Summary When it comes to deep reading, our brains were not wired to be good at deep reading it is a skill. But with practice, discipline, no distraction, and going at your own pace, reading might actully get deep.

Tracking This is how Wolf is trying to wrap her points she is making her most main points in the text and making it sound very powerful so it stays in our head.

Restatment For the thrid takeway it is all about discipline. It takes practice to get good at deep reading it just does not come like that.

Restatmemt Us humans are not wired for reading it is a skill. A skill that you have to practice with real good stratagies.

Restatment When you are trying to deep read it is important to note that you need to keep the districations down to a minimium.Its really hard to not have any distractions but try your best.

Tracking During the middle of the transcirpt it is the takways that I will be learning throuout the text. I am going to go back and re read the beggning part of the text because I can tell it was not sticking with me.

Reaction Adding in here the takeways that is making it so much more easy for me to understand the main ideas and really understand what I was really supposed to get out of the reading.

Reaction It might just be becuase im not a creative thinker like this but the way she is saying this it is very confusing to understand.

Tracking I can tell this is the start to the introduction. Maryannne wolf is setting up her introduction to inform the lisennters what the text is all about.

Reaction Very relatable. Hooked me in very fast which is great so then I want to stay for the rest of the text.

y si revisamos la correlación escolaridad x ingresos a la luz de estas limitaciones?

Entiendo la lógica del ejercicio pero me parece que para que funcione habría que pasarles un libro de código para identificar las variables que nos interesan

Queremos que los estudiantes sigan esto en R o no es necesario? Porque si es así habría que mostrar como crear el objeto datos_no_lineales

Me parece bien está dinámica pero no sé si me queda tan claro como se aplica en el resto del práctico

Revisar

It is notable that the author uses Christian values to denounce slavery. This subverts the very basis on which slavery was justified. And through storytelling and narrative, the cruelty of the slave owners is directly contrasted with Christian values, both through the narrator’s words and through what can be inferred from the text by the reader. This strengthens the core message of the story. The author challenges the readers to reconsider what it means to be Christian, and how contradictory the current society is with those ideas.

This passage shows the dehumanizing nature of slavery. Enslaved people are being sold off after being gambled away. This 'uncertainty of a slave's position' is something that usually seems to be overlooked when discussing the cruelty of slavery. The fact that enslaved people are seen as commodities, and therefore sold without their knowledge or consent, deprives them of home, both physically and mentally. In this sense, enslaved people have no security in the sense of home, which is the basis for personal identity. Slavery is directly undermining the enslaved person's identity, as well as their freedom.

Clotel is described as 'beautiful' in an extremely degrading manner. Her beauty is considered comparable to, or even superior to, that of white women. White beauty standards are presented as the only measure of beauty and are therefore applied to Clotel. She is described as being 'as white as' the people who want to buy her and is referred to as an 'albino'. This implies that a person of color can only be considered beautiful when they have features that are similar to white features. The suggestion that beauty is not inherent in a person of color but is only possible when their heritage is mixed with white slave owners reveals the cruel racial hierarchy of white supremacy that permeates everyday life of this time, even shaping beauty standards. It is also worth noting that people like Clotel are called 'mulatto', which derives from the word for 'mule'. This offensive origin of the word also shows the degradation of colored people.

It is not only that we get there, but how we get there. Dealing with human lives that rely on contamination to exist in order to pay their bills still deserve a "just transition". That means making their transition to having a life without making climate change worse but doing so in a peaceful manner.

From reading this in print it creates hope. Let us study the ways how this can be possible.

This is a fact that shows up over and over. But because of better data and the repetitive nature of this issue we can find these locations and work on them.

Labyrinthe Scolaire : Défis, Pressions et Réalités du Système Éducatif Français

Résumé Exécutif

à mettre en lien avec le débat https://www.youtube.com/watch?v=Ws-jcTT8ZWc&t=337s&pp=ugUEEgJmcg%3D%3D

L'analyse du contexte source révèle un système éducatif français perçu comme un « petit tuyau » étroit où chaque enfant doit s'insérer, souvent au prix d'une pression intense.

Le document met en lumière une crise multidimensionnelle : une complexité administrative croissante (Parcoursup, Affelnet), une stratification sociale marquée entre le public et le privé, et un manque chronique de moyens humains (enseignants, AESH).

Les parents, transformés en véritables gestionnaires de flux et de carrières (« The Organizer »), pallient les défaillances de l'institution par un investissement personnel et financier massif, tout en naviguant dans un labyrinthe d'orientation qui s'avère anxiogène tant pour les familles que pour les élèves.

I. L'Expérience Parentale : Entre Gestion de Projet et Pression Sociale

Le rôle des parents a évolué vers une fonction de gestionnaire de haut niveau pour garantir la compétitivité de l'enfant.

• L'organisation intensive : Les parents structurent l'emploi du temps de leurs enfants de manière quasi ministérielle.

Un exemple type montre une semaine saturée par le conservatoire (violon), le cirque, l'escalade, le tennis et la piscine, laissant le dimanche comme unique moment de « relâche ».

• La lutte contre les écrans : L'occupation du temps par des activités extrascolaires est explicitement utilisée comme une stratégie pour éviter la « guerre des portables » et la tension liée au temps d'écran.

• Le traumatisme des lacunes : Certains parents, marqués par leur propre échec scolaire (notamment en mathématiques dès le CP), s'engagent dans une « rééducation » personnelle pour accompagner leurs enfants, accumulant manuels et fiches pour prévenir tout décrochage.

• Le poids administratif : Les parents dénoncent une montagne de dossiers et la nécessité de se connecter à des heures précises sur des plateformes saturées pour les inscriptions (conservatoire, centres de loisirs).

II. Le Labyrinthe de l'Orientation et de l'Élite

L'orientation est vécue comme un processus opaque et précoce, dicté par des algorithmes et une hiérarchie rigide des filières.

1. La stigmatisation de la filière professionnelle

Le système tend à utiliser la voie professionnelle comme une variable d'ajustement pour les élèves en difficulté plutôt que comme un choix de vocation.

• Orientation par défaut : Dès le collège (parfois dès la 5ème), les élèves ayant des résultats inférieurs à 10 sont orientés vers le Bac Pro, assimilant cette filière à un échec.

• Méconnaissance des métiers : Les jeunes en Bac Pro (ex: coiffure) découvrent parfois la réalité difficile du métier (hygiène des clients, routine) seulement lors des stages, faute d'une préparation adéquate.

2. La complexité des plateformes et des choix

• Affelnet et les lycées de secteur : Le passage au lycée est marqué par une stratégie complexe.

Le retrait de lycées d'élite (comme Charlemagne) des secteurs géographiques modifie radicalement les plans des familles, les forçant à « post-rationaliser » des choix vers des lycées moins prestigieux mais jugés plus « bienveillants » (ex: Hélène Boucher, Sophie Germain).

• Parcoursup : Décrit comme une période mouvementée, Parcoursup impose aux jeunes de 18 ans une réflexivité prématurée.

On leur demande de rédiger des lettres de motivation pour des filières qu'ils choisissent parfois « au pif », tout en subissant l'angoisse des listes d'attente.

| Étape Scolaire | Principal Défi Identifié | | --- | --- | | CP | Apprentissage de la lecture et pression du "bachotage". | | 6ème | Transition vers le collège, peur des professeurs et de la charge de travail. | | 3ème | Premier palier d'orientation critique et gestion du Brevet. | | Terminale | Pression de Parcoursup et nécessité de spécialisation précoce. |

III. Une Institution en Tension : Moyens et Inégalités

Le service public d'éducation est décrit comme étant « à l'os », tenant principalement par le dévouement de son personnel.

• Pénurie de personnel spécialisé : L'obtention d'une AESH (Accompagnement des Élèves en Situation de Handicap) est un combat judiciaire.

Dans un cas cité, un parent a dû saisir le tribunal administratif via un avocat pour obtenir seulement 3 heures d'accompagnement par semaine sur les 9 initialement prévues.

• Fermetures de classes : Des mobilisations de parents et d'enseignants ont lieu contre les suppressions de postes dans des collèges pourtant identifiés comme étant « en difficulté ».

• Évitement scolaire : Dans certains secteurs parisiens, jusqu'à 40 % des familles sectorisées évitent le collège public local au profit du privé, craignant la mixité sociale ou des problèmes de discipline fantasmés ou réels.

• Le "Bricolage" permanent : L'éducation nationale est qualifiée de « plus grande enseigne de bricolage du pays » en raison du manque d'enseignants (absents dans plus de la moitié des établissements à la rentrée) et de personnels de santé (RAZED).

IV. Citations Clés et Perceptions

Le climat scolaire est résumé par des témoignages incisifs des différents acteurs :

« L'éducation nationale c'est un tout petit tuyau et tout le monde doit rentrer dans ce tout petit tuyau... c'est très compliqué. »

« On ne propose pas à un jeune qui a des bons résultats d'aller en bac pro... on leur fait croire que si tu vas en pro c'est que tu es en échec. »

« Les plateformes sont complètement obscures... même quand on est français, c'est hyper complexe et à chaque fois qu'on clique, on n'est pas sûr de cliquer correctement. »

« L'école en France n'est certainement pas égalitaire... elle est créée pour l'élite, pour sélectionner les meilleurs au prix d'énormément de jeunes laissés sur le côté. »

V. Conclusion de l'Analyse

Le document source dépeint un système où la réussite dépend de plus en plus de la capacité des parents à naviguer dans la complexité administrative et à compenser les manques de l'État.

Si l'idéal de « Liberté, Égalité, Fraternité » demeure un projet, la réalité du terrain montre une école qui sélectionne par la pression et la compétition, laissant les familles les plus démunies ou les élèves ne rentrant pas dans le « tuyau » standard face à un sentiment d'injustice et d'abandon.

Iago reveals his plan: he will use Othello's biggest quality, his honesty, against him. Othello trusts people who only seem honest, so Iago can lead him "by th' nose" like an animal — the same kind of animal image he used against him in scene 1.

Back to the first line of the play: Iago uses Roderigo to make money for himself. Repeating the phrase over and over works like brainwashing — Roderigo ends up selling all his land.

Brabantio's rhyme (see/thee) sounds like a prophecy: Othello will be deceived, but not by the person we'd expect. Calling him "honest Iago" right after is ironic since we know he isn't. "My life upon her faith" — it will actually cost him his life.

In front of everyone, she chooses Othello, but without rebelling: she respects her father and uses her mother's example to justify it. Far from the shy girl Brabantio described.

Their love comes from the story of his dangerous past: she admires him, he loves being admired. Built on a story, it can be broken by another one — Iago's.

He uses false modesty: he says he speaks badly to win over the senators, then gives one of the most eloquent speeches of the play.

This false attack mirrors the whole play: illusion and diversion are its main themes. It introduces Othello and Iago right after — but while the Senate sees through the Turks' trick, Othello won't see through Iago's.

Copilot says that the Turks valued Cyprus's strategic position in the Mediterranean near the Ottoman territories and thought it dangerous to leave it in the hands of the Venetians, their territorial rivals.

He talks about him like he's not human ("thing", "sooty"), and thinks she was bewitched into leaving her family and protection for a stranger — even though he used to welcome Othello as a guest.

It shows his calm and trust in his own capacities: he stops a fight with one slightly mocking line instead of drawing his sword. This is the controlled Othello Iago will later break.

Again, Othello seems confident and refuses to hide — he has nothing to hide. But Iago swears by Janus, the two-faced god, which fits him perfectly.

He is confident about his power and doesn't think Brabantio is a real threat, but it also shows he is naive: he believes his actions will speak for him, while he's saying it to the man who just betrayed him.

After betraying Othello by telling Brabantio about the secret marriage, Iago now tries to gain Othello's trust by pretending he almost stabbed Roderigo to defend him.

Iago presence on both sides of this debacle could outline one of two things. It could be a foreshadowing of how Brabantio is actually far worse than the other characters in terms of discrimination. It could also mean that Iago is a more complex character, siding with both out of whatever aversion for the wrath of one.

This line is taken from the preface of Baudelaire's Les Fleurs du Mal which is a early 20th century work that discusses the rapidly industrializing world at that time. Elliot alludes to this work because he as well is writing a little bit about how the industrialization of London has turned it into something of a wasteland. This line translates to hypocrite reader - my fellow, - my brother. This line that breaks the fourth wall is a way to connect with the reader and say that everybody is connected and feels the same way. He is calling out the reader who says that the industrialization is bad, but doesn't do anything about it.

Brabantio is anxious but also furious. "Oppresses" shows the news is an emotional weight, not just anger — and since he already dreamed it, he believes it before checking. That's how Iago will get Othello too: by feeding fears people already have.

Iago uses an openly racist animal metaphor (old black ram / white ewe) to tell Brabantio about Othello and his daughter and make him furious — "now, now, very now" makes it feel urgent. Smart again: he disappears before anyone sees him.

He talks like a true mastermind: for him showing your real feelings just makes you prey, and "I am not what I am" flips God's "I am that I am".

Marriage was obviously more of an official matter than an emotional one at this time. For his daughter to get married to Othello without the permission of the family would be considered a great disservice to her father and house. For Brabantio to not know at all that his daughter is gone and married to Othello should be considered an act of stupidity considering how important marriage is for politics.

some

删除

不要让女主脱光，留着内衣内裤

这个是在男配说女主不要和不三不四的鸭子在一起的时候男主知道的，这个时候男主只是被接吻的感觉吸引到了，最好找个时候让男主眼睛变红；

让电视剧时有时无一直发出奇怪声音

让女主不要全裸，不要裸体，但是可以写被拉开衣服，一定要有内衣，可以是黑色蕾丝内衣；

应该是感慨是男主的血给她带来了能力提升；

设置女主用枪射出的是蓝色的光，增加画面效果，另外帮我想一些能体现女主的能力进步的画面；

承接上文情节，加一个警报的声音通知有紧急活动，打断女主和女配的对峙

eLife Assessment

This is an important study addressing a debated question in brain repair research by testing whether NeuroD1 can convert brain immune cells into nerve cells using a virus-free genetic approach and live imaging. The compelling evidence presented here supports that, under the conditions tested, NeuroD1-expressing cells do not become nerve cells and instead retain their original identity. This work will be of interest to neuro-immunologists.

Reviewer #1 (Public review):

Summary:

This study revisits an important and controversial question in brain repair: whether NeuroD1 can convert brain immune cells into nerve cells in vivo. Using a virus-free genetic system, in vivo imaging, injury experiments, and single-cell profiling, the authors provide convincing evidence that NeuroD1-expressing cells do not become nerve cells under the tested conditions. Instead, these cells largely retain their original immune-cell identity, and some appear to undergo cellular stress or loss.

