Schools still enforce the use of Standard English, code meshing is rarely utilized. There's a myth that learning multiple languages/dialects confuse student and prevents them from learning Standard English.

The authors explain that Jayda’s grammar is wrong due to the rules but not "wrong". DAE (Dominant American English) is introduced to be more inclusive and to show that "standard" English is socially constructed.

The teacher lets the students personality shine through by writing her words exactly spoken, instead of correcting her and limiting her identity.

This frames the story early on around prominence and timeliness. The story puts an emphasis on the historic nature of his election to the papacy, while immediately contrasting him with the late Pope Francis. The word "wildcard" here suggests unpredictability. Nobody expect him, an American pope, to be selected for this huge role.

The emphasis on "first US leader" puts an emphasis on the unusualness of the matter and proximity for American readers.

The personal testimony here appeals to the news value of human interest. It portrays Prevost as approachable and resilient. It gives him a sense of warmth and stability...something very easing to the Catholic population.

Invokes conflict, one of the news values we talked about in class. It shows his ability to navigate division...something that pope will need to be able to do. The phrase "moderating influence” frames him as a key peacemaker.

The simplicity of “Peace be with you” resonates with audiences and sets a tone for his gentle leadership narrative.

Prominence mentioned...links the papacy to global politics. Including President Trump's reaction paints more of a political narrative, showing how the papal leadership can intersect with secular power of foreign leaders like Trump.

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Deeper Learning: It is important to elevate the need of teachers to bring practices, instruction that can replicate real life situation that will benefit the students. We need to figure out how to support students thinking, by brining prior knowledge, building background knowledge. Also another support is scaffolds that support students thinking and language. We also need to be monitoring along the the way to see how students are progressing. I do want to acknowledge that it is not easy and it requires lots of preparation and practice.

long-term memory of music stuff is stored in different places of brain than long lasting memory, which is why it is still recalled

found that implicit muscial memory can be preserved in patients with AD

Dementia= umbrella term for memory loss alzheimer's = no dev by aging, abnormal buildup of proteins around brain cells - the actual disease

mild = appear unhealthy, confusion, poor judgement, misplacing things moderate = incr memory loss, diff speech/lang/read/write, diff rec fam n friends, agitation, anxi severe = need caregivers for daily living. inability to comm, weight loss, loss of bowel/bladder contr, and incr sleep

This just prompt me to think about our new IM curriculum, that I know asking level 3 or 4 DOK questions is essential for students, yet if we do not model how to respond or provide students with time to grapple with learning, then students will not have the ability to think and being able to engage in the conversation of the class, the metacognitive skills are essential part of students leanring.

The tight feedback loop (edit story → run test → see results in Storybook) is excellent for DX. It encourages testing early and often, basically from the same place you develop components.

Given that Storybook 9 is built for this testing paradigm, this seems like a forward-looking model for UI development + testing.

For user experience designers, Storybook is a convenient tool as you can work seamlessly when you develop a site, which let you know if your code is working or failing, and has a feature that lets you review an error.

This is brilliant. I haven't learned how to do testing properly with frontend development and this tool seems very useful for visualizing interactions with the application. Since these interactions with storybook are all recorded, they are automatically programmed into the code for you.

• A 30-year study found that childhood friendships have a bigger impact on adult attachment styles than relationships with parents.
• Attachment theory originally emphasized parental influence, but this study shows mothers influence general attachment style only slightly (2-3% variance).
• Early friendships significantly influence adult romantic and friendship attachment anxiety and avoidance (4-11% variance).
• Quality childhood friendships teach give-and-take dynamics that shape how adults form and maintain relationships.
• The study followed 705 participants from childhood through age 26-31, analyzing parent-child and peer relationships.
• Positive early friendships correlate with more secure adult romantic and platonic relationships.
• The research highlights the importance of peer relationships in social and emotional development over family interactions.
• Choosing positive and supportive friends during childhood contributes to healthier adult attachments.

Needs citation

Test comment

• Myopia (nearsightedness) is increasingly common, with serious risks for high myopia such as legal blindness, retinal detachment, and cataracts.
• It results primarily from the eyeball growing too long, causing light to focus in front of the retina, leading to poor distance vision.
• Environmental influences, especially increased indoor time and near work with screens, have driven the rapid rise of myopia in recent decades.
• Digital screens stress the eyes by forcing continuous near focus and lack of depth cues, causing accommodative spasms and vision strain.
• Preventative actions include spending at least 2 hours outdoors daily, regularly focusing on distant objects, and practicing the 20-20-20 rule during screen time.
• Corrective solutions such as glasses, contacts, and LASIK improve vision but do not address underlying eye structure changes or risks associated with high myopia.
• Claims of myopia reversal exist but lack widespread scientific validation.
• The prevalence of myopia and reliance on corrective lenses is projected to grow drastically, making vision protection urgent in the digital age.
• Ongoing research and awareness about vision health and behavioral changes are crucial to mitigate the myopia epidemic.

