would also improve the manuscript.

Major Concerns

Central TRF1-COUP-TF2-LBD result lacks quantification and clarity: the tethering of COUP-TF2's LBD to telomeres via TRF1 is a core result of the paper. This experiment demonstrates that this domain is sufficient to induce weak H3K9me3 enrichment and ALT features (APBs and ATDS). However, the supporting ALT data are presented only in Supplementary Figures S1A and S1B, and are not quantified. These data should be quantified with appropriate statistics and moved to a main figure.

Response: The current study builds upon our recent publication (PMID: 38752489), which comprehensively analyzed ALT induction (APBs, ATDS, C-circles, T-SCEs) by orphan NR-TRF1 expression (COUP-TF1, COUP-TF2, TR2, and TR4; full-length and LBD) in various human fibroblast cell lines. To avoid potential duplicate publication concerns, particularly regarding APB and ATDS results for COUP-TF2LBD-TRF1 in BJT cells, we have put the data with revised quantification results in Supplementary Figure 1D-E. We will follow the reviewer's suggestion and move this data to the main figures if the editors agree.

Furthermore, the broader functional implication is not explored. Does this tethering induce a fully functional ALT pathway? For example, can telomerase knockout cells expressing TRF1-COUP-TF2-LBD maintain long-term proliferation? Such evidence would significantly strengthen the impact of the study.

Response: While COUP-TF2LBD-TRF1 expression rapidly induces key ALT phenotypes, we acknowledge that this alone is insufficient to directly promote telomere lengthening and long-term proliferation of primary fibroblasts, as discussed in Gaela et al., 2024 (PMID: 38752489). However, our ongoing, unpublished studies indicate that COUP-TF2LBD-TRF1 can drive immortalization of primary BJ fibroblasts expressing SV40LT by promoting ALT-mediated telomere elongation (Attached Figure A-C; additional data not shown). These findings suggest that COUP-TF2 may cooperate with additional genetic or epigenetic alterations to facilitate ALT development. We appreciate the reviewer's recognition of this critical aspect. As our immortalization study is still in progress and will be the subject of a separate manuscript, we hope the reviewer understands that the data shown in this letter will not be included in the revised manuscript.

Chromatin manipulation experiments lead to ambiguous conclusions: the authors propose that telomeric heterochromatin promotes ALT activity, but their own experiments (e.g., Figure 2) show that both heterochromatin-inducing (KRAB-TRF1) and euchromatin-inducing (VP64-TRF1) tethering can trigger ALT-like features. This makes it difficult to conclude that heterochromatin is specifically required.

To clarify:

-Did the authors express TRF1-VP64 in an ALT cell line? According to their model, this should suppress ALT activity.

-More broadly, do chromatin alterations per se (regardless of direction) trigger ALT features? Clarifying these points is important for interpretation.

Response: In response to the reviewer's suggestion, we expressed VP64-TRF1 and KRAB-TRF1 in WI38-2RA/VA13 cells to investigate telomere chromatin changes and ALT activity. Our data indeed revealed that VP64-TRF1 suppresses telomere H3K9me3 and ALT activity, while KRAB-TRF1 increases both (Supplementary Figure 2E), suggesting that heterochromatin triggers ALT activation.

The observation that VP64-TRF1 reduces ALT activity in WI38-2RA/VA13 cells contrasts with findings in BJT cells. Of note, studies from the Azzalian and Lingner groups demonstrated that experimentally induced TERRA expression promotes ALT activity in ALT and non-ALT cells (PMID: 36122232, PMID: 40624280). Therefore, we propose that TERRA upregulation may contribute to the ALT induction observed in BJT cells (Figure 2A, Supplementary Figure 2A, B). Given the high basal TERRA expression, expression of VP64-TRF1 and KRAB-TRF1 did not result in a consistent change in TERRA levels (Supplementary Figure 2F). Thus, the ability of VP64-TRF1 to suppress ALT activity in WI38-2RA/VA13 cells could be attributed to the reduction of telomere H3K9me3 and heterochromatin loss. Altogether, our results support the hypothesis that heterochromatin formation, rather than euchromatin triggers ALT.