Strengths:

The main strength of the work is that it tests this question with a cleaner genetic strategy, avoiding some of the concerns associated with viral delivery and unintended cell labeling. Although the overall conclusion is consistent with the authors' previous work, the current study adds useful independent evidence, particularly through the virus-free fate-mapping system and live imaging in the brain.

Weaknesses:

The tested time window cannot fully exclude the possibility of very delayed or incomplete neuronal differentiation.

Overall, this is a useful and careful study that supports the conclusion that NeuroD1 does not drive brain immune cells to become nerve cells in the tested settings. It should be valuable for researchers studying brain repair, cell fate conversion, and genetic fate mapping, and it provides a clear caution against overinterpreting reprogramming results based only on viral labeling.

Reviewer #2 (Public review):

Summary:

In vivo glia-to-neuron conversion emerges as a potential regeneration-based therapeutic strategy for neural injuries and diseases. However, controversies exist in this exciting field, largely arising from the non-stringent methods use to analyze in vivo neuronal conversions. The study by Li et al. directly tackled such a controversy on Neurod1-mediated microglia-to-neuron conversion. They took advantage of transgenic mouse lines to specifically express Neurod1 in microglia of adult mouse brains. Results from immunohistology, in vivo live cell imaging, and scRNA-seq convincingly demonstrate that microglia cannot be converted in vivo to neurons by ectopic Neurod1 expression under the specified normal or injury conditions. Instead, it induces microglia death, consistent with their earlier findings. These solid results, though negative, are critical additions to the research field and further support that stringent lineage tracing methods are essential for studying in vivo cell reprogramming. Overall, the studies are rigorously designed and executed.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study revisits an important and controversial question in brain repair: whether NeuroD1 can convert brain immune cells into nerve cells in vivo. Using a virus-free genetic system, in vivo imaging, injury experiments, and single-cell profiling, the authors provide convincing evidence that NeuroD1-expressing cells do not become nerve cells under the tested conditions. Instead, these cells largely retain their original immune-cell identity, and some appear to undergo cellular stress or loss.

Strengths:

The main strength of the work is that it tests this question with a cleaner genetic strategy, avoiding some of the concerns associated with viral delivery and unintended cell labeling. Although the overall conclusion is consistent with the authors' previous work, the current study adds useful independent evidence, particularly through the virus-free fate-mapping system and live imaging in the brain.

Weaknesses:

There are some limitations. In the injury experiment, the labeled cells may include both resident brain immune cells and blood-derived immune cells recruited after injury, so the authors should be cautious when referring to all labeled cells as microglia. The level of NeuroD1 expression achieved by the genetic system is also not fully defined, which matters because the effects of such a cell-fate regulator may depend on expression level. Finally, the tested time window may not fully address very delayed or incomplete neuronal differentiation.

Overall, this is a useful and careful study that supports the conclusion that NeuroD1 does not drive brain immune cells to become nerve cells in the tested settings. It should be valuable for researchers studying brain repair, cell fate conversion, and genetic fate mapping, and it provides a clear caution against overinterpreting reprogramming results based only on viral labeling.

We’d like to thank the reviewer for providing these thoughtful suggestions and positive assessment of our study. We agree that CX3CR1 lineage can label both microglia and blood-derived immune cells after injury, To more specifically assess microglial lineage tracing and minimize the potential contribution of infiltrating peripheral myeloid cells, we performed additional experiments using TMEM119-CreER::LSL-NeuroD1-EGFP mice. These experiments showed no evidence of microglia-to-neuron conversion following TBI. To evaluate the expression level of NeuroD1, we performed qPCR to verify the NeuroD1 expression level in the CD11b⁺ cell population of the NeuroD1-expressing mice, and found that NeuroD1 expression was increased approximately 6.21-fold compared with control mice, indicating robust NeuroD1 expression. For the question about the time window, we have revised our Discussion and Conclusion parts to avoid overgeneralizing our findings beyond the experimental conditions tested. We agree that our data cannot fully exclude the possibility of delayed neuronal differentiation or neuronal conversion under other pathological conditions. Besides, we have also revised the manuscript accordingly to address the concerns raised by the reviewer.

Reviewer #2 (Public review):

Summary:

In vivo glia-to-neuron conversion emerges as a potential regeneration-based therapeutic strategy for neural injuries and diseases. However, controversies exist in this exciting field, largely arising from the non-stringent methods employed for analyzing in vivo neuronal conversions. The study by Li et al. directly addressed this controversy regarding Neurod1-mediated microglia-to-neuron conversion. They took advantage of two transgenic mouse lines to specifically express Neurod1 in the microglia of adult mouse brains. Results from immunohistochemistry, in vivo live-cell imaging, and scRNAseq convincingly demonstrate that microglia cannot be converted in vivo to neurons by ectopic Neurod1 expression under both normal and injury conditions. Instead, it induces microglia death, consistent with their earlier findings. These solid results, though negative, are critical additions to the field and further support that stringent lineage tracing methods are essential for studying in vivo cell reprogramming. Overall, the studies are rigorously designed and executed. Only minor issues need to be dealt with.

We’d like to thank the reviewer for providing these thoughtful comments and for the positive assessment of our study. We are glad that the reviewer agrees that our results from immunohistochemistry, in vivo live-cell imaging, and scRNA-seq support the conclusion that microglia cannot be converted into neurons by NeuroD1 expression under the tested conditions. We have carefully addressed the minor issues raised by the reviewer, including rechecking the grammar throughout the manuscript, revising the description of the TBI behavioral results, discussing the limitations of scRNA-seq for neuronal detection, adding the information of the promoters used in the study, and carefully revising the references. Besides, we have carefully revised the manuscript according to the reviewer’s suggestions.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Major comments

(1) TBI lineage tracing may label both microglia and infiltrating macrophages. Because tamoxifen was administered after TBI, CX3CR1-CreER may label not only resident microglia but also injuryrecruited CX3CR1+ monocytes/macrophages. The authors should avoid referring to all reporterpositive cells as microglia unless this is clearly supported. They should clarify the composition of reporter-positive cells in the TBI setting, ideally using existing scRNA-seq data or markers distinguishing microglia from infiltrating macrophages.

The current data still support the conclusion that CX3CR1-lineage myeloid cells do not show obvious neuronal conversion in this TBI model, but the wording should be more precise.

Thank you for pointing this out. We agree that, because TBI can recruit CX3CR1-expressing monocytes/macrophages, CX3CR1-CreER lineage tracing after injury may label both resident microglia and infiltrating myeloid cells. To more specifically assess the contribution of resident microglia, we therefore performed additional lineage-tracing experiments using TMEM119CreER::Ai14 (TMEM119-Ai14) and TMEM119-CreER::LSL-NeuroD1-EGFP(TMEM119-ND1) mice, with tamoxifen administration before or after TBI. In both experimental settings, we did not detect reporter-positive cells co-expressing the neuronal marker NeuN in either the lesion core or distal regions. These results provide no evidence of microglia-to-neuron conversion in the TBI model within the examined time window. We have also revised the text throughout the manuscript to distinguish resident microglia from broader CX3CR1-lineage myeloid cells where appropriate.

Author response image 1.

TMEM119-CreER::LSL-NeuroD1-EGFP animal also show no microglia-to-neuron conversion in TBI. (A)Tamoxifen induction after TBI shows there is no GFP⁺NeuN⁺ cells in both lesion core and distal region. (B)Tamoxifen induction before TBI shows there is no GFP⁺NeuN⁺ cells in both lesion core and distal region.

(2) NeuroD1 expression level should be better characterized. The authors show that GFP-positive cells co-express NeuroD1, but the approximate NeuroD1 expression level is not clear. This is important because the effect of a fate-determining transcription factor may be dose-dependent. The authors should provide, if possible, a quantitative estimate of NeuroD1 expression using available IF, scRNA-seq, qPCR, or other data. This would help interpret the negative reprogramming result and would also be useful for future studies using this Rosa26-LSL-NeuroD1-IRES-GFP mouse line.

Thank you for this valuable suggestion. To quantitatively assess NeuroD1 expression in the myeloid compartment, we isolated CD11b⁺ cells from CX3CR1-CreER::Ai14 (CX3CR1-Ai14) and CX3CR1-CreER::LSL-NeuroD1-EGFP (CX3CR1-ND1) mice using MACS and performed qPCR analysis of Neurod1 expression. Neurod1 expression was increased approximately 6.21-fold in CD11b⁺ cells from CX3CR1-ND1 mice compared with the corresponding control mice (Author response image 2). These results confirm robust NeuroD1 expression in the CD11b⁺ cell population of the NeuroD1-expressing mice. This level of induction is broadly comparable to the approximately 4-fold increase in Neurod1 expression reported following NeuroD1 induction in the intestine, in which the same LSL-NeuroD1-EGFP transgenic mouse line was used [1].

Author response image 2.

Using qPCR verify the relative expression of Neurod1.

(3) Figure 4 scRNA-seq should show Neurod1 expression. The scRNA-seq data show that reporterpositive cells retain microglial markers and lack neuronal markers, but Neurod1 expression itself is not shown. The authors should consider adding Neurod1 feature plots, violin plots, or average expression comparisons between control and NeuroD1-expressing groups. This would help validate the expression system and estimate the level achieved in sorted reporter-positive cells.

Thank you for your suggestion. We examined the expression of Neurod1 in our scRNA-seq database, but the Neurod1 transcripts were rarely detected (not just in this database, also our previous dataset). This might because of Neurod1 is a transcription factor and single-cell RNA-seq method is not suitable for detect these low-expression transcript factor genes, so we utilized qPCR to evaluate the expression of Neurod1, as shown in Author response image 2.

(4) The microglial loss phenotype may be related to NeuroD1/EGFP dosage. In Figure 3, the loss of GFP-positive microglia may reflect a specific effect of NeuroD1 in microglia, but it could also be related to excessive transgene expression or expression-system toxicity. The authors should discuss this possibility and, if possible, examine whether stress/death markers correlate with NeuroD1 or GFP expression intensity.

Thanks for your suggestion. We agree with that, so we performed TUNEL staining on CX3CR1-CreER::Ai14 (CX3CR1-Ai14 for short) and CX3CR1-CreER::LSL-NeuroD1EGFP(CX3CR1-ND1 for short) mice at D4 and D18. The data shows that TUNEL⁺ Reporter⁺ cells were higher in NeuroD1-expressing microglia, so we’d like to say death cells (or apoptotic cells) are positively correlated with NeuroD1 expression. Besides, we also discussed more about this. See Figure 3 E, F, G and Fig. S3E

(5) The time window and injury context should be more cautiously discussed. The study follows the genetic model up to 45 days. This is informative, but delayed or incomplete neuronal differentiation cannot be fully excluded, especially if conversion would require a longer repair phase after injury. In addition, except for TBI, the study does not include another neuronal-loss context that might provide a permissive regenerative niche. The authors do not necessarily need additional long-term experiments, but they should avoid overgeneralizing beyond the tested time window and injury model.

Thank you for this thoughtful comment. We agree that our study evaluated the effects of NeuroD1 expression within a defined experimental time window (up to 45 days) and under the specific physiological and injury conditions examined. Therefore, our data cannot exclude the possibility that neuronal conversion might occur at later time points or under other pathological conditions that provide a more permissive regenerative environment. In response to the reviewer's suggestion, we have revised the Discussion and Conclusion to avoid overgeneralizing our findings beyond the experimental conditions tested. We now emphasize that NeuroD1 expression alone did not induce microglia-to-neuron conversion within the time frame and injury paradigms examined in this study, rather than concluding that such conversion can never occur under other conditions.

Minor comments

(1) In Figure 2, two panels are labeled "D"; the second should likely be "E."

Thank you for pointing this out, we have corrected it in figure legend.

(2) Several spelling and grammar errors should be corrected, such as "Represent images," "illutrating," and "GFP-postive."

Thank you for pointing this out, we have corrected it in our manuscript.

(3) More scRNA-seq details should be provided, including cell numbers per group, QC metrics, and cluster annotation information.

Thanks for your suggestion, we have added more details about scRNA-seq data as shown in Figure S4.

(4) The authors should maintain cautious wording throughout, especially when referring to "microglia" versus broader CX3CR1-lineage myeloid cells.

Thanks for your suggestion, we added the data about we utilize TMEM119-ND1 mouse line to trace microglia-to-neuron conversion, which is specific to microglia not myeloid cell. Besides, we also carefully revise these minor comments in our manuscript.

Reviewer #2 (Recommendations for the authors):

The following are some required minor edits.

(1) Please recheck the grammar throughout the manuscript.

Thank you for pointing this out, we have carefully revised our manuscript.

(2) Figure 4B-C: "indicating that NeuroD1 expression in microglia does not rescue TBI induced motor dysfunction". This should be rephrased, since dysfunction was not detected when comparing TBI and sham.

Thank you for pointing this out, we changed our expression into “indicating that NeuroD1 expression in microglia does not improve the performance in TBI model”.

(3) Interpretation of the scRNA-seq data needs to be cautious, since neurons normally do not survive well during this procedure.

Thank you for your suggestion, we discussed about this in our discussion part.

(4) It will be informative to list the sources or sequences of the promoters used.

Thanks for your advice, all the virus information are provided in our method part.

(5) Some of the references are repetitive.

Thank you for your suggestion, we have carefully revised the reference.

Reference

(1) Li, H. J. et al. Intestinal Neurod1 expression impairs paneth cell differentiation and promotes enteroendocrine lineage specification. Sci Rep 9, 19489 (2019). https://doi.org/10.1038/s41598-019-55292-7

New paragraph

interesting

Aunque no aparece información sobre elles es interesante ver qué datos nos da Internet.

Shouldn't this electron configuration end in 6 ? Shouldn't it be 1s2 2s2 2p6 3s2 3p6 ?

Shouldn't this electron configuration end with a 5 instead of a 7 ? Shouldn't it be 1s2 2s2 2p6 3s2 3p5 ?

The - log10(pH) thing..... Hydrogen ion concentration.... Which just so happens to be a measure of an acid's.......... Strength..... Guh.....

The more stable the conjugate base (i.e. No H+ proton), the stronger the acid.

Why is this so? Perhaps one thinks that fluorine is best able to attract negative charges, as it is the most electronegative element. But in fact, it is the size that begins to matter.

eLife Assessment

This study characterises the heterogeneity and developmental origins of macrophages in the thymus and demonstrates a role for Csf1r-expressing myeloid cells in the first step of T cell development. The state-of-the-art experiments provide convincing evidence, and the assessors concerns were addressed in the revised submission. These results provide an important advance that furthers our understanding of thymus biology, especially in view of the contribution of heterogenous thymic macrophage subpopulations.