The Duke's final words being about another artpiece he has demonstrates how little he cared for the Duchess. The need to brag about more art being made for him not only shows his ability to display power, but it also shows a reflection of his true intentions. The bronze cast is of Neptune (a god) taming a seahorse--this reflects how the Duke views himself: a god taming a lesser creature; as he sees himself as a god, he will inevitably treat the new duchess similarly. There was never going to be a dual-respect and understanding between him and the Duchess as she was as useful as a seahorse to him. His calculated shift from a painting of his "beloved" wife, to a bronze cast displaying a feat of dominance demonstrates the Duke's ability for social politics and directs attention away from the gruesome end of the Duchess.

While looking up the Duchess, Lucrezia de' Medici, came a poem called "My Next Duchess" by a priest named Lawrence Jones in which a member of the envoy warns of the Duke as a means to save the next duchess from his grasp. While the poem does not follow all the same writing conventions as Browning's poem such as form and tone, it is effective in the way that provides a secondary perspective on the story within this poem. The break of such conventions lends a sort of response that is more human that the facade that the Duke puts on.

The poem aims to explore how the envoy reacted to the Duke's monologue, and the horror at which is deemed worthy enough to become a cautionary tale to future noblewomen about the Duke of Ferrara.

To build off the previous annotation, the Duke's mask slips for a second as he admits that he would, in fact, "stoop"--but in doing so, it shows he does not have as much control as he fronts to his envoy. However, the Duke regains said control (and re-masks) through the use of others' fear of what he is capable of by stating that he chose "never to stoop" to the Duchess's level of behavior or intellect.

This point is mentioned by Garratt regarding Browning's writing strategy using "masks" in his poetry, "The envoy is meant to be impressed by this graciousness, this taste, manners, and above all, command of life; the Duke hopes desperately that the envoy will carry that impression back to the count, and to the new duchess” (117). Garratt's point shows that the Duke's intentions in telling this story is so that the next Duchess will not behave the same way as the previous one, and she'll have no excuse as she'd already been warned; he is molding his new duchess through fear.

The Duke's glee at showing off the painting is an example of the facade of dominance he establishes throughout the poem. Robert F. Garratt's article reinforces this stating, “In fact, there is a safety about the duchess' looks now that they are frozen on canvas, and the Duke can truly enjoy them because he controls the strings to the curtain” (117). By being able to show when she is allowed to "smile" at anyone using drawstrings illustrates that above else, the Duke's need to dominate and impress are more important than the life of another person, regardless of how close they are to him. His insistence that Fra Pandolf has created such a masterpiece "by [his] design" alludes to the notion that the portrait does not actually capture the essence of the Duchess, but rather a version he demanded be created, displaying his need for control.

HD Part# is FRH

0.6/1kV

This article talks about: In the early stages of the Industrial Revolution, new textile machinery began to be widely used. These machines could be operated by workers with fewer or lower skills, thus replacing or weakening the position of traditional skilled workers. Simultaneously, Britain was experiencing the Napoleonic Wars, and economic hardship exacerbated working-class discontent. In this context, workers believed that factory owners were using new machines to replace skilled labor "in a fraudulent and deceptive manner," suppress wages, and produce inferior goods.

This class was quite different from what I initially imagined. We not only discussed the basics of Python, but also many ethics-related topics, such as moral schools of thought, cyber ethics, and colonialism. It connected history with modern life. This was also the first time I'd looked at programming languages ​​from an ethical perspective.

This is very goofy. I have a hard time believing any of this is true, although perhaps he did witness some court cases and felt inspired to craft a tale. Its interesting that he mentions Arabian Nights because this reads so much like an old tale typical of such works.

The mirrored words here make it almost certain this is a fabrication. This reminds me of passages from the bible where phrases are repeatedly identically.

Apparently Couret is so cool and the Naqib respects him

This a funny conundrum in the subject of Europeans discussing other societies. A lot of people wrote about places they had never been to and the people who actually went to these places were inclined to embellish or even tell outright tall tales.

The summary of this website is : Modern colonialism, especially since the 16th century, has enabled European countries to export populations and establish permanent colonies to multiple continents (the Americas, Australia, Africa, and parts of Asia) and exert political, economic, and territorial domination over these regions, thanks to technological advancements in maritime navigation and transoceanic migration. The details in it: The difference between colonialism and imperialism: Imperialism can refer to direct control (governance) or indirect control (through agents or economic dependence) — both can be expansionist and exploitative.