We thank the reviewer's insightful comments, which have allowed us to resolve the ambiguity of our results and strengthen the notion that heterochromatin formation promotes ALT. We think that the heterochromatin features and high TERRA expression represent two independent, coexisting mechanisms within ALT cancer cells to guarantee ALT activation. We have modified the main text on page 4-5 accordingly.

TERRA downregulation contradicts current models: while TERRA upregulation is often observed in ALT cells and is thought to contribute to replication stress and recombination at telomeres, the authors show that TRF1-KAP1 expression induces ALT features while TERRA is downregulated. This observation is not addressed in the manuscript. The authors should at least discuss this discrepancy and propose whether this reflects a cell line-specific phenomenon or a decoupling between TERRA levels and ALT induction in this context.

Response: We thank the reviewer for the comments. As mentioned above (Major Concerns 2), heterochromatin formation and TERRA expression are two mechanisms that can independently promote ALT. Unlike ALT cell lines that have high TERRA levels, human fibroblasts BJ cells have low TERRA that does not induce ALT phenotypes. Thus, the effect of KRAB-TRF1 on ALT induction in BJ cells could be attributed to the heterochromatin formation, but not reduction of TERRA. We have modified the main text on page 5 to clarify the result.

Minor Comments

Introduction (p. 3): The authors cite Episkopou et al. as showing increased H3K9me3 at ALT telomeres. This is incorrect; that paper suggests the opposite. The first study to clearly demonstrate H3K9me3 enrichment at ALT telomeres is Cubiles et al., 2018 and should be cited instead. Results (p. 5, first paragraph): The manuscript should cite Déjardin and Kingston, 2009 as the first to report COUP-TF2 and TR4 localization at ALT telomeres. The studies by Conomos et al., 2012 and Gaela et al., 2024 build on this prior evidence. Please also include this citation in the bibliography.

Response: We appreciate the reviewer's careful reading and for pointing out these errors. The citation errors on pages 2 and 3 have now been corrected.Broader relevance of TRIM28-OR interaction: TRIM28 is a complex protein with roles in SUMOylation, heterochromatin formation, and transcriptional initiation/elongation regulation.

The authors should explore whether similar COUP-TF2/TRIM28 interactions occur at other genomic loci. Public ChIP-seq data for COUP-TF2, TR4, and TRIM28 could be mined to investigate whether these factors co-occupy regulatory regions elsewhere in the genome, and how this relates to gene expression states.

Response: We appreciate the reviewer's insightful suggestion regarding a potential genome-wild functional interaction between TRIM28 and COUP-TF2. To address this, we analyzed public ENCODE ChIP-seq data from K562 cells (TRIM28: ENCSR000BRW; COUP-TF2: ENCSR000BRS). This analysis revealed 3,326 co-binding sites for TRIM28 and COUP-TF2 (Attached Figure A). Interestingly, these co-binding sites were preferentially located within gene bodies (70.7%) and promoter regions (4.3%) (Attached Figures B-D), suggesting a potential cooperative role in gene regulation that aligns with our observation of physical interaction. While the finding is intriguing, a full exploration is beyond the scope of this manuscript, which focuses on ALT telomere regulation. We consider this is an important insight and have briefly noted it in the discussion (p. 9), although the corresponding analyses are not included in the revised manuscript.

Reviewer #2 (Significance (Required)):

This work contributes mechanistic insight into how heterochromatin is established at ALT telomeres-an important and timely question in telomere biology and cancer research. It offers a noncanonical recruitment mechanism for TRIM28, independent of KRAB-ZNFs, and highlights the functional role of orphan nuclear receptors in telomeric chromatin regulation. The study has potential implications for understanding ALT regulation and for identifying new intervention points in ALT-positive cancers. The work is conceptually interesting, but the conclusions are currently limited by insufficient quantification, some interpretative ambiguities, and a few overlooked references. Addressing the concerns listed above would significantly enhance the rigor and impact of the manuscript.

Response: We appreciate the reviewer's recognition of the significance of our work in elucidating the molecular basis of ALT regulation through COUP-TF2-TRIM28-mediated heterochromatin formation. We also thank the reviewer for the valuable feedback, which has significantly strengthened our manuscript.