Reviewer #1 (Public review):

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

Summary:

The current manuscript characterizes in detail the macrophages in the thymus. The authors identify two distinct populations of thymic macrophages and describe their surface marker expression and transcriptional signatures. They also explore their ontology and kinetics of settling and persistence in the thymus and find that the TIMD4+ macrophages are derived from embryonic progenitors and self-maintain in the thymus, while the TIMD4- macrophages are derived from monocytes. Most importantly, the authors test the functional importance of thymic macrophages for T cell development using an in vitro depletion system, from which they conclude that macrophages are important for one of the earliest selection steps in T cell development - the beta selection.

Strengths:

The authors use state-of-the-art techniques, such as multiple genetically modified mice, multi-color flow cytometry, single-cell RNA sequencing, genetic fate mapping, and fetal thymic organ culture (FTOC) combined with depletion. Their work is in good agreement with prior published studies on the subject, such as Tacke et al. (PMID: 26091486) and Zhou et al. (PMID: 36449334). In addition to reproducing prior knowledge, the authors uncover novel and unexpected facets of thymic macrophage biology, such as their SpiC independence and the fact that TIMD4- thymic macrophages depend on CCR2 (Tacke et al. have shown that the overall thymic macrophage compartment is normal in CCR2-/- mice). Most surprisingly, the authors claim that thymic macrophages control an early checkpoint in T cell development, the beta selection. This has not been reported before, as beta selection is usually considered a cell-autonomous process in thymocytes that does not require input from other cells.

Reviewer #2 (Public review):

This manuscript from Zuniga-Pflucker laboratory describes that thymic macrophages are heterogeneous in flow cytometric and transcriptomic profiles, containing two major populations characterized by TIMD4 and CX3CR1 expression. These macrophage populations are both parenchymal in the thymus but are unequal in developmental ontogeny, Flt3 expression history, and CCR2 dependency. The manuscript further reports the interesting findings that the depletion of thymic macrophages impairs thymocyte development at the DN3 beta-selection checkpoint. These results provide an important advance for further understanding of thymus biology, especially in view of the contribution of heterogenous thymic macrophage subpopulations.

Author response:

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

We thank the editors and reviewers for their thoughtful evaluation and helpful recommendations. These comments helped us distinguish more clearly between two complementary advances in the study. First, the phenotypic, transcriptomic, and ontogenetic analyses refine the organization of the thymic macrophage compartment and identify CCR2 dependence of the TIMD4- VCAM1+ population. Second, the MaFIA fetal thymus organ culture (FTOC) experiments reveal that an intact Csf1r-expressing myeloid compartment is required for efficient progression across the DN3-to-DN4 checkpoint. The revised manuscript now presents both advances directly while matching the cellular specificity of each conclusion to the experimental system that supports it.

The central functional result is supported by a coordinated set of observations: depletion of Csf1r expressing cells reduces CD4⁺CD8⁺ double-positive (DP) ab-lineage thymocyte production, preserves γδ T cell output, causes reciprocal accumulation of DN3 and loss of DN4 cells, and reduces CD27 expression within the DN compartment. We now emphasize that convergence, which localizes the phenotype to the b-selection transition. At the same time, because the MaFIA system targets Csf1r-expressing myeloid cells rather than a single macrophage subset, the manuscript assigns the demonstrated requirement to the thymic myeloid compartment, this now reflected on the revised title as well. This framing preserves the biological importance of the result without attributing more cellular specificity than the experiment provides.

The major revisions include:

- A revised Title and an Abstract that leads with the principal conclusions: TM specialization, unequal developmental contributions, CCR2 dependence, and a requirement for Csf1r expressing myeloid cells during DN3-to-DN4 progression.

- A clearer account of what Zhou et al. established and how the present work adds VCAM1-based prospective resolution, intravascular-labeling information, SpiC analysis, CCR2 dependence, and functional fetal thymus organ culture (FTOC) data.

- A Results section that explains the convergence of DN3, DN4, CD27, DP cells, and γδ T cell measurements as part of developmental transition.

- More precise interpretation of relative marker expression, pseudotime, SpiC deficiency, Tomato/GFP double-positive events, and thymocyte-associated transcripts in the scRNA-seq results.

- A focused Discussion paragraph that acknowledges the cellular breadth of the MaFIA model while retaining the conclusion that the Csf1r-expressing myeloid niche supports early ab T-lineage development.

- Expanded figure legends that make the gating, reporter, heatmap, MaFIA construct, and developmental interpretations easier to follow.

The revision is based on fuller analysis and clearer presentation of the results and datasets.

eLife Assessment

The macrophage characterisation is interesting, although the evidence for the specific involvement of macrophages in beta-selection is incomplete, as alternative explanations have not been ruled out.

We agree that the depletion experiment resolves a requirement for the Csf1r-expressing thymic myeloid compartment rather than for macrophages alone. We have revised the manuscript with this distinction in mind. Importantly, the developmental conclusion remains strong: DN3 accumulation, DN4 loss, reduced CD27 expression, and diminished CD4⁺CD8⁺ double-positive (DP) output all point to impaired progression at the b-selection checkpoint, while preserved γδ T cell output and epithelial-cell numbers argue against nonspecific failure of the entire fetal thymus organ culture (FTOC). The revised text therefore states that Csf1r-expressing myeloid cells support this early ab T cell checkpoint, while discussing the relative contributions of TMs, monocytes, and DCs as the next level of cellular resolution.

The Title, Abstract, final Results section, Discussion, Conclusion, and Figures 8-10 Legends were revised to make the positive compartment-level conclusion explicit and consistent.

Public Reviews:

Reviewer #1 (Public review):

The thymic macrophage depletion experiments are not well controlled; DCs are also depleted, the fetal thymus has little or no medulla, and direct AP20187 toxicity to DN thymocytes in MaFIA mice has not been excluded.

The reviewer identifies the key issue of cellular attribution. We have recast the experiment according to what the MaFIA system directly tests: the functional contribution of Csf1r-expressing myeloid cells within an intact FTOC. This interpretation is supported by efficient loss of both TM populations, preservation of EpCAM+ epithelial-cell numbers, and absence of TM depletion in AP20187treated non-transgenic C57BL/6 FTOCs. We no longer use adult cortical-versus-medullary anatomy to infer that macrophages must be the sole responsible population in the fetal thymus. Instead, we emphasize the experimentally secure result that perturbing the Csf1r-expressing myeloid niche produces a selective and internally consistent defect in ab T cell development at the DN3-to-DN4 transition. The possibility of contributions from DCs, monocytes, or direct transgene expression in a thymocyte fraction is addressed once, in a focused Discussion paragraph, as the rationale for assigning the conclusion at the compartment level.

The MaFIA Results section now leads with the transgene-dependent depletion and the convergent developmental phenotype; the Discussion contains a balanced statement of cellular resolution; the Title and Legends consistently refer to Csf1r-expressing myeloid cells.

Reviewer #2 (Public review):

Zhou et al. previously reported similar TM heterogeneity, localization, and developmental characteristics. The manuscript should distinguish prior findings from new findings and address localization in relation to beta-selection.

We have made this distinction explicit throughout. Zhou et al. established the two-population framework, their cortical versus medullary/cortico-medullary localization, their broad embryonic versus adult hematopoietic origins, and their age-associated remodeling. Building on that foundation, the present study contributes: (i) a prospective TIMD4/VCAM1 gating strategy linked to MafB and Csf1r reporters; (ii) intravascular-labeling evidence that both VCAM1+ populations are predominantly parenchymal; (iii) transcriptomic definition of efferocytic versus antigen-presentation/interferon programs; (iv) evidence that total TM abundance is maintained independently of SpiC; (v) selective CCR2 dependence of TIMD4- VCAM1+ macrophages and thymic monocytes; and (vi) functional evidence that the Csf1r-expressing thymic myeloid compartment supports DN3-to-DN4 progression. We use the anatomical localization established by Zhou et al. as the relevant biological context and reserve our new conclusions for the endpoints measured here. Accordingly, the FTOC phenotype is assigned to the Csf1r-expressing myeloid compartment rather than specifically to cortical TIMD4+ macrophages.

The Introduction now clearly separates established knowledge from the questions addressed here, and the Results and Discussion explicitly identify the study-specific advances. Reference 27 has also been corrected to the final eLife publication.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) Trajectory analysis

The Abstract mentions trajectory analysis, but there is no corresponding Results section.

We added a Results paragraph describing the Monocle3 analysis and integrated its interpretation with the fate-mapping and CCR2 experiments. Rooting the trajectory in Ly6c2+ Ccr2+ monocytes produces a transcriptional continuum toward macrophage states, supporting progressive acquisition of macrophage programs from a monocyte-like state. We also explain why pseudotime is complementary to, but not a substitute for, genetic lineage information: it models transcriptional relationships and therefore is not used to infer direct conversion between the two mature TM populations. This rationale allows the analysis to contribute meaningfully without asking it to resolve ontogeny on its own.

The trajectory Methods were clarified and a new Results paragraph was added after the scRNA-seq specialization analysis; the Abstract now summarizes the result at the appropriate level.

(2) Comparison with the Zhou et al. gating strategy

Compare the TIMD4/VCAM1 gating strategy with the CD64/F4/80/TIMD4 definition used by Zhou et al., including the identity of TIMD4-VCAM1- cells.

We now map the two VCAM1+ gates directly onto the established TM framework. TIMD4+ VCAM1+ cells correspond to the TIMD4+ cortical population, whereas TIMD4- VCAM1+ cells show the CX3CR1 enrichment expected of the medullary/cortico-medullary population. VCAM1 therefore adds a useful prospective discriminator within the CD64+ F4/80+ parent gate. The TIMD4- VCAM1- gate is MafB-low/negative, Csf1r-EGFP-high, and enriched for Ly6C, CCR2, and CX3CR1, supporting its designation as monocyte-enriched. We use “monocyte-enriched” because it accurately captures the dominant phenotype without implying that every event in the gate is developmentally identical.

The first Results section and Figures 1-2 Legends now explain this correspondence and terminology.

(3) Difference in IV-CD45 labeling

Why are approximately 40% of Ly6C+CD11b+ cells labeled, but only approximately 10% of TIMD4-VCAM1- cells?

The percentages arise from different denominators. Ly6C+ CD11b+ is a broad myeloid gate that contains both blood-exposed and parenchymal cells. TIMD4- VCAM1- is a narrower population defined within the CD64+ F4/80+ parent gate and therefore samples a different compartment. We now make that gating relationship explicit. The i.v.-labeling experiment consequently supports two positive conclusions: both VCAM1+ macrophage populations are predominantly parenchymal, and the broad Ly6C+ CD11b+ compartment contains a substantially larger blood-exposed component.

The i.v.-labeling Results paragraph and Figure 2 Legend now describe the two gates and their distinct denominators.

(4) SpiC requirement in individual TM populations

Determine proportions and numbers of TM subpopulations in Spic-/- mice.

The biological rationale for this suggestion is strong because Spic is enriched in TIMD4+ VCAM1+ macrophages. Figure 3C, however, quantifies the aggregate CD64+ F4/80+ TM compartment. We therefore revised the conclusion to the level directly supported by that experiment: total TM abundance is maintained in Spic-/- mice despite the expected loss of splenic red pulp macrophages. This is an informative distinction because it shows that the overall thymic macrophage compartment does not share the obligate SpiC dependence of red pulp macrophages, even though a subset-selective quantitative or functional effect remains a question for future work.

The Figure 3 Results paragraph, Discussion, and legend now state preservation of total TM abundance rather than making a population-by-population claim.

(5) Tomato/GFP double-positive cells

Many cells appear to express both GFP and tdTomato. What does this mean?

We expanded the explanation of the mTmG reporter. Flt3-Cre-mediated recombination initiates a switch from membrane Tomato to membrane GFP, but the pre-existing membrane Tomato protein need not disappear instantaneously. Tomato+ GFP+ events are therefore consistent with recent/incomplete reporter transition or persistence of stable Tomato protein after recombination. We do not treat them as a third ontogenetic lineage. The key comparative result is the distribution of reporter histories across populations: TIMD4+ VCAM1+ macrophages retain a large FLT3-independent fraction, whereas TIMD4- VCAM1+ macrophages and monocytes show substantially greater FLT3 history.

The fate-mapping Results paragraph and Figure 6 Legend now define the reporter transition and the interpretation of double-positive events.

(6) Direct AP20187 toxicity in DN thymocytes

Test AP20187 toxicity in MaFIA and control DN thymocytes, for example by active caspase-3 staining.

The proposed caspase-3 analysis is designed to determine whether a DN thymocyte fraction expresses sufficient MaFIA transgene to be directly affected. The existing controls establish two important features of the result: AP20187 does not reduce TM numbers in non-transgenic C57BL/6 FTOCs, demonstrating transgene dependence, and the developmental response is patterned rather than global, with preserved γδ T cell output, DN3 accumulation, DN4 loss, and reduced CD27 expression. These convergent observations support the conclusion that integrity of the Csf1r-expressing myeloid compartment is required for efficient DN3-to-DN4 progression. We now state that conclusion prominently and note the remaining question of thymocyte-intrinsic transgene activity once, in the focused Discussion paragraph.

The MaFIA Results now emphasize the transgene-dependent control and convergent developmental measurements; the Discussion states the remaining cellular-resolution issue in one focused paragraph.

Reviewer #2 (Recommendations for the authors):

(1) Figure 1 color annotation

Clarify whether the color annotation in panel C is applied to panel F.

Yes. Panel F evaluates MafB-mCherry and Csf1r-EGFP reporter expression within the same TIMD4/VCAM1-defined populations shown in panel C. We now state this explicitly so the reporter patterns can be interpreted as independent support for the identity of each prospectively defined gate. We also removed the previous reference to immunofluorescence localization because localization data are not displayed in Figure 1.

Figure 1 Legend revised.

(2) Figure 2 controls, thresholds, and population identities

Provide staining controls and thresholds; reconsider categorical “lack” statements; clarify Ly6C+CD11b+ and CD64+F4/80+ populations and the apparent IV-CD45 discrepancies.

The underlying interpretive point is whether marker expression is categorical or relative. We revised the text to describe Ly6C, CCR2, and CX3CR1 comparatively, which more faithfully represents continuous flow-cytometric measurements. This improves the biological conclusion: both VCAM1+ populations are Ly6C-low/negative and CCR2-low relative to the TIMD4- VCAM1- monocyte-enriched population, while CX3CR1 is enriched in TIMD4- VCAM1+ relative to TIMD4+ VCAM1+ macrophages. We also define the broad Ly6C+ CD11b+ comparison gate, the CD64+ F4/80+ macrophage parent gate, and the narrower TIMD4/VCAM1 subgates. With those denominators made explicit, the iv-CD45 measurements become internally consistent rather than apparently contradictory.

The Figure 2 Results paragraph and legend now use relative marker language and explain the gate hierarchy.

(3) Figure 3 colors, VCAM1, and SpiC populations

Explain panel B colors and the apparent Vcam1 difference; provide individual TM-population data in Spic-/- mice.