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Referee #2

Evidence, reproducibility and clarity

Summary

This manuscript investigates the role of orphan nuclear receptors (ORs), specifically COUP-TF2 and TR4, in promoting H3K9me3 enrichment at ALT telomeres via recruitment of TRIM28 (KAP1). The authors propose that the AF2 domain of COUP-TF2, located in its ligand-binding domain (LBD), is sufficient to recruit TRIM28 to telomeres. This, in turn, promotes heterochromatinization and induces hallmarks of the Alternative Lengthening of Telomeres (ALT) pathway, including APB formation and telomeric DNA synthesis outside of S-phase. This study addresses one important and unresolved question in the field: by what mechanism is the heterochromatic state established at ALT telomeres? Another timely question, not addressed here is: how is heterochromatin (specifically H3K9me3) functionally linked to ALT? The findings are potentially novel and mechanistically insightful. However, key elements of the study, particularly the central tethering experiments, require stronger quantification and clarity. Additional mechanistic tests and literature adjustments would also improve the manuscript.

Major Concerns

1. Central TRF1-COUP-TF2-LBD result lacks quantification and clarity: the tethering of COUP-TF2's LBD to telomeres via TRF1 is a core result of the paper. This experiment demonstrates that this domain is sufficient to induce weak H3K9me3 enrichment and ALT features (APBs and ATDS). However, the supporting ALT data are presented only in Supplementary Figures S1A and S1B, and are not quantified. These data should be quantified with appropriate statistics and moved to a main figure. Furthermore, the broader functional implication is not explored. Does this tethering induce a fully functional ALT pathway? For example, can telomerase knockout cells expressing TRF1-COUP-TF2-LBD maintain long-term proliferation? Such evidence would significantly strengthen the impact of the study.
2. Chromatin manipulation experiments lead to ambiguous conclusions: the authors propose that telomeric heterochromatin promotes ALT activity, but their own experiments (e.g., Figure 2) show that both heterochromatin-inducing (KRAB-TRF1) and euchromatin-inducing (VP64-TRF1) tethering can trigger ALT-like features. This makes it difficult to conclude that heterochromatin is specifically required. To clarify:
3. Did the authors express TRF1-VP64 in an ALT cell line? According to their model, this should suppress ALT activity.
4. More broadly, do chromatin alterations per se (regardless of direction) trigger ALT features? Clarifying these points is important for interpretation.
5. TERRA downregulation contradicts current models: while TERRA upregulation is often observed in ALT cells and is thought to contribute to replication stress and recombination at telomeres, the authors show that TRF1-KAP1 expression induces ALT features while TERRA is downregulated. This observation is not addressed in the manuscript. The authors should at least discuss this discrepancy and propose whether this reflects a cell line-specific phenomenon or a decoupling between TERRA levels and ALT induction in this context.

Minor Comments

Introduction (p. 3): The authors cite Episkopou et al. as showing increased H3K9me3 at ALT telomeres. This is incorrect; that paper suggests the opposite. The first study to clearly demonstrate H3K9me3 enrichment at ALT telomeres is Cubiles et al., 2018 and should be cited instead. Results (p. 5, first paragraph): The manuscript should cite Déjardin and Kingston, 2009 as the first to report COUP-TF2 and TR4 localization at ALT telomeres. The studies by Conomos et al., 2012 and Gaela et al., 2024 build on this prior evidence. Please also include this citation in the bibliography. Broader relevance of TRIM28-OR interaction: TRIM28 is a complex protein with roles in SUMOylation, heterochromatin formation, and transcriptional initiation/elongation regulation. The authors should explore whether similar COUP-TF2/TRIM28 interactions occur at other genomic loci. Public ChIP-seq data for COUP-TF2, TR4, and TRIM28 could be mined to investigate whether these factors co-occupy regulatory regions elsewhere in the genome, and how this relates to gene expression states.

Significance

This work contributes mechanistic insight into how heterochromatin is established at ALT telomeres-an important and timely question in telomere biology and cancer research. It offers a noncanonical recruitment mechanism for TRIM28, independent of KRAB-ZNFs, and highlights the functional role of orphan nuclear receptors in telomeric chromatin regulation. The study has potential implications for understanding ALT regulation and for identifying new intervention points in ALT-positive cancers.

The work is conceptually interesting, but the conclusions are currently limited by insufficient quantification, some interpretative ambiguities, and a few overlooked references. Addressing the concerns listed above would significantly enhance the rigor and impact of the manuscript.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #1

Evidence, reproducibility and clarity

Summary

This manuscript builds upon the authors' prior findings that targeting COUP-TF2 to TRF1 induces ALT-associated phenotypes and G2-mediated synthesis in telomerase-immortalised BJT human fibroblasts. In this study, the authors show that telomere-coupled COUP-TF2 promotes H3K9me3 enrichment in these cells, and that this effect is blocked by TRIM28 depletion. Furthermore, TRIM28 depletion also suppresses the formation of ALT phenotypes in VA13 ALT cells. Given that TRIM28 has been implicated in regulating H3K9me3 deposition via SETDB1, and has been reported to co-purify with TR2 and TR4 (though not previously in the context of ALT telomeres), these findings add mechanistic depth to how heterochromatin regulators contribute to ALT activity. Overall, the manuscript's conclusions are generally supported by the presented data, but several aspects require clarification or additional experimental validation.

• The authors report a modest reduction in telomeric H3K9me3 following COUP-TF2 and TR4 depletion in U-2 OS and VA13 cells (Figure 1B). To strengthen the claim that these orphan receptors specifically regulate H3K9me3, the authors should 1) Assess additional heterochromatic histone marks (e.g., H4K20me3) at telomeres, 2) Normalize telomeric signals to both parental histone levels and input, and 3) Evaluate whether global H3K9me3 levels also decrease upon receptor depletion
• Most experiments explore chromatin changes in telomerase-positive BJT fibroblasts (Figure 2, Figure 4D). It remains unclear whether similar manipulations in ALT cells yield consistent effects, which would give a broader context for ALT phenotype induction. Are ALT phenotypes similarly induced in ALT cells? Does altered chromatin status affect telomere length or telomerase recruitment/activity? Can these pathways drive ALT phenotypes in non-immortalised cells?
• When referring to Figure 3G, the authors state that that telomeric H3K9me3 was abolished upon depleting TRIM28 from the U2OS and WI38-VA13/2RA cells. Abolished is a strong word for a 50% decrease, and this sentence should be revised. The reduction appears greater than that seen with COUP-TF2/TR4 depletion. Are the effects additive? If so, might TRIM28 act, at least in part, independently of COUP-TF2/TR4?
• VA13 cells consistently exhibit stronger effects than U-2 OS (e.g., Figures 1 and 3). This discrepancy could be linked to the high content of variant repeats in VA13 cells. The authors should assess whether variant repeat content underlies the differential response. Repeating key experiments in additional ALT lines with varied repeat compositions would be informative.
• In line with the previous point, it would be useful to show whether TRIM28 telomeric enrichment is affected by COUP-TF2/TR4 depletion in U-2 OS cells (Figure 4C). To improve confidence in these findings, the authors should perform telomeric ChIP assays, especially with the COUP-TF2^LBDΔAF2-TRF1 mutant construct.
• The immunoprecipitation experiments showing TRIM28 association with orphan receptors should include benzonase treatment to rule out DNA-mediated co-association (Figure 4F-G).
• The study would benefit from a direct assessment of whether COUP-TF2LBDΔAF2-TRF1 fails to induce ALT phenotypes in BJT fibroblasts.
• The experiments performed in Figure 5E-H lack a vector-only + siCtrl control.
• In Figure 5E, the observation that APB formation is restored in siTRIM28 + Vector-treated cells is unexpected. The authors should address this finding and clarify whether this reflects biological noise or a compensatory effect.

Significance

This work offers valuable mechanistic insight into how COUP-TF2 and TRIM28 coordinate to regulate heterochromatin deposition and ALT phenotype formation. It adds to the growing understanding of chromatin-mediated telomere regulation. What remains unclear is how important this interaction is for ALT maintenance, as H3K9me3 is only moderately altered upon TRIM28 depletion in ALT cells. Depletion of TRIM28 has been shown previously to induce APB formation and telomere elongation in U-2 OS ALT cells (Wang et al., 2021), the opposite to what the authors observed here in VA13 cells (Figure 5E-H). Clarifying whether these differences are variant repeat-dependent, or reflect intrinsic features of specific ALT cell lines, would substantially elevate the study's impact.