Panel B is a row-scaled expression heatmap: yellow and purple indicate relatively higher and lower scaled expression for each gene, respectively. These colors should not be read as absolute expression or compared directly with antibody fluorescence, because transcript abundance and cell-surface protein are regulated at different levels and have different dynamic ranges. The legend now makes this distinction explicit. Vcam1 transcript enrichment is nevertheless consistent with VCAM1 protein being a useful surface discriminator in our gating scheme. For SpiC, we now limit the conclusion to the measurement displayed in panel C, as preservation of total CD64+ F4/80+ TMs, while explaining the biological significance of their divergence from SpiC-dependent splenic red pulp macrophages.

Figure 3 Results, Discussion, and Legend revised.

(4) Figure 4 genes not shown

H-2K, H-2D, and Runx3 are described but not shown in the figure.

We clarified the division of information between Figure 4 and Table 1. Figure 4 displays pathway-level GO enrichment, which supports the higher-order conclusion that TIMD4- VCAM1+ macrophages are enriched for antigen-presentation and interferon-response programs. Individual genes contributing to the subset signatures, including H2-K1, H2-D1, and Runx3, are reported in Table 1. The revised wording no longer implies that those individual genes are plotted in Figure 4.

Figure 4 Results paragraph and Legend revised.

(5) Figure 6 double-positive reporter cells

Explain Tomato+GFP+ cells and their relationship to single-positive cells.

As described in our response to Reviewer 1, we now explain the kinetics of the mTmG reporter switch and interpret double-positive events as reporter-transition/persistence events rather than a separate lineage. This interpretation focuses the analysis on the biologically informative comparison— the different balance of FLT3-independent and FLT3-history labeling among TIMD4+ VCAM1+, TIMD4- VCAM1+, and monocyte-enriched populations.

Fate-mapping Results and Figure 6 Legend revised.

(6) CX3CR1 phenotype in Figures 6 and 7

The text describes CX3CR1 phenotype without showing the data.

We now describe Figures 6 and 7 using the markers actually displayed in those panels: TIMD4 and VCAM1. The relationship to CX3CR1 is established independently in Figure 2 and in the integrated scRNA-seq analysis. This separation makes the evidentiary chain clearer: Figure 2 links the surface-defined populations to CX3CR1 phenotype, Figure 6 compares their FLT3 reporter histories, and Figure 7 tests their CCR2 dependence.

Corresponding Results language and Figures 6-7 Legends revised.

(7) Figure 8 Csf1r-EGFP in thymocytes/DCs and “45.2”

Show Csf1r-EGFP expression in thymocytes and DCs; determine whether their reduction is indirect or direct; clarify 45.2.

The reviewer highlights why the MaFIA result should be interpreted at the Csf1r-expressingcompartment level. The reporter is demonstrably expressed by both TM populations and thymic monocytes, and AP20187 causes transgene-dependent TM depletion in FTOC. DC reduction may reflect direct transgene activity, dependence on the altered myeloid niche, or both; similarly, direct transgene expression was not measured in fetal DN thymocytes. We now make that cellular resolution explicit while emphasizing the developmental conclusion supported by the complete phenotype. We also clarify that CD45.2 denotes the congenic allele of the C57BL/6 control and not a distinct treatment or numerical value.

MaFIA Results, Discussion, and Figure 8 Legend revised.

(8) Figure 10 CD27 profiles

Show CD27 flow-cytometric profiles.

We revised the text and legend to make clear that Figure 10 reports quantified frequencies of CD27+ DN3 and DN4 cells. The CD27 measurement is interpreted in conjunction with, rather than in isolation from, the DN-stage data. Reduced CD27 among DN3 cells, reciprocal DN3 accumulation and DN4 loss, and reduced downstream DP output form a coherent sequence that localizes the developmental impairment to the b-selection-associated transition. We therefore use CD27 as a correlate of successful pre-TCR-associated progression rather than claiming direct biochemical measurement of pre-TCR signaling.

Final Results paragraph, Discussion, and Figure 10 Legend revised.

(9) Supplemental Figure 2 schemes

Improve the schemes and explain dLNGFR and AP20187.

The revised Figure Legend now explains each functional element. dLNGFR is the membrane-targeting low-affinity nerve growth factor receptor segment within the MaFIA fusion construct and is not used here as a lineage marker. AP20187 is a synthetic homodimerizer that binds the engineered FKBP domains, bringing the Fas intracellular domains together and initiating apoptosis in transgene-expressing cells. These definitions make the logic of the depletion system understandable without requiring familiarity with the original MaFIA construct.

Supplemental Figure 2 Legend revised.

Closing statement

The revised manuscript now presents the study in clear, evidence-matched terms. It identifies the advances in TM phenotypic organization, transcriptional specialization, developmental contribution, SpiC independence of total TM abundance, and CCR2 dependence of the TIMD4- VCAM1+ population. It also emphasizes the convergent evidence that Csf1r-expressing myeloid cells support progression through the DN3-to-DN4 checkpoint, while accurately defining the cellular resolution of the MaFIA experiment. We thank the editors and reviewers for helping us sharpen both the significance and the precision of these conclusions.

eLife Assessment

AIRE has been well known to contribute to immune self-tolerance in the thymus by expressing auto-antigens. In this manuscript, the authors describe unexpected findings about the interaction of AIRE with AID in B cells, and its function in the immune system, thereby contributing to a fundamental understanding of the broader functions of AIRE. The strength of this manuscript is that, by employing biochemical and genetic experiments, the authors convincingly show interaction between AIRE and AID and subsequent AIRE's function in the GC responses.

Reviewer #1 (Public review):

Summary:

The authors provide in vivo and in vitro evidence for an interaction between AIRE and AID. This has implications for the dynamics of the germinal center response and autoimmunity related to the APSI disease.

Strengths:

Several both biochemical and in vivo experiments to show interaction and the function of AIREs regulation of AID activity in the GC response.

Comments on revised version.

I believe the manuscript is improved.

Reviewer #2 (Public review):

Summary:

In this study, Zhou et al investigated the expression and function of AIRE in B cells in peripheral lymphoid tissues. First, they found the expression of AIRE protein in mature B cells in the follicles in human tonsils and spleens from healthy donors. Flow cytometry analyses using human samples as well as Aire-reporter mice demonstrated AIRE expression in germinal center B cells. The expression of Aire in B cells was induced by CD40 signals. Then, to investigate the impact of AIRE deficiency on B cell function, the authors used a method of transplanting bone marrow cells from Aire-KO and WT mice into B-cell-deficient mice, comparing B cell development and function reconstituted in the recipient mice. Their results showed that Aire-deficient B cells strongly responded to immunization with antigens, exhibiting enhanced class switching and somatic hypermutation of antibodies compared with WT B cells. The same phenomena were observed in CRISPRed B cell lines lacking Aire. The authors successfully utilized the Aire-deficient B cell line to demonstrate that Aire suppresses antibody class switching and somatic hypermutation via its interaction with AID. Finally, using B cell transfer into B cell-deficient mice demonstrated that mice harboring Aire-deficient B cells produced high levels of autoantibodies against Th17 cytokines and exhibited reduced resistance to Candida infection. This mirrors characteristic symptoms in AIRE-deficient patients. The findings of this study not only reveal an unexpected function of AIRE in B cells but also have the potential to contribute to understanding the pathogenesis of APECED and offering a new direction for developing therapies.

Strengths:

The strength of this study lies in demonstrating the expression of function of AIRE in B cells in both mice and humans. It also revealed the direct interaction between AIRE and AID, along with its binding mode (requiring CARD and NLS domains of AIRE), and showed that this interaction is crucial for AIRE function in B cells. It is also significant that the study demonstrated how B cell-intrinsic dysfunction of AIRE leads to autoantibody production against cytokines.

Comments on revised version.

My previous concerns have been properly addressed.

Author response:

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

eLife Assessment

AIRE has been well known to contribute to immune self-tolerance in the thymus by expressing auto-antigens; in this manuscript, the authors describe unexpected findings about the interaction of AIRE with AID in B cells, and its function in the immune system, thereby contributing to a fundamental understanding of the broader functions of AIRE. The strength of this manuscript is that, by employing biochemical and genetic experiments, the authors convincingly show interaction between AIRE and AID and subsequent AIRE's function in the GC responses. However, two weak points exist: first, the connection between AIRE, auto-anti IL17 Abs, and IL17-positive effector T cells, and second, like the thymus, expression of auto-antigens by AIRE in the GC B cells has not been tested.

We thank the journal editors for the thoughtful and constructive evaluation of our data and for recognizing the significance, strengths and weaknesses of our study. We have addressed these major weak points in the responses below and have also provided discussion on the important aspect of whether AIRE regulates autoantigen expression in GC B cells analogous to its role in mTECs, which certainly warrants future investigation.

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors provide in vivo and in vitro evidence for an interaction between AIRE and AID. This has implications for the dynamics of the germinal center response and autoimmunity related to the APSI disease.

The manuscript describes an unexpected function of AIRE, which is more well known for its function to regulate negative selection of T cells in the thymus. Here, the gene has also been shown to be expressed by B cells (Immunity 2015: 26070482). They describe that AIRE interacts with AID, and in its absence, B cells acquire more hypermutations and also produce autoantibodies against IL-17. These autoantibodies have been described previously.

Strengths:

The study is interesting and provides some additional information about how AIRE regulates immune cell function. Several biochemical and in vivo experiments show the interaction and the function of AIREs in the regulation of AID activity in the GC response.

Weaknesses:

Some of the hypothetical consequences of this regulation are not investigated. This includes responses to model antigens and dynamics of the germinal center related to kinetics.

We are grateful for the reviewer’s interest and careful and thoughtful review of the manuscript.

Major Comments:

(1) AID regulates both switch and somatic hypermutation. Switch is easier to achieve, so which of these processes does AIRE influence the most? Also, the switch is thought to occur before the B cell enters the GC. Looking at the histology, is AIRE also expressed at the early proliferative stage that has been described by Ann Haberman?

We thank the reviewer for raising this provocative point. Yes, it has been recently shown that CSR occurs earlier after B cell activation largely prior to GC entry (Roco et al. 2019), and CSR is generally easier to induce in vitro than SHM. These data and observations further support well-established findings that CSR and SHM involve distinct mechanisms and pathways to resolve DNA lesions (Frossi et al., 2019; Masani et al., 2013; Roco et al., 2019; Schrader et al., 2023), even though both require AID. We observed a strong upregulation of AIRE expression upon CD40 signaling (Fig. 2C–G) and that AIRE in B cells negatively regulates both CSR and SHM (Fig. 3E– J and Fig. 4A–H), but it would be difficult to directly compare the magnitude of AIRE’s influence between them because they are mechanistically distinct and occurs at different stages and locations during antigen-specific B cell responses.

We observed some AIRE-expressing B cells outside of the GC (Fig. 1A–C, J), some of which could represent newly activated cells about to enter the GC reaction, but we did not perform imaging or flow cytometry experiments or track their Ki67 or Bcl6 expression in the Aire^Adig reporter mice. However, these cells appear to be largely IgD^-, suggesting that they may have already undergone CSR and are perhaps not (entirely) the early proliferative B cells described by Dr Haberman.

(2) In experiments determining anti-CD40-dependent upregulation of AIRE, naïve resting B cells were used from mice. A proportion of the B-cells got activated. Are these MZB or FOB cells as MZBs are more easily activated?

We thank the reviewer for this careful interpretation of our data. Indeed, the methods used to purify naïve B cells do not exclude MZBs and, as they are more sensitive to activation, may also express AIRE upon stimulation with CD40L. However, although we cannot exclude MZBs in these cultures, they represent less than 10% of the total B cells in these cultures whereas we observed approximately 30% of the cells upregulating AIRE expression.

Interestingly, Yamano et al. (Yamano et al., 2015) suggested that GC B cells may not express AIRE due to strong BCR signaling; however, it is known that GC B cells have attenuated BCR signaling to promote LZ to DZ transition, consistent with the timing of AIRE upregulation postCD40-CD40L engagement (Davidzohn et al., 2020; Khalil et al., 2012). In contrast, MZBs are known to exhibit greater baseline activation of pathways downstream of BCRs (Hampel et al., 2011), which may provide inhibitory signals for the upregulation of AIRE.

(3) In the BM chimeric experiments in Figure 3. Do the AIRE+ and AIRE - populations distribute equally among B cell subpopulations?

We thank the reviewer for this interesting comment. In our chimera experiment, although we did not analyze specific subsets such as B-1 or MZB, we observe that AIRE-deficient B cells outcompeted WT B cells in lymphoid organs as well as in the blood in spite of prior publications showing equivalent reconstitution of CD45.2 mice with CD45.1/CD45.2 B cells (Kalari Kandy et al., 2023).

(4) Furthermore, in the NP-KLH experiments, one would expect that B cells with increased affinity would leave the GC earlier and become plasma cells. Thus, the kinetics of the AIRE+ vs AIRE- B cells within the GC would be different? Also, would they maybe take over at some point, as the increased affinity would favor help from Tfh cells that are known to be limited?

We thank the reviewer for these insightful comments. Yes, we would expect to see that Aire^-/- B cells will dominate GCs over time. Indeed, our chimera experiments (Fig. 3A–D) indicated that Aire^-/- B cells were of higher frequency in the GCs compared to WT post-immunization, consistent with the expansion of higher affinity clones. However, although we did not quantify the development of PCs in these mice, our adoptive transfer experiments showed that mice receiving Aire^-/- B cells developed higher affinity antibodies compared to those receiving WT B cells (Fig. 3G), indicating a higher affinity plasma cell pool compared to controls.

(5) Given the previous studies on AIRE's function in regulating transcription (PMID: 34518235), how does this interaction fit into this picture?

We thank the reviewer for raising this important point. Although we did not directly test the role of AIRE in the regulation of tissue-restricted antigens, we observed a clear interaction between AIRE and pSer5 Pol II (Fig. 6F), consistent with its function as a broad-spectrum transcriptional regulator (Fang et al., 2024; Giraud et al., 2012; Oven et al., 2007). This is particularly important for antibody diversification in B cells, as it is known that AID is targeted to sites of Pol II pausing (Chaudhuri et al., 2003; Pavri et al., 2010). Further, recent reports have shown that the CARD domain of AIRE promotes its polymerization and the formation of nucleation sites at which a positive feedback loop to create transcriptional hubs (Huoh et al., 2024). Interestingly, the CARD domain is also necessary for the interaction between AIRE and AID (Fig. 5F), indicating that either these condensates are also critical for preventing AID from being recruited to Pol II or that AIRE may perform alternative functions in B cells compared to mTECs. Nevertheless, in this current manuscript, we focus on the capacity of AIRE to utilize this interaction with pSer5 Pol II to prevent AID localization to its DNA substrates, however, future studies may focus on how this interaction may impact the expression of peripheral tissue antigens to promote T cell tolerance.