for - Medium article - cogress - Part 1 - progress trap - James Gien Wong - definition - cogress - to - Medium article cogress - Part 2 - progress trap - James Gien Wong - https://hyp.is/t8FhpDGAEfC4J7f0NEFujg/medium.com/@gien_SRG/human-cogress-part-2-d6fd075a55c7 - to - Stop Reset Go hypothesis annotations - progress trap - Ronald Wright - https://jonudell.info/h/facet/?max=100&expanded=true&user=stopresetgo&exactTagSearch=true&any=ronald+wright - General - https://jonudell.info/h/facet/?max=100&expanded=true&user=stopresetgo&exactTagSearch=true&any=progress+trap - from - youtube - Planet Critical interview - Samuel Miller MacDonald - The Myth of Progress - https://hyp.is/r-hmFtjKEfCd8odATbINbA/www.youtube.com/watch?v=BEhmWEDkZUQ

for - book - A Short History of Progress (2004) - author - Ronald Wright - progress trap - Ronald Wright - A Short History of Progress (2004) - to - movie - Surviving Progress (2011) - https://hyp.is/sRPYJtjLEfCwuDdwG2xNnw/www.nfb.ca/film/surviving-progress/ - to - book - Progress: A History of Humanity's Worst Idea - from - youtube - Planet Critical interview - Samuel Miller MacDonald - The Myth of Progress - https://hyp.is/r-hmFtjKEfCd8odATbINbA/www.youtube.com/watch?v=BEhmWEDkZUQ

for - progress traps - movie - Surviving Progress (2011) - from - book - A Short History of Progress (2004) - https://hyp.is/93k5CtjLEfC1UpPEi59BHA/archive.org/details/shorthistoryofpr0000wrig - from - youtube - Planet Critical interview - Samuel Miller MacDonald - The Myth of Progress - https://hyp.is/r-hmFtjKEfCd8odATbINbA/www.youtube.com/watch?v=BEhmWEDkZUQ

for - youtube - How the rich took over the economy - from - youtube - interview - Thomas Piketty - can't blame the top, so demonize the bottom - https://hyp.is/10dTvtheEfC_-8OXfzSTJA/www.youtube.com/watch?v=XeZoNTJgBZs

• SRG comment - A great explainer video of how the Volcker Shock started the whole chain reaction of modern day wealth inequality - to Thatcher and Reagan down to Trump

for - economic history - Volcker Shock - 2 political allies - Thatcher (1979) and Reagan (1980) came to power - cast taxes, social programs and regulation as the bogeyman

• SRG comment - Reagan and Thatcher policies - advocating for inequality - against the sacred

for - economic history - Volcker Shock - IMF Structural adjustment program - privatize public assets, - cut spending of welfare, - austerity across the board - deregulation, - open domestic markets to foreign corporations, - remove protection of local businesses and workers - IMF - a deal with the devil

for - economic history - Volcker Shock - defaulted countries turn to IMF

for - economic history - Volcker Shock - first casualties - Latin American debt crisis - Mexico, Brazil, Argentina defaulted on loans

for - economic history - 1979 - Paul. A. Volcker appointed chairman of Federal Reserve - Volcker Shock - shift - from employment to inflation - raised interest rates to an astounding 20%, intentionally causing a recession

for - economic history - Volcker Shock - used Milton Friedman's theory to provide cover to stop Keynesian commitment to full employment and instead protect capital from inflation. - Volcker raised interest rates to 20%,, causing massive plant shutdowns and unemployment to surge above 10%. - The recession closed shops, and labor lost its bargaining power when plants are shut down.

for - economic history - Milton Friedman - represented business elites - Monetarism - inflation seen as ultimate threat to elites

for - economic history - 1972 - Business Round Table established

for - economic history - powell memo - Lewis Powell - inequality - corporate lawyer who became supreme court judge - memo that started a long term political campaign to exploit the elite crisis for corporations to take control of universities, media, law and public opinion FOR THE ELITES

for - inequality - Thomas Piketty - opinion - middle class can't get tax relief from the elites - so they take it out on those below them - to - youtube - economic history - what started the chain reaction of modern day inequality - https://hyp.is/SIBPoNjHEfCxI8N7cC7ntw/www.youtube.com/watch?v=gAV0bkTHui8

DOI: 10.3390/ijms22083884

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DOI: 10.1016/j.cmet.2025.11.010

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DOI: 10.1016/j.celrep.2025.116574

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DOI: 10.6084/m9.figshare.30595343

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DOI: 10.6084/m9.figshare.30595343

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DOI: 10.6084/m9.figshare.30595343

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What is this?