(6) In the uracil experiments, the readout for AID to induce double-stranded breaks could be tested.

We thank the reviewer for this suggestion for complementary data to our uracil analyses. In our manuscript, we tested the generation of uracil in Aire^+/+ and Aire^-/- CH12 cells since this is a downstream function of AID’s activity. Therefore, our data focused on an immediate and direct impact of AIRE on AID’s activity. Further, although we observe an interaction between AIRE and AID as well as a downstream functional consequence, it is unclear whether AIRE may also impact other pathways in these cells after activation that may confound the results of downstream analyses, such as DSBs. We agree that DSBs would be an interesting readout further downstream, and future work may focus on the function of AIRE in these additional processes.

(7) The candida experiments are a nice connection to the situation in patients. However, why is it mostly auto-antibodies against IL-17? How about other immune responses, as well as T cellindependent type I and II responses?

We thank the reviewer for raising this important point. Indeed, we observed a significant increase in the generation of autoreactive, neutralizing antibodies against Th17-associated cytokines (Fig. 7D, E). However, although AIRE-deficient patients produce neutralizing antibodies against type 1 interferons as well, clearance of the fungal pathogen Candida albicans relies heavily on IL-17 to promote the upregulation of antimicrobial peptides and neutrophil infiltration (Conti et al., 2014), which may be a particularly pronounced response induced in our mouse models. Therefore, we targeted our assay towards these cytokines, but do not rule out possible production of autoantibodies against other factors. It would be interesting for additional studies to determine the production of autoreactive antibodies against cytokines in other infection models, such as viral infections, as APS-1 patients have been reported to display an increased susceptibility to viral infections as well (Bastard et al., 2021; Hetemaki et al., 2021; Oikonomou et al., 2021).

These important discussions have been included in the revised manuscript.

Reviewer #2 (Public review):

Summary:

In this study, Zhou et al investigated the expression and function of AIRE in B cells in peripheral lymphoid tissues. First, they found the expression of AIRE protein in mature B cells in the follicles in human tonsils and spleens from healthy donors. Flow cytometry analyses using human samples as well as Aire-reporter mice demonstrated AIRE expression in germinal center B cells. The expression of Aire in B cells was induced by CD40 signals. Then, to investigate the impact of AIRE deficiency on B-cell function, the authors used a method of transplanting bone marrow cells from Aire-KO and WT mice into B-cell-deficient mice, comparing B-cell development and function reconstituted in the recipient mice. Their results showed that Aire-deficient B cells strongly responded to immunization with antigens, exhibiting enhanced class switching and somatic hypermutation of antibodies compared with WT B cells. The same phenomena were observed in CRISPRed B cell lines lacking Aire. The authors successfully utilized the Aire-deficient B cell line to demonstrate that Aire suppresses antibody class switching and somatic hypermutation via its interaction with AID. Finally, using B cell transfer into B cell-deficient mice demonstrated that mice harboring Aire-deficient B cells produced high levels of autoantibodies against Th17 cytokines and exhibited reduced resistance to Candida infection. This mirrors characteristic symptoms in AIRE-deficient patients. The findings of this study not only reveal an unexpected function of AIRE in B cells but also have the potential to contribute to understanding the pathogenesis of APECED and to offering a new direction for developing therapies.

We are grateful for the reviewer’s careful and thoughtful review of the manuscript and appreciation of the significance of our findings.

Strengths:

The strength of this study lies in demonstrating the expression of the function of AIRE in B cells in both mice and humans. It also revealed the direct interaction between AIRE and AID, along with its binding mode (requiring CARD and NLS domains of AIRE), and showed that this interaction is crucial for AIRE function in B cells. It is also significant that the study demonstrated how B-cell-intrinsic dysfunction of AIRE leads to autoantibody production against cytokines.

Weaknesses:

As for loss-of-function analysis of Aire in B cells, in addition to the B cell transfer from Aire-KO mice performed in this study, generating B cell-specific Aire-deficient mice using Aire-flox mice (Dobes et al, Eur J Immunol 2018) would further reinforce the conclusions of this study. Furthermore, the relationship with Aire function in thymic B cells reported by previous studies remains unclear, posing an unresolved challenge. This study also failed to address whether Aire deficiency affects gene expression in GC B cells, in particular, whether it induces the expression of various self-antigens as reported in thymic B cells or mTECs.

We thank the reviewer for these thoughtful critiques of our manuscript. As this study was largely carried out prior to the development of Aire-flox mice, we took advantage of the adoptive transfer model to generate mice with AIRE deletion specifically in B cells. Since Aire-flox mice were developed, these mice would be the new gold standard for analysis and would provide further strength to our existing data. Unfortunately, these mice are not readily available, and we do not currently have the resources to reconstitute this line and are unable to obtain them.

In addition, although we do not directly test the role of AIRE in the regulation of tissue-restricted antigens, we observed a clear interaction between AIRE and pSer5 Pol II (Fig. 6F), consistent with its function as a transcriptional regulator (Fang et al., 2024; Giraud et al., 2012; Oven et al., 2007). This is particularly important for antibody diversification in B cells, as it is known that AID is targeted to sites of Pol II pausing (Chaudhuri et al., 2003; Pavri et al., 2010). Further, recent reports have shown that the CARD domain of AIRE promotes its polymerization and the formation of nucleation sites at which a positive feedback loop creates transcriptional hubs (Huoh et al., 2024). Interestingly, the CARD domain is also necessary for the interaction between AIRE and AID (Fig. 5F) indicating that either these condensates are also critical for preventing AID from being recruited to Pol II or that AIRE may perform alternative functions in this subset of B cells compared to mTECs. Nevertheless, in this study, we focused on the capacity of AIRE to utilize this interaction with pSer5 Pol II to reduce AID targeting to its DNA substrates. However, future studies may focus on how this interaction may impact the expression of peripheral tissue antigens to promote T cell tolerance.

The discussion related to these important aspects raised by the reviewer are included in the original and revised manuscript.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) How do these findings connect to IgA responses and the ongoing GC response in the peyers patches?

We thank the reviewer for raising this additional point. Indeed, we also observed AIRE expression in GC B cells from Peyer’s patches (Fig. S1J) and found that CSR to IgA in our in vitro models was increased by the deletion of AIRE in B cells (Fig. 4A–C). These data suggest that, in AIRE-deficient B cells, there may be an increase in IgA production, however, we did not directly test mucosal IgA levels in our adoptive transfer models. Future work may focus on the role of B cell intrinsic AIRE and its impact on the capacity of Peyer’s patch-associated B cells to produce IgA and its role on mucosal barrier immunity and microbiota coating.

These important discussions have been included in the revised manuscript.

(2) Some of the histology is rather dark, and it would be nice to see some more examples in the supplement.

We thank the reviewer for bringing this to our attention. We have included an additional staining of a whole follicle with GC in the supplement as requested (Fig. S1C, bottom panel).

Reviewer #2 (Recommendations for the authors):

(1) In the Discussion section, could the authors explore Aire's functions and biological significance in greater depth? Although Aire was originally identified as a regulator of tissue-restricted antigen expression in mTECs, it has subsequently shown to play crucial roles in diverse processes, including germ cell development, antigen processing, and B cell function regulation (this study). In light of these recent findings, it is conceivable that Aire did not evolve specifically as an mTECassociated gene. Rather, the reverse may be possible: Aire may have originally emerged as a regulator of germ cell development and/or immune responses, with its expression and functions later co-opted by mTECs (analogous to how other transcription factors have been adopted for differentiation of mimetic mTECs). I would welcome the authors expanding on this evolutionary perspective in future work or revisions.

We thank the reviewer for this insightful and fascinating perspective on the evolutionary and ancestral functions of AIRE as a transcription factor. We have incorporated additional discussion in the revised manuscript.

(2) Personally, I knew that Aire is expressed in B cells, as indicated by several gene expression databases. But the ImmGen database shows that Aire mRNA expression is detected at higher levels in plasma cells than in GC B cells. What do the authors think?

We sincerely thank the reviewer for this question and appreciate the personal comment in support of our results. Indeed, the ImmGen database shows little Aire expression in GC B cells, with most of the signal coming from plasma cells in the B cell compartment. Further, as the Reviewer suggested, reanalysis of previously published scRNA-seq (Duan et al., 2021) has shown Aire transcript in some, albeit a lower percentage of, GC B cells (Author response image 1), which was more abundant near the peak time of the immune response after immunization, in line with our and the Reviewer’s observations.

Author response image 1.

Analysis of Aire expression in the day 7 (upper row) and day 14 (lower row) post-immunization scRNA-Seq datasets (Duan et al., 2021), showing 200 Aire⁺ GC B cells out of 3542 GC B cells (5.65%) on day 7 and 183 Aire⁺ GC B cells out of 19757 GC B cells (0.93%) on day 14, and the distribution of these Aire⁺ GC B cells in both dark zone (DZ) and light zone (LZ) B cells.

A myriad of technical factors can impact the detection of lower-expressed genes by bulk RNA-seq and even scRNA-seq, and that looking at individual cells may give better resolution. This is the case for a considerable number of other genes we have studied or that have been reported in other investigators’ publications. Many biological factors can also have an important effect (e.g. protein levels may vary depending on cell states and do not necessarily correlate with transcript levels). Significant impact of the strain background, diet, microbiome and the living environment of the animals cannot be excluded either. Similarly, recent reports have shown that CGRP from spleen-innervating nociceptors signals to splenic B cells to promote germinal center reactions (Wu et al., 2024). However, Immgen showed no Ramp1 expression in splenic B cell compartments. In addition, Nur77/Nr4a1 has also been shown to be highly expressed by a small number of light zone cells and is critical for regulating GC reactions (Brooks et al., 2021; Mueller et al., 2015) even though ImmGen shows very little expression of this gene in its bulk sequencing. Therefore, we think that much of the data from ImmGen should be carefully validated with additional experimental methods. In this case, we were able to detect AIRE protein using methods including immunofluorescence, immunoprecipitation, western blot, and flow cytometry.

(3) Line 343: "donor Aire-/-" may be a mistake for "donor Aire+/+"?

We thank the reviewer for bringing this to our attention and have adjusted the manuscript accordingly.

(4) Line 346: Is "data now shown" a spelling mistake? Including this, there are multiple occurrences of "data not shown" in the Results and Discussion sections. If the authors describe or discuss the data, it should be shown.

We thank the reviewer for bringing this to our attention and have revised the manuscript accordingly.

(5) Line 346-350: The authors should investigate more thoroughly whether Aire influences gene expression in GC B cells, particularly the expression of a set of self-antigen genes, as demonstrated in thymic B cells by Yamano et al (Immunity 2015). Regardless of the outcome, such data would be highly significant for this study and the broader research community.

We thank the reviewer for raising this important point. Although we did not directly test the role of AIRE in the regulation of tissue-restricted antigens, we observed a clear interaction between AIRE and pSer5 Pol II (Fig. 6F), consistent with its function as a transcriptional regulator (Fang et al., 2024; Giraud et al., 2012; Oven et al., 2007). This is particularly important for antibody diversification in B cells, as it is known that AID is targeted to sites of Pol II pausing (Chaudhuri et al., 2003; Pavri et al., 2010). Further, recent reports have shown that the CARD domain of AIRE promotes its polymerization and the formation of nucleation sites at which a positive feedback loop to create transcriptional hubs (Huoh et al., 2024). Interestingly, the CARD domain is also necessary for the interaction between AIRE and AID (Fig. 5F) indicating that either these condensates are also critical for preventing AID from being recruited to Pol II or that AIRE may perform alternative functions in B cells compared to mTECs. Nevertheless, in this current study, we focused on the capacity of AIRE to utilize this interaction with pSer5 Pol II to prevent AID targeting to its DNA substrates, and future studies may focus on how this interaction may impact the expression of peripheral tissue antigens to promote T cell tolerance.

(6) Line 1282-1293: The number of B cells used for Western blotting should be clearly indicated. Since the expression levels of Aire mRNA in B cells are lower than those in mTECs, detecting Aire protein in B cells is also likely difficult. It is important to specify the number of B cells required so that other researchers can reproduce the Western blotting results presented in this paper.

We thank the reviewer for raising this important point and have revised the manuscript accordingly.

References

Bastard, P., E. Orlova, L. Sozaeva, R. Levy, A. James, M.M. Schmitt, S. Ochoa, M. Kareva, Y. Rodina, A. Gervais, T. Le Voyer, J. Rosain, Q. Philippot, A.L. Neehus, E. Shaw, M. Migaud, L. Bizien, O. Ekwall, S. Berg, G. Beccuti, L. Ghizzoni, G. Thiriez, A. Pavot, C. Goujard, M.L. Fremond, E. Carter, A. Rothenbuhler, A. Linglart, B. Mignot, A. Comte, N. Cheikh, O. Hermine, L. Breivik, E.S. Husebye, S. Humbert, P. Rohrlich, A. Coaquette, F. Vuoto, K. Faure, N. Mahlaoui, P. Kotnik, T. Battelino, K. Trebusak Podkrajsek, K. Kisand, E.M.N. Ferre, T. DiMaggio, L.B. Rosen, P.D. Burbelo, M. McIntyre, N.Y. Kann, A. Shcherbina, M. Pavlova, A. Kolodkina, S.M. Holland, S.Y. Zhang, Y.J. Crow, L.D. Notarangelo, H.C. Su, L. Abel, M.S. Anderson, E. Jouanguy, B. Neven, A. Puel, J.L. Casanova, and M.S. Lionakis. 2021. Preexisting autoantibodies to type I IFNs underlie critical COVID-19 pneumonia in patients with APS-1. The Journal of experimental medicine 218:

Brooks, J.F., C. Tan, J.L. Mueller, K. Hibiya, R. Hiwa, V. Vykunta, and J. Zikherman. 2021. Negative feedback by NUR77/Nr4a1 restrains B cell clonal dominance during early Tdependent immune responses. Cell Rep 36:109645.

Chaudhuri, J., M. Tian, C. Khuong, K. Chua, E. Pinaud, and F.W. Alt. 2003. Transcription-targeted DNA deamination by the AID antibody diversification enzyme. Nature 422:726-730.

Conti, H.R., A.R. Huppler, N. Whibley, and S.L. Gaffen. 2014. Animal models for candidiasis. Curr Protoc Immunol 105:19.16.11-19.16.13.

Davidzohn, N., A. Biram, L. Stoler-Barak, A. Grenov, B. Dassa, and Z. Shulman. 2020. Syk degradation restrains plasma cell formation and promotes zonal transitions in germinal centers. The Journal of experimental medicine 217:

Duan, L., D. Liu, H. Chen, M.A. Mintz, M.Y. Chou, D.I. Kotov, Y. Xu, J. An, B.J. Laidlaw, and J.G. Cyster. 2021. Follicular dendritic cells restrict interleukin-4 availability in germinal centers and foster memory B cell generation. Immunity 54:2256-2272 e2256.

Fang, Y., K. Bansal, S. Mostafavi, C. Benoist, and D. Mathis. 2024. AIRE relies on Z-DNA to flag gene targets for thymic T cell tolerization. Nature 628:400-407.

Frossi, B., G. Antoniali, K. Yu, N. Akhtar, M.H. Kaplan, M.R. Kelley, G. Tell, and C.E.M. Pucillo. 2019. Endonuclease and redox activities of human apurinic/apyrimidinic endonuclease 1 have distinctive and essential functions in IgA class switch recombination. J Biol Chem 294:5198-5207.

Giraud, M., H. Yoshida, J. Abramson, P.B. Rahl, R.A. Young, D. Mathis, and C. Benoist. 2012. Aire unleashes stalled RNA polymerase to induce ectopic gene expression in thymic epithelial cells. Proceedings of the National Academy of Sciences of the United States of America 109:535-540.

Hampel, F., S. Ehrenberg, C. Hojer, A. Draeseke, G. Marschall-Schroter, R. Kuhn, B. Mack, O. Gires, C.J. Vahl, M. Schmidt-Supprian, L.J. Strobl, and U. Zimber-Strobl. 2011. CD19independent instruction of murine marginal zone B-cell development by constitutive Notch2 signaling. Blood 118:6321-6331.

Hetemaki, I., S. Laakso, H. Valimaa, I. Kleino, E. Kekalainen, O. Makitie, and T.P. Arstila. 2021. Patients with autoimmune polyendocrine syndrome type 1 have an increased susceptibility to severe herpesvirus infections. Clin Immunol 231:108851.

Huoh, Y.S., Q. Zhang, R. Torner, S.C. Baca, H. Arthanari, and S. Hur. 2024. Mechanism for controlled assembly of transcriptional condensates by Aire. Nature immunology 25:15801592.

Kalari Kandy, R.R., X. Fan, and X. Cao. 2023. CD45.1/CD45.2 Congenic Markers Induce a Selective Bias for CD8+ T Cells during Adoptive Lymphocyte Reconstitution in Lymphocytopenia Mice. Immunohorizons 7:755-759.

Khalil, A.M., J.C. Cambier, and M.J. Shlomchik. 2012. B cell receptor signal transduction in the GC is short-circuited by high phosphatase activity. Science 336:1178-1181.

Masani, S., L. Han, and K. Yu. 2013. Apurinic/apyrimidinic endonuclease 1 is the essential nuclease during immunoglobulin class switch recombination. Mol Cell Biol 33:1468-1473.

Mueller, J., M. Matloubian, and J. Zikherman. 2015. Cutting edge: An in vivo reporter reveals active B cell receptor signaling in the germinal center. Journal of immunology 194:29932997.

Oikonomou, V., T.J. Break, S.L. Gaffen, N.M. Moutsopoulos, and M.S. Lionakis. 2021. Infections in the monogenic autoimmune syndrome APECED. Curr Opin Immunol 72:286-297.

Oven, I., N. Brdickova, J. Kohoutek, T. Vaupotic, M. Narat, and B.M. Peterlin. 2007. AIRE recruits P-TEFb for transcriptional elongation of target genes in medullary thymic epithelial cells. Mol Cell Biol 27:8815-8823.

Pavri, R., A. Gazumyan, M. Jankovic, M. Di Virgilio, I. Klein, C. Ansarah-Sobrinho, W. Resch, A. Yamane, B. Reina San-Martin, V. Barreto, T.J. Nieland, D.E. Root, R. Casellas, and M.C. Nussenzweig. 2010. Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5. Cell 143:122-133.

Roco, J.A., L. Mesin, S.C. Binder, C. Nefzger, P. Gonzalez-Figueroa, P.F. Canete, J. Ellyard, Q. Shen, P.A. Robert, J. Cappello, H. Vohra, Y. Zhang, C.R. Nowosad, A. Schiepers, L.M. Corcoran, K.M. Toellner, J.M. Polo, M. Meyer-Hermann, G.D. Victora, and C.G. Vinuesa. 2019. Class-Switch Recombination Occurs Infrequently in Germinal Centers. Immunity 51:337-350 e337.

Schrader, C.E., T. Williams, K. Pechhold, E.K. Linehan, D. Tsuchimoto, and Y. Nakabeppu. 2023. APE2 Promotes AID-Dependent Somatic Hypermutation in Primary B Cell Cultures That Is Suppressed by APE1. Journal of immunology 210:1804-1814.

Wu, M., G. Song, J. Li, Z. Song, B. Zhao, L. Liang, W. Li, H. Hu, H. Tu, S. Li, P. Li, B. Zhang, W. Wang, Y. Zhang, W. Zhang, W. Zheng, J. Wang, Y. Wen, K. Wang, A. Li, T. Zhou, Y. Zhang, and H. Li. 2024. Innervation of nociceptor neurons in the spleen promotes germinal center responses and humoral immunity. Cell 187:2935-2951 e2919.

Yamano, T., J. Nedjic, M. Hinterberger, M. Steinert, S. Koser, S. Pinto, N. Gerdes, E. Lutgens, N. Ishimaru, M. Busslinger, B. Brors, B. Kyewski, and L. Klein. 2015. Thymic B Cells Are Licensed to Present Self Antigens for Central T Cell Tolerance Induction. Immunity 42:1048-1061.

他と文体を合わせるなら「使い分けてください」

なにが、なにに対して送る量??

単位時間あたりの送信量ってこと??

スレッドセーフの方が一般的かなぁ https://ja.wikipedia.org/wiki/%E3%82%B9%E3%83%AC%E3%83%83%E3%83%89%E3%82%BB%E3%83%BC%E3%83%95

どの公式ドキュメントを引用しているのか、リンクをはってほしい

など、の手前が多いので読みにくい。全部述べたいのであれば「以下の要素の影響により~~~。◯◯の管理コスト~~~」みたいな

あと、イベント〜〜管理コスト、の管理コストがその前3つにかかっていると思うけど、全部「、」で区切られると係り受けがわかりにくいです。

たとえば

イベント〜〜の管理コストの他に、メモリ~~

のように書くとわかりやすくなると思います。

測定して、適切なイベントループ数を~~とか

整数演算回数を変えることで、CPU処理がより多い◯◯をシミュレートしてみました。

みたいな目的の説明がほしい

ここで件数を変えることはとくに意味がないので、数を合わせてほしい。

64件、合計256件とするか、一つ前のセクションを5,000リクエストとするか

これは上で「rps」と書いたもの?用語を統一してほしい

決まります。

と区切った方がいいかな。前後の文で主語が変わっているかな。

リクエストの処理時間の中央値、とか?

中央の、がよくわからなかった

このコードはどこかにないのかな? githubとかで公開してくれたらうれしいかも

これとかも io_delay で定義してあるけど呼び出し側が存在しないのでわからない。コード上でコメントを書くかデフォルト値を指定してほしい

ちょっと古い https://www.python.org/downloads/release/python-3147/

コードあってますか?コードを見ても総タスク32とか5ステップとか読み取れないです

急にネットワークの話ができたので、目的をもうちょい明確にした方がいいかなと思いました。

まずは◯◯の部分のみの速度を比較するために、小さなベンチマーク（マイクオｒベンチマーク）を作成します。このコードでは外部ネットワークやWebフレームワークなどは使用せず~~

みたいな

実行時間しか比較してなさそうなので

「実行時間を比較します」と書いてほしい。他に比較するものがあるならそれを明示してほしい

ここに書いてあるような説明をコード中にも書いてほしい。

これ多分意味のある処理じゃなくて、ただCPUを使いたいから計算しているだけですよね。ただ、そういうことをdocstringに書いてほしい。ちょっと読者に委ねすぎだと思います

日本語の方が親切だと思います。っていうかコードのcaptionと同じだったら同じ文章入れておいた方がよいかと

MUST: コードがそこそこ長さがあるので、以下のコードではこういうことをやっています、という説明が先に欲しい。 そしてコードにコメントを入れてほしい

ベンチマークじゃなくても、実際にGILなしで動いているか確認するためには重要かなと思いました

nits: Python 3.14なのでダブルクォートでおいかと。このあとの文章中での表記とあわせる

可能であれば、何を比較するか書いてほしい。

eLife Assessment

The manuscript is important in that it has the potential to advance our understanding of how liver regeneration is orchestrated after injury of pericentral hepatocytes, which are a target of many drugs and toxins. The findings are solid and suggest that midlobular hepatocytes are protected from toxin-induced injury by a genetic program delaying proliferation to prevent cell death. The effects of individual actors in the proposed program are convincingly shown but whether they act in concert and whether cell cycle arrest is indeed the protective mechanism remains to be determined.

Reviewer #2 (Public review):

The manuscript reports protection of midlobular hepatocytes from APAP toxicity by activation of Atf4-CHOP (Ddit3)-mediated cell cycle arrest and stress response. The authors acknowledge that their finding is unexpected because CHOP typically induces cell death. Therefore, they functionally validate several aspects of the proposed Atf4-CHOP mechanism. Along these lines, the mitigation of APAP toxicity by AAV expression of Atf4 or Btg2, the latter identified as CHOP effector, is impressive. Whether Atf4 indeed acts through CHOP and whether midlobular hepatocytes are protected because of cell cycle arrest is less clear. These and other criticisms are described in the following.

Major points:

(1) Starting with the basics, one wonders why midlobular hepatocytes manage to mount a defensive response to APAP but PC hepatocytes don't. Is this because midlobular hepatocytes express the relevant Cyps (2e1 but also 1a2 and 3a11) at lower levels, which mitigates toxicity and buys them time? This would be supported by F2A but not by F3B, at least not for the most important Cyp2e1. A moderate difference is shown for Cyp1a2 expression in F3D but is that enough to explain the different fates? Or are additional post-transcriptional effects on these Cyps at work? In the re-revised manuscript, it was clarified in the legends of F2A and F3B that they visualize the same data in different ways.

(2) The evidence presented in support of cell cycle arrest of midlobular hepatocytes is not fully convincing: there is no overt difference in S and G2/M gene scores in F2F; the marker genes used for S phase and G1 to S progression in F2G are unusual. Along these lines, one wonders if spatial transcriptomics confirmed the Ki67 immunostaining results in F1 also for specific zones, not only overall, as shown in F2E? The discussion and abstract of the re-revised manuscript acknowledge that spatial transcriptomics did not independently confirm cell cycle arrest in midlobular hepatocytes.

(3) The authors conclude in line 364 that halting of proliferation by Btg2 favors survival, which raises the question of whether Btg2 knockout causes death in midlobular hepatocytes in F6K. Data addressing this question, that is, localization and extent of tissue necrosis and ALT levels after APAP, are missing. The efficiency of knockout of Btg2 is also not given. Additional Btg2 knockout data support its proposed role in the revised manuscript.

(4) Related to the previous question, the BTG2 immunostaining in F6F is not convincing when compared to F6D. One also wonders if it is necessary to apply APAP to find induction of BTG2 by AAV-Ddit3? The text of the re-revised manuscript reflects that issues with immunostaining did not allow for concluding that APAP promotes nuclear localization of BTG2.

(5) Related to the previous question, the proposed Atf4-Ddit3 axis is challenged by the lack of midlobular induction of Atf4 in the APAP scRNA-seq data published by another group presented in S4F and G. Further analysis of AAV-Atf4 samples generated for F5 could address if it is really Atf4 that acts on Ddit3 in APAP toxicity. The extended list of transcription factors (from 30 to 50) includes Atf4 but direct evidence for an interaction with Ddit3 is missing from the revised manuscript. The re-revised manuscript acknowledges this limitation by referring to a Atf4-Chop (Ddit3) axis and defining that as a functional pathway, not direct interaction of the proteins.

(6) Related to the previous question, the ATF4 immunostaining in F5A doesn't look convincing, with many brown pigments appearing to be outside of the nucleus, which was addressed by adding high-magnification images to F5A of the re-revised manuscript.

(7) It is not ruled out that AAV expression of Atf4 or Btg2 reduces hepatocyte sensitivity to APAP by affecting expression of the Cyps needed for activation. In other words, does AAV-Atf4 or AAV-Btg2 change the expression of any of the Cyps relevant to APAP in the 3 weeks before APAP application (F5B)? S5A of the revised manuscript rules out loss of Cyp2e1 expression as a confounding factor.

(8) It is laudable that the authors tried to extend their findings to human by using snRNA-seq data from a published study (line 391) but it is unclear why they didn't analyze all 10 patients in that study but instead focused on 2 and stated that this small sample number prevented drawing definitive conclusions and could therefore only be mentioned in the discussion. The re-revised manuscript includes analysis of the more substantial snRNA-seq dataset (in addition to the limited spatial transcriptomics) of patients with APAP toxicity in S4-3E, which confirms the midlobular expression of stress-response genes observed in mice but differs from mice in activation of proliferation genes, which may be due to sampling at later stages of the injury response as explained in the text.

Minor points:

(1) What is the functional classification of DEG in F2A based on? GO terms? Clarified in revised manuscript.

(2) The rationale for focusing on CHOP is not clear because Ddit3 is not shown in the spatial transcriptomics in F2A and not significant in F2B, contradicting what is stated in line 206. F2A includes Ddit3 in the revised manuscript and although it is not significant in S1G (former F2B), it is among the most highly expressed transcription factors in F4B and S3B.

(3) The term "redistribution" used in line 197 to describe expression of Cyp2e1 and other Cyps in the midlobular zone seems inappropriate considering that they just continue to be expressed there whereas PC hepatocytes are dying in F3B; the same applies to "Gene Expression Shift" in F3H. Clarified in revised manuscript.

Comments on revised version.

The revised manuscript addressed many of the original points and the re-revised manuscript clearly describes remaining uncertainties, which may be technical in nature such as lack of cell cycle arrest of midlobular hepatocytes in spatial transcriptomics or could be addressed in follow-up studies such as the nature of the interaction between Atf4 and CHOP.

Author response:

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

eLife Assessment

This study addresses an important question in liver biology: how zonal hepatocytes balance survival and proliferation following injury? The authors propose that a midzone Atf4-Chop axis to Btg2 program temporarily suppresses proliferation to promote survival after a variety of chemical and surgical liver injury models. The authors provide evidence that some zones mount tailored stress responses, which ultimately promote regeneration and liver healing; however, the "mid-zone" changes with different injury models, making it difficult to conclude that the ATF4-CHOP response is specific to this zone in all injury contexts. In addition, it is possible that Atf4 and Btg2 overexpression could lead to Cyp2e1 suppression, which could reduce the extent of injury after CCl4 or APAP. To some extent, these points make the strength of the evidence incomplete, but do not entirely detract from the significance of the study, which is underscored by the helpful observation that there are zone-specific stress responses that mediate liver regeneration and survival.