为什么不是 the main problem schools face

在这个语境中，"problem" 像一个障碍物，它主动地“面对”或“朝向”了学校，因此使用主动语态的 "facing" 是最自然和常见的表达。

Grouping your categories may give clarity when writing.

Keep these components in mind

Keep these key features in mind when it comes to writing. Make a checklist and check your boxes as you go

Try to keep this in mind when asked to write a synthesis essay. Connect ideas and information and don't forget to explain how it all connects.

Definition of synthesis, keep in mind.

It was very interesting that this article was literally just the monologue of what Jimmy Kimmel said. There's not really any "think pieces" that sway any opinions

This is interesting that the dramatic imbalance in forces, showing how a small defending army relied on massive, historic walls to withstand a much larger invading army.

The sentence shows that Europe didn’t just recover after the plague, but also experienced major growth in learning and creativity.

High-Level Summary

The kidneys are bean-shaped organs protected by three outer layers and organized internally into the cortex, medulla, and renal pelvis. Nephrons in the cortex filter blood supplied by a highly branched vascular network that enters and exits through the renal hilum. Urine formed by nephrons flows through the renal pyramids into calyces, then the renal pelvis, and finally the ureter. Each kidney contains over one million nephrons, which are either cortical or juxtamedullary, depending on their position relative to the medulla.

Study Notes: Kidney Structure 1. External Kidney Structure

The kidney is surrounded by three protective layers (outer → inner):

• Renal fascia Tough connective tissue Anchors kidney to surrounding structures

• Perirenal fat capsule Cushions and stabilizes the kidney

• Renal capsule Thin, tough layer directly covering kidney surface

• Internal Kidney Regions

The kidney has three main internal regions:

Renal Cortex (outer region) Granular appearance Contains nephrons (functional units of the kidney) Site of blood filtration

Renal Medulla (middle region) Made of renal pyramids (cone-shaped tissue masses) Each kidney has ~8 pyramids Renal columns lie between pyramids and carry blood , vessels Pyramid tips = renal papillae, which point toward the , pelvis

Renal Pelvis (inner region) Located at the hilum Funnel-shaped urine collection area Drains urine into the ureter

1. Hilum of the Kidney

2. Concave region of the kidney

3. Entry/exit point for: Renal arteries Renal veins Nerves

4. Exit point for the ureter

5. Urine Flow Pathway

6. Minor calyces → Major calyces → Renal pelvis → Ureter → Urinary bladder

7. Renal Lobes

8. A renal lobe = one renal pyramid + surrounding cortical tissue

9. Functional subdivision of the kidney

Blood Supply of the Kidney (In Order) 1. Aorta 2. Renal arteries 3. Segmental arteries 4. Interlobar arteries (run through renal columns) 5. Arcuate arteries (arch at cortex–medulla boundary) 6. Cortical radiate arteries 7. Afferent arterioles 8. Glomerular capillaries (nephrons)

Venous return: Veins follow the same path in reverse. Same names as arteries except no segmental veins. Drain into the inferior vena cava.

Nephrons (Functional Units) Each kidney contains >1 million nephrons Located mainly in the renal cortex

Types of Nephrons Cortical nephrons (≈85%) Located deep in cortex Short loops of Henle Juxtamedullary nephrons Located near cortex–medulla boundary Long loops of Henle Important for urine concentration

Parts of a Nephron Renal corpuscle Renal tubule Associated capillary network

This passage connects to feminist frameworks that value lived and embodied knowledge. It reflects Moraga and Anzaldúa’s concept of “theory in the flesh,” where knowledge emerges from material experience, and aligns with feminist critiques of abstract, detached theory discussed in class.

This challenged my understanding of activism. I used to associate political action mainly with protests, but the authors show that decolonization also happens through everyday relational practices care, responsibility, and accountability to land and community.