We appreciate the editor’s thoughtful assessment. Regarding regional specificity, we agree that "mid-zone" patterns differ across injury models, particularly in HPx; to maintain consistency, we have removed the PHx data and now focus our conclusions on the APAP and CCl₄ models. Concerning the potential confound of Cyp2e1 suppression, our AAV overexpression resulted in only modest reductions in Cyp2e1 protein (Revised Figure 4-figure supplement 3A). Although enzymatic activity was not directly measured, basal CYP2E1 protein levels generally correlate with enzymatic activity. Furthermore, published studies indicate that protection from APAP toxicity requires >50% CYP2E1 suppression (PMID: 35145060; PMID: 30151903), whereas the reduction observed here was substantially smaller and therefore unlikely to account for the marked decrease in liver injury.

Reviewer #2 (Public review):

The manuscript reports protection of midlobular hepatocytes from APAP toxicity by activation of Atf4-CHOP (Ddit3)-mediated cell cycle arrest and stress response. The authors acknowledge that their finding is unexpected because CHOP typically induces cell death. Therefore, they functionally validate several aspects of the proposed Atf4-CHOP mechanism. Along these lines, the mitigation of APAP toxicity by AAV expression of Atf4 or Btg2, the latter identified as CHOP effector, is impressive. Whether Atf4 indeed acts through CHOP and whether midlobular hepatocytes are protected because of cell cycle arrest is less clear. These and other criticisms are described in the following.

Major points:

(1) The difference in baseline Cyp2e1 expression between F2A and F3B remains unexplained after revision.

We apologize for the confusion. The apparent discrepancy reflects different visualization methods rather than a biological inconsistency. Figure 2A and Figure 3B are derived from the same spatial transcriptomic dataset but are visualized using different normalization strategies. Figure 2A uses row-wise Z-score normalization to emphasize the relative spatial distribution of each gene across liver zones. Consequently, even a moderate spatial gradient (e.g., ~1.5–2-fold) is visually enhanced to facilitate comparison of zonation patterns between genes. In contrast, Figure 3B presents the absolute log-normalized transcript abundance without rowwise scaling. Because Cyp2e1 is highly expressed in both the pericentral and adjacent mid-zone hepatocytes, the absolute difference between these regions is relatively modest, resulting in a less pronounced visual contrast. Thus, the different appearance of Cyp2e1 between Figures 2A and 3B reflects the visualization strategy rather than conflicting expression data. To avoid further confusion, we have added this clarification to the legends of both Figures 2A and 3B.

(2) In contrast to the revised discussion, the abstract does not reflect that limited evidence for a cell cycle arrest in pericentral hepatocytes was found.

We thank the reviewer for the continued effort in improving our manuscript. To reflect the limited support of ST data for cell cycle arrest, we have revised the Abstract (Revised manuscript, page 1, line 13-26)

(3) Additional Btg2 knockout data support its proposed role in the revised manuscript.

We appreciate the reviewer’s acknowledgement of the new functional data supporting the role of Btg2.

(4) The BTG2 immunostaining remains weak, not only in in F6F but now also in F6D of the revised manuscript, which together with lack of high-resolution immunostaining of AAV-Ddit3-induced BTG2 in the absence of APAP results in limited support for the conclusion that APAP promotes nuclear localization of BTG2.

We agree that the current immunostaining does not provide definitive evidence for nuclear localization. In response, we have deliberately avoided any claims regarding nuclear translocation in the revised manuscript, instead framing our findings strictly around increased BTG2 expression and accumulation in the peri-necrotic mid-zone region following APAP injury.

(5) The extended list of transcription factors (from 30 to 50) includes Atf4 but direct evidence for an interaction with Ddit3 is missing from the revised manuscript.

We thank the reviewer for this important point. We agree that our current dataset does not provide direct evidence for a physical interaction between Atf4 and Ddit3 proteins. Throughout the manuscript, we use the term "Atf4–Chop axis" to denote a functional regulatory pathway rather than a direct protein–protein interaction. To avoid ambiguity, we have revised the manuscript to characterize Atf4 and Ddit3 as co-activated transcriptional co-regulators and to restrict our conclusions to transcriptional co-regulation and pathway convergence, as supported by SCENIC, Cut&Run, and GO analyses. We now explicitly define "axis" as a functional pathway (revised manuscript, page 5, line 182–184; page 6, line 260–261; page 8, line 331333).

(6) The ATF4 immunostaining after APAP challenge remains weak.

We agree that the endogenous ATF4 immunostaining is relatively weak. This is consistent with the low basal abundance and transient induction of ATF4 protein during the integrated stress response. To improve visualization, we have added higher-magnification insets to Revised Figure 5A, which more clearly show nuclear ATF4 staining in hepatocytes adjacent to the necrotic region following APAP treatment. We hope these enlarged images better illustrate the spatial distribution of ATF4 while accurately reflecting its endogenous expression level.

(7) S5A of the revised manuscript rules out loss of Cyp2e1 expression as a confounding factor.

We thank the reviewer for acknowledging that Revised Figure S5A addresses this concern.

(8) The revised manuscript continues to focus on rare spatial transcriptomics analyses of patients with APAP toxicity although more snRNA-seq analyses of such patients are available which should also allow for analysis of hepatocyte zonation.

We thank the reviewer for this constructive suggestion and apologize for not describing our analyses more clearly in the previous revision. In response, we analyzed both the snRNA-seq and spatial transcriptomics (ST) datasets from GSE223561, which includes snRNA-seq profiles from 9 healthy and 10 APAP explants together with ST data from 3 healthy and 2 APAP patients (Revised Figure 4-figure supplement 3).

The snRNA-seq data readily resolved hepatocyte zonation using established pericentral (Glul, Cyp2e1, Cyp2a5), periportal (Alb, Cyp2f2, Sds), and mid-zonal (Igfbp2, Hamp) markers (Figure 1A–D). Consistent with our mouse data, mid-zonal hepatocytes in APAP patients showed increased expression of stress-response genes (DDIT3, ATF4, and HMOX1). However, unlike the early mouse injury model, proliferation-associated genes (MKI67 and CCNB1) were also elevated (Revised Figure 4-figure supplement 3E), likely reflecting the end-stage nature of liver explants, in which regenerative responses are already well established rather than the early (3–12 h) injury phase examined in our study.

The ST dataset provided complementary spatial information that cannot be obtained from dissociated nuclei alone. Although limited in sample number, one APAP specimen closely recapitulated our murine observations, showing mid-zonal stress enrichment accompanied by reduced proliferation, whereas the second exhibited a predominantly proliferative profile consistent with a later regenerative stage (Revised Figure 4-figure supplement 3F).

Together, these analyses indicate that human mid-zonal hepatocytes exhibit a conserved stress-response program, while the relationship between stress signaling and proliferation is highly dependent on the stage of injury. We have incorporated both the snRNA-seq and ST analyses into the revised manuscript and explicitly discuss the temporal limitations of the available human datasets (page 5-6, lines 225239; page 9, lines 368-379).

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

(1) F2A includes Ddit3 in the revised manuscript and although it is not significant in S1G (former F2B), it is among the most highly expressed transcription factors in F4B and S3B.

We thank the reviewer for this careful observation. As noted, Ddit3 is differentially expressed in the mid-zonal hepatocytes at both 3 and 6 h after APAP treatment (Revised Figure 2A). We also agree that Ddit3 does not reach statistical significance in the differential expression analysis shown in Revised Figure 2-figure supplement 1G under our predefined thresholds. However, our rationale for prioritizing Ddit3 was based primarily on transcription factor activity rather than differential expression alone. Differential expression analysis evaluates changes in the abundance of an individual transcript, whereas SCENIC regulon analysis infers the functional activity of a transcription factor from the coordinated expression of its downstream target genes. Consequently, transcription factors with relatively modest changes in mRNA abundance may nevertheless exhibit high regulon activity if their downstream regulatory network is broadly activated.

Consistent with this, Ddit3 was identified as the highest-activity regulon in the mid zone, while Atf4 ranked seventh (Figures 4B and Figure 4-figure supplement 1B). Given the well-established cooperative roles of ATF4 and DDIT3 in the integrated stress response, these unbiased network analyses identified the ATF4–DDIT3 axis as a biologically relevant pathway for further investigation. To avoid overinterpretation, we have revised the manuscript to clarify that DDIT3 was prioritized based on its consistently high regulon activity and the activation of its downstream transcriptional network, rather than on differential expression alone (Revised manuscript, page 5, line 188-192).

We thank the reviewers for their rigorous critique again. We thank eLife for fostering an environment of fairness and transparency that enables authors to communicate openly and present their data honestly.

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Uncover the real ROI of call tracking for Indian automotive dealerships. See how this call monitoring solution boosts dealer performance at every touchpoint.

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A national U.S. survey found that in 2014, most children overall were not spanked in the previous year. The estimated rate was about 37% overall and 49% for children ages 0-9. Researchers concluded that corporal punishment had declined compared with earlier decades. Another long-running study reported a decline from about 50% of parents reporting spanking in 1993 to about 35% in 2017.

Thankfully not the majority anymore.

Uni MH500 2mm Lead Holder - One Cautionary Note, One Defeated Expectation - Mechanical Pencil Review<br /> by Unnecessary Detail

https://www.youtube.com/watch?v=cRAh0vF2WEk

Surely AI generated, but an interesting paean to a slower life with evening hobbies.

Case#: Patient #35, male, 35yo at report, 14yo at onset,

DiseaseAssertion: STGD

FamilyInfo: family 30, segregation was noted as "yes" but no other details provided

CasePresentingHPOs:

CaseHPOFreeText:

CaseNotHPOs:

CaseNotHPOFreeText:

GenotypingMethod:

PreviouslyPublished: n/a

Variant: allele 1: A1038V;L541P allele 2: G818E

ClinVar: 99135

CAID: CA227000

SupplementalData: supplemental table 1

This paper is listed in LOVD as containing this variant. Unable to find the variant in the main text and unable to locate the supplemental info. At the end of the paper, it says supplemental info can be found online, so used the DOI linked at the end of the article but it just leads to a PDF again with no supplemental

Case#: 19 year old woman

DiseaseAssertion: Stargardt disease (STGD)

FamilyInfo: no family history of ocular disease

CasePresentingHPOs:HP:0025158

CaseHPOFreeText:20/25 in the right eye and 20/25-1 in the left eye, small irregular perifoveal lesions of both increased and decreased autofluorescence

CaseNotHPOs:na

CaseNotHPOFreeText:na

Genotyping Method: next gen sequencing

PreviouslyPublished: na

Variant: c.6079C > T, p.(Leu2027Phe) c.4139C > T, p.(Pro1380Leu)

ClinVar: not found not found

CAID: not found not found

SupplementalData: “black shadow” in the right eye after getting hit by a volley ball

PMID: 10874631

Gene: ABCA4

HGNC ID: 34

Case#: patient 34, female

DiseaseAssertion: RP19

FamilyInfo: paternal first cousin with STGD, healthy father heterozygous for 1938-1 G>A splice mutation, mother homozygous for normal allele

CasePresentingHPOs: HP:0000662, HP:0007663, HP:0007737, HP:0001133

CaseHPOFreeText: choriocapillaris atrophy, severe concentric reduction of the visual field, abrogation of rod function

Genotyping Method: PRISMTM Ready Reaction Sequencing Kit on an automatic fluorometric DNA sequencer

PreviouslyPublished: N/A

Variant: NM_000350.3(ABCA4):c.1938-1G>A

ClinVar: 99106 https://www.ncbi.nlm.nih.gov/clinvar/variation/99106/?term=%22ABCA4%22%5BGENE%5D+AND+%22(c.1938-1G%3EA)%22%5BVARNAME%5D

gnomAD: 0.000002488 https://gnomad.broadinstitute.org/variant/1-94060760-C-T?dataset=gnomad_r4

This variant was mentioned only as a previously published report.

PMID: 22661473

Gene: ABCA4

HGNCID: HGNC:34

Stage I disease was characterized by central macular atrophy with parafoveal or perifoveal flecks. Where flecks were more numerous and extended anterior to the vascular arcades and/or nasal to the optic disc, then patients were classified as having stage II disease. Although partial resorption of flecks may be present in this stage, more complete resorption of flecks was indicative of stage III disease with choriocapillaris atrophy also within the macula. Widespread RPE and chorioretinal atrophy throughout the fundus defined stage IV disease.27 Based on this classification system, the patients in our study were subdivided into 2 groups, that is those with milder disease (stage I or II) and those with more severe disease (stage III or IV) phenotypes.

Case#: Male, Family #1, Patient #1, II:1 in pedigree

DiseaseAssertion: Hypomorphic Stargardt disease

FamilyInfo: Paternal female cousin also has hypomorphic Stargardt disease and both of them carry the complex allele p.[L541P; A1038V] and p.N1868I. Additionally, their paternal aunt was diagnosed with Stargardt disease. This information can be found on Fig.1.

CasePresentingHPOs: HP:0000622, HP:0025010

CaseHPOFreeText: This proband developed blurred vision at age 30. The foveal atrophy affects the left eye. In the first visit at 33.9 years old patient presents with 20/25-3 Snellen VA OD, 20/70-2 Snellen VA OS, 0.16 LogMAR VA OD, 0.58 LogMAR VA OS, and stage 1. In the last visit at age 40.7, the patient had a 20/40-2 Snellen VA OD, a 10/80-1 Snellen VA OS, 0.34 LogMAR VA OD, 0.92 LogMAR VA OS, and was now in stage 2. In a Goldmann Visual Field test, they found central scotomas II4e; mild-moderate constriction II2e.

CaseNotHPOs: N/a

CaseNotHPOFreeText: The proband maintained some relative foveolar sparing in his right eye and had a BCVAs of 20/4022 (test done at 40 years old).

Genotyping Method: Genetic testing was performed at Columbia University. It was not stated which method this proband underwent, so the genetic testing could have been one of the following: "The entire ABCA4 gene locus was sequenced in 17 patients; the ABCA4 gene, including all exons and intron/exon boundaries were sequenced in 4 patients. In the remaining 6 cases representing family members, only targeted testing was performed."