This connects to class discussions on women of colour feminism, particularly critiques raised in This Bridge Called My Back, where Moraga and Anzaldúa show how white feminism has historically erased women of colour’s lived experiences. Similarly, Hunt and Holmes demonstrate how white queer politics can erase Indigenous queer and Two-Spirit perspectives, reinforcing the need to center those historically marginalized rather than expanding existing colonial frameworks.

This challenged my understanding of activism. I used to associate political action mainly with protests, but the authors show that decolonization also happens through everyday relational practices care, responsibility, and accountability to land and community.

The authors explain that mainstream queer theory and activism often overlook settler colonialism. In my own words, queer politics can reproduce colonial assumptions when it treats gender and sexuality as separate from land, Indigenous history, and sovereignty.

This helped me reflect on my own position as someone living on Indigenous land in Canada. The authors make clear that decolonization is not metaphorical it is about land, treaty responsibilities, and recognizing Indigenous sovereignty in everyday life.

A redação = The essay

early genetic and prenetal distrubances later stressful events that trigger the psychosis

it is more based on the populations of European contries and don't cover all aspects

1-Apperance: how does the person look like wearing and pysical 2-mood: how emotions show itself 3- Cognition: aware of the time and location 4-insight and judgement: aware of the illness itself 5- intellectual functioning: the expression of the toughts are not distrupted and has a flow

instinct and desires:id social restraints: super ego ego tryies to find the middle between superego and id

• danger: danger to you and people around you(ex: suicidal thoughts) deviance: things considered unnatural and unacceptable, against the social norms(staying bed all day) distress: having negative thoughts and stress (ı am not going to get thought his) *disfunction: this behaviours limits your daily habits your sleep work job etc(not being able to concentrate or go to work) They are important because they complete each other and four of them together is the definiton of an abnormality

條約法

權利法案

Volcanic Ash?

Gas exchange relies on Fick’s Law of Diffusion, which states that the rate of diffusion is directly proportional to the surface area of the membrane. Oxygen cannot enter the bloodstream fast enough to meet the body's demands, and carbon dioxide cannot exit efficiently. The alveolar walls contain the dense network of capillaries where the actual exchange takes place. When the walls are destroyed, the capillaries are destroyed with them. This creates a "dead space" effect where there is air in the lungs, but insufficient blood flow to pick up the oxygen. Healthy alveoli are elastic—they snap back to push air out during exhalation. Smoking destroys the elastin fibers that provide this recoil. When the smoker inhales fresh air, it mixes with this stale, trapped air.10 This lowers the partial pressure of oxygen (PO2) within the alveoli, reducing the driving force that pushes oxygen into the blood.

for: - MET - METI - Major Evolutionary Transition in Individuality - definition - Major Evolutionary Transition in Individuality (METI) - two conditions for METI - 1. living forms that were capable of independent replication before the replication can only replicate as part of a larger unit after the MET - 2. there is a lack of within-group conflict such that the larger unit can be thought of as a fitness-maximizing individual in its own right. - When these 2 conditions are met, evolution lea a new higher level organism. - The new individual acts with a single purpose where the interests of the previously independent individuals are now aligned.

Devices using a deprecated operating system

API key allows access to ArcGIS data

Note: "RGS" in the speech-to-text is "ArcGIS"

Flock camera saves recordings locally

for - definition - intelligence - Michael Levin the ability to adaptively navigae a problem space with some degree of competency to get your needs met (to fulfill your goal seeking activity)

for - stories - unity - cells - humans - Michael Levin - whether you are a cell or a human, you need to buy into the same story in order to solve problems together.

for - language - shifts - dominance hierarchy to egalitarianism

for - paper - The emergence of egalitarianism in a model of early human societies (2017) - author - Guillaume Calmettes - James N. Weiss

Gordon and Chloe swimming unclothed in the deep ocean together

for - ETI - sociocultural systems as ETI - critique - lack autonomous reproduction at the group level - lack operation of natural selection in the reproductive mode

• paper - Human socio-cultural evolution in light of evolutionary transitions: introduction to the theme issue (2023)
• author
  • Yohay Carmel
  • Ayelet Shavit
  • Ehud Lamm
  • Eors Szathmary