PreviouslyPublished: N/a

**Variant: ** M1) NM_000350.3(ABCA4):c.5603A>T M2) NM_000350.3(ABCA4):c.1622T>C (p.Leu541Pro)

ClinVar: M1) 99390 M2) 99067

CAID: N/a

gnomAD: M1) The highest minor allele frequency was 0.05787 (https://www.ncbi.nlm.nih.gov/clinvar/variation/99390/) M2) The highest minor allele frequency was 0.00017 (https://www.ncbi.nlm.nih.gov/clinvar/variation/99067/)

SupplementalData: Table 1. provided patient information for those with p.N1868I ABCA4 Stargardt disease and the associated ABCA4 mutations. Fig.1. shows the pedigrees of the families. Fig.3. shows Macular SD-OCT line profiles for some of the patients. Table 2. describes the onset/symptoms of the patients with p.N1868I ABCA4 Stargardt Disease. Table 3. shows Visual Acuity and Stage at Baseline and Most Recent Follow-up in Patients With p.N1868I ABCA4 Stargardt Disease. Fig.4. shows BCVA better eye vs Duration since first examination for the patients. Table 4. shows clinical findings in the patients.

This variant is reported to be found in 1 individual in heterozygous or compound heterozygous form, but there is no patient identifier, so impossible to know whether they have a second variant

As shown in other publications, the ATPase activity of this variant appears to be drastically reduced compared to WT when transfected into HEK293 cells, but expression is comparable to WT.

L541P was purified, reconstituted into membranes, and tested for basal and retinal-stimulated ATPase activities. L541P was shown to have reduced basal ATPase, and no stimulation by retinal (Fig 4a)

Case#: Patient 4, Female, Caucasian, 33yo

DiseaseAssertion: STGD1

FamilyInfo: One of eight siblings; four affected. Disease segregates with ABCA4 variants consistent with autosomal recessive inheritance. Parents are deceased and can't be tested.

CasePresentingHPOs: HP:0007663, HP:0007754, HP:0025147, HP:0007924, HP:0011507

CaseHPOFreeText: gradual decline in visual acuity over 9 years, BCVA 20/300 OU, bilateral beaten-bronze appearance of the macula, numerous perimacular yellow flecks, fluorescein angiography showing hyperfluorescence in the posterior pole and dark choroid in the periphery

CaseNotHPOs: N/A

CaseNotHPOFreeText: absence of central hypofluorescence on fluorescein angiography (present in affected siblings but not this patient)

Genotyping Method: SSCP analysis; Taq Dyedeoxy Terminator Cycle Sequencing kit

PreviouslyPublished: N/A

Variant: NM_000350.3(ABCA4):c.2588G>C (p.Gly863Ala), NM_000350.3(ABCA4):c.161G>A (p.Cys54Tyr)

ClinVar: ClinVarID:7879, ClinVarID:99065

CAID: N/A

SupplementalData: Segregation and sequencing data (Figures 1, 4)

Case#: STGD87, 10-14yo at onset, German

DiseaseAssertion: STGD

FamilyInfo: segregation in family

CasePresentingHPOs:

CaseHPOFreeText: "The diagnosis of STGD was based on the demonstration of bilateral impairment of central vision and the appearance of perimacular and/or peripheral yellow-white flecks, with or without atrophy of the central retinal-pigment epithelium and a normal or only mildly abnormal flash electroretinogram when recorded in early stages of the disease."

CaseNotHPOs:

CaseNotHPOFreeText:

GenotypingMethod: denaturing gradient gel electrophoresis, dHPLC, and SSCP analysis, PCR amplification of individual coding exons and flanking intron sequences, direct DNA sequencing

PreviouslyPublished: n/a

Variant: Q1750X; 2588G→C in trans "Correct segregation of disease alleles was demonstrated in all 39 cases in which family samples were available for study"

ClinVar: 7879

CAID: CA119128

SupplementalData: n/a

Case#: Female, 6 years old, presenting with vision loss and behavioral changes.

Disease Assertion: After testing she was diagnosed with Stargardt disease

FamilyInfo: No family history of vision loss in childhood

CasePresentingHPOs: HP:0000572, HP:0000708, HP:0007988, HP:0008001, HP:0030609

CaseHPOFreeText: Showed changes in behavior through increased reliance on parents, and "Over the past six months, she had become increasingly emotional, anxious, frustrated, with difficulty concentrating on simple tasks". Additionally, beyond just the visual loss, she presented with 20/200 OU on her visual acuity test, as well as 1/14 Ishihara color plates with either eye. She also would overlook the top of objects, showed retinal arteriolar narrowing, had degeneration in the ellipsoid zone, and multiple hyperreflective granular deposits.

CaseNotHPOs: HP:0000648, HP:0000486, HP:0000639, HP:0000613, HP:0001336, HP:0011145 (just seizures in general, this was the closest I could find)

CaseNotHPOFreeText: Beyond the HPOs above, she also demonstrated a lack of seizures and had no other neurologic dysfunction. Additionally, she demonstrated brisk pupillary responses without paradoxical pupillary constriction to darkness. She also had normal results for the slip lamp biomicroscopy and tonometry.

CasePreviousTesting: Previously tested for myoclonus, seizures, and neurologic dysfunction with no history of any (did not describe the testing methods for such).

Genotyping Method: Although the genetic testing came up negative in relation to neuronal ceroid lipofuscinosis and the mutations associated, Stargardt disease was confirmed through whole genome sequencing. This revealed "compound heterozygosity for 2 pathogenic variants in the ABCA4 gene (c.3007 C > T p.Q1003X and c768 G > T PV256 = )".

Previously Published: n/a

Variant: NM_000350.3(ABCA4):c.3007C>T (p.Gln1003Ter) & NM_000350.3(ABCA4):c.768G>T (p.Val256=)

ClinVarID: 4538557 & 99505

CAID: n/a

gnomAD: For the first ID the data for this one was absent from gnomAD (https://www.ncbi.nlm.nih.gov/clinvar/variation/4538557/?term=%22NM_000350.3(ABCA4)%3Ac.3007C%3ET+(p.Gln1003Ter)%22%5BVARNAME%5D). While the other ID had the minor allele frequency of 0.00009 (highest compared to others available) (https://www.ncbi.nlm.nih.gov/clinvar/variation/99505/?term=%22NM_000350.3(ABCA4)%3Ac.768G%3ET%22%5BVARNAME%5D+AND+%22(p.Val256%3D)%22%5BVARNAME%5D)

Supplemental Data: Introduction was section in this article that discusses symptoms present as well as the re-diagnosis of Stargardt after the initial incorrect diagnosis of Batten disease. Additionally, Fig.1, Fig.2, and Fig.3 showed some of the phenotypes that appeared with this patient's condition.

Case#: Liu Proband A034, Chinese, female

DiseaseAssertion: Stargardt

FamilyInfo: Mother has the Pro68Leu variant, but the father was not genotyped to know the phase of the second variant. Proband's daughter also has the Pro68Leu variant, but is unaffected.

CasePresentingHPOs:

CaseHPOFreeText: no individual details provided

CaseNotHPOs:

CaseNotHPOFreeText: patients with other ocular diseases, such as choroidal neovascularization, glaucoma, and diabetic retinopathy, or were undergoing treatments/therapeutic trials were excluded.

GenotypingMethod: Either eye gene-enriched (from 36 to 450 target genes) panel-based next-generation sequencing (NGS). Sanger bi-directional sequencing was conducted to confirm the rare candidate variants (allele frequency: less than 1.0% of the general population) and to perform the co-segregation analysis

PreviouslyPublished: n/a

Variant: c.203C>T p.(Pro68Leu); c.3883_3884del p.(Glu1295Lysfs*126)

ClinVar: 99113

CAID: CA226972

SupplementalData: S1 only provides group demographics for the different clinical classifications (fundus grade, FAF type, etc)

Case#: Patient 11, male, age 53

DiseaseAssertion: STGD

FamilyInfo: diagnosis of autosomal recessive STGD based on the pedigree and clinical phenotype of fleck deposits with or without genetic testing

CasePresentingHPOs: HP:0000608, HP:0000007, HP:0030610, HP:0030500

CaseHPOFreeText: Macular degeneration. autosomal recessive, Photoreceptor outer segment loss on macular OCT, Yellow/white lesions of the macula

CaseNotHPOs: n/a

CaseNotHPOFreeText: n/a

Genotyping Method: n/a

PreviouslyPublished: n/a

Variant: NM_000350.3:c.5461-10T>C

ClinVar: NM_000350.3(ABCA4):c.5461-10T>C

CAID: CA220687

SupplementalData: composite mask analysis shown in figure 3 for patient 11, show large areas of matched degeneration and isolated IS/OS loss

Case#: Patient 14, female, age 42

DiseaseAssertion: STGD

FamilyInfo: diagnosis of autosomal recessive STGD based on the pedigree and clinical phenotype of fleck deposits with or without genetic testing

CasePresentingHPOs: HP:0000608, HP:0000007, HP:0030610, HP:0030500

CaseHPOFreeText: Macular degeneration. autosomal recessive, Photoreceptor outer segment loss on macular OCT, Yellow/white lesions of the macula

CaseNotHPOs: n/a

CaseNotHPOFreeText: n/a

Genotyping Method: n/a

PreviouslyPublished: n/a

Variant: Allele 1: NM_000350.3:c.4222T>C Allele 2: NM_000350.3:c.4918C>T

ClinVar:Allele 1: NM_000350.3(ABCA4):c.4222T>C (p.Trp1408Arg) Allele 2: NM_000350.3(ABCA4):c.4918C>T (p.Arg1640Trp)

CAID:Allele 1: CA227166 Allele 2: CA227253

SupplementalData: composite mask analysis shown in figure 3 for patient 14, show diffusely intact IS/OS and RPE with central area of mixed types of degeneration. Both patient 2 and 14 show foveal preservation of IS/OS and RPE

Case#: DNAID 072888/Pat258, female

DiseaseAssertion: STGD1

FamilyInfo: n/a

CasePresentingHPOs:

CaseHPOFreeText: "In classic STGD1, loss of central vision starts around the second decade of life, but both early- and late-onset subtypes have been extensively described." No patient-specific details provided

CaseNotHPOs:

CaseNotHPOFreeText:

GenotypingMethod: smMIPs sequencing of the complete ABCA4 locus

PreviouslyPublished: n/a

Variant: c.6416G>C (p.Arg2139Pro); c.3323G>A (p.Arg1108His)

CAID: CA10611614

SupplementalData: tables s9 and s11

A ClinVar entry claimed this variant showed "impaired substrate binding capacity and limited substrate release after ATP binding," but it is not found in the text or figures and there is no supplementary data.

Case#:case 1 II:4

DiseaseAssertion: Stargardt disease (STGD1)

FamilyInfo: mother has identical phenotype as proband, dad and sister asymptomatic, brother was symptomatic at 8 years old, other brother symptomatic at 15 years old.

CasePresentingHPOs: HP:0000007

CaseHPOFreeText: at age 50, with central visual imparement in right eye, 20/40 right, 20/20 left, linear and branching hyperautofluorescent subretinal deposits and extrafoveal RPE atrophy in both eyes,

CaseNotHPOs: n/a

CaseNotHPOFreeText: n/a

Genotyping Method:

PreviouslyPublished: n/a

Variant: c.6031_6044delins18M/p.(Ile2003LeufsTer41)

ClinVar: not found

CAID: not found

SupplementalData:

Case#: Sporadic Case #16, Mexican

DiseaseAssertion: Stargardt

FamilyInfo: n/a

CasePresentingHPOs:

CaseHPOFreeText: STGD diagnosis based on: "onset of symptoms in childhood or early adulthood (before 20 years of age), bilateral central vision loss (central and peripheral visual field computerized testing), a retinal “beaten-bronze” foveal appearance and/or yellow-whitish flecks from the posterior pole to the mid periphery, normal caliber of the retinal vessels, no pigmented bone spicules in the retinal periphery, a normal to subnormal electroretinogram, and a typical dark choroid in fluorescein angiography."

CaseNotHPOs:

CaseNotHPOFreeText:

PreviouslyPublished: n/a

Variant: allele 1: c.2453G>A (p. G818E); c.5824G>C (p. E1942Q) allele 2: c.6384A>G (p.H2128R) direct sequencing of exons of ABCA4

ClinVar: 867010

CAID: CA957117

SupplementalData: n/a

HB Lead Grades Explained + How to Select Right Pencil for Writing & Art<br /> by [[Unnecessary Detail]] on YouTube<br /> accessed on 2026-09-20T23:58:33

HB is typically the mid-point for most manufacturers <br /> H (hardness) tends to go lighter and scratchier (less graphite, more clay)<br /> B (darkness) tends to go darker and smoother (more graphite, less clay)<br /> F is typically a grade (fine/firm) between H and HB

For general writing look in the range of H / F / HB / B / 2B

Charcoal pencils tend to be much darker that clay/graphite pencils.

In the United States, pencils are often graded from #1 to #5 with the #2 being equivalent to HB.

其中： - @Before：只能在目标方法执行前做一些事。 - @Around：可以决定目标方法是否执行。 - pjp.proceed()：代表调用原来的目标方法；如果不调用它，目标方法就不会执行。 所以，@Around 常用于日志、权限检查、事务、耗时统计等功能

Bioavailability is the fraction of a medication dose that reaches systemic circulation; it is 1 (100%) for intravascular administration and less than 1 for other routes when some drug fails to enter circulation.

Extravascularly means a drug is given outside the vascular system, so it must be absorbed before reaching systemic circulation.

aka gen z gender role

observed/recorded. By definition it was not "assigned"

5 Stages of Lead Holder Usage & Collection - 2mm Mechanical Pencil Buying Guide<br /> by [[Unnecessary Detail]] on YouTube<br /> accessed on 2026-09-20T23:56:00

Lead Holders Explained in 5 Levels of Detail - 2mm Mechanical Pencils<br /> by [[Unnecessary Detail]] on YouTube<br /> accessed on 2026-09-20T23:37:00

Why You Must Try 2mm Lead Holders vs. Wooden Pencils, Fountain Pens & Mechanical Pencils<br /> by [[Unnecessary Detail]] on YouTube<br /> accessed on 2026-09-20T23:24:15

I was not aware of the repeated incidents of racism on google as a search engine. It makes me wonder why its just now I'm hearing about this, it makes me suspect the algorithim hides controversial issues like these away from public view.

This would strengthen assumptions that the company purposely desgined the system in that sort of way because this incident has already occured before.

Users might assume that google is denying accusations of creating a racially influenced algorithimns in their keyword planner to avoid public backlash.

The evidence of no correlation with white with pornography differing from other racial groups leads to evidence of racial bias concerning adult media. Their previous research supports their conclusion as they displayed search categories not including white pornography.

When not payment of money, interest on the amount of damages (discretion of court).

Interest runs from time claim if made judicially or extrajudicially. (if claim is quantified or reasonably certain) OR From the date the judgement of the court is made. (if without quantification)

When obligation is payment of money, interest is paid from judicial demand.

Interest on interest will also be computed from default.

September 19, 2026<br /> by [[Heather Cox Richardson]] in Letters From an American<br /> accessed on 2026-09-20T22:51:34