I really understand of colonialism, since during the world war II, some cities in China had been colonized by other countries. For example, like Hong Kong was colonized by Britain, and Macau was colonized by Portugal. Colonization is more likely to occur when a country is weaker than others, but once they become powerful, the colonized territories may be returned.

https://www.reddit.com/r/pencils/comments/p8w070/wooden_pencils_with_red_lead/

Some people probably want to leave a city to raise kids, but for many it's the only option.

https://musgravepencil.com/products/hermitage-510-wood-cased-hexagonal-pencil-with-red-core-musgrave-pencil-company

https://www.reddit.com/r/pencils/comments/1nd7rvp/red_pencils_for_sketching_and_drawing/

Exactly

I did not find this out until I was an adult

https://www.reddit.com/r/pencils/comments/1g925ar/colored_pencils_for_writing/

local first

from

to

ici il y a juste une erreur avec l'article

move the pull quote to the bottom so it kinda resembles the whole of her dreams

yep

digital public infrastructure

и это тоже нужно взять на вооружение для отзывов.... (или в СПОРАХ!)

нужно взять на заметку для отзывов на работы студентов

ноумен

Google the results of efficacy. It's disproportionately priveleging kids who are already speaking the dominant language. Many immigrant kids do not participate.

Look up the definition between "Positive Right" and "Negative Right." Maybe they should decree a "moral commitment" instead.

Ummmm and what about resources for Urdu, Pashto, and Tagalog? Can these lazy teachers be given infinite resources so we can heal the world at last?? Literally get it together, teachers.

And what is the policy if there are 12 mother tongues in the classroom?

Also, a simple google search will find a trove of evidence that this program is struggling.

Are they taking this seriously though? Will they halt an entire classroom for one somali refugee? What percent of support do they get - prorated or One Hundred percent? This article speaks in Nobility but fails to address the logical conclusion of opening Pandora's Box.

I sure hope it's important, the EU is spending One BILLION / $1,000,000,000 / $1,000,000 x1,000 per year on translations alone.

This is the definition of capitalism, from the webster dictionary, Capitalism is an economic system whose main characteristics include: Capital goods (such as factories, equipment, land, and other means of production) are owned by private individuals or corporations. Investment decisions are primarily made by private individuals, rather than by the government. The prices, production, and distribution of goods are mainly determined through a competitive free market. One detail is that Capitalism is often compared to economic systems such as communism or socialism.

Due to the incentive for innovation being profit, ultimately it will come eventually at the expense of the quality of the platform. The design of Tik Tok is specifically rigged to maximize the engagement of the user and that maximizes their profit but comes at the expense of that user's experience on the platform. I think that a similar thing has happened to Twitter, Instagram, and really all other social media platforms that make their money from engagement.

https://www.makerscabinet.com/products/hovel

important

I totally agree with it, I am majoring in economic, and nowadays, the market is driven by private or corporate ownership. Many countries are capitalism, and as long as we want economic to grow, it easily became a capitalist country.Capitalism may lead to increasingly wider class distinctions and a growing gap between the rich and the poor. However, it appears to be a major trend at present.

take any peergos Custom App create a new folder with some appropriately chosen Utf8Icon as the name of the folder - in this case 📝 for pad

from the CID of the asset folder

Rely on the fact that IPFS deduplicates

ou can have arbitrarily large and complex folder structures

accessed from any other folder with any name!

instead of

I wanted to say

Next Steps:

• add the ability to download the changes one makes to the document when opened by the edit link
• that can be shared again via IPFS
• when 1 uses the editor, a unique pseudo identity is created for the individual/browser combination without any possibility of personal identification or tracking and those changed versions of the documents could be shared back to the author of the document

downloaded thehtml file uploaded to IPFS

and now it is available via dweb.link

and editable!

📝next/0.0.2/index16.html

The beauty of it all is that the document is shared ready to be edited

and reshared attributed to the editor eventually

thes IndyWeb becaones the

The Permanent yet Editable Web

📝next/0.0.2/index16.html

explain how the financial institutions DIRECTLY HELP to these farmers. Like the literal way how they do it.

is there any link that refers to the program?

who is she then?

cite where did you get the data

asdasd

repeating

I looked this up, it means "relating to signs and symbols."

Although I am against this action on practical levels, there is something we can all agree on: Teachers MUST work more. They don't do enough, and it's time to start treating them like the accountability slaves they dreamed of becoming. MUST.

This just popped into my head: If a classroom is discussing the concept of "souls," dont some (unnamed) cultures strictly believe women don't have souls? How do we justify changing what is the linguistic standard without also logically opening up the interpretation for everyone? And do we REALLY want to validate a movement that preaches women should have no autonomy or mention of a soul?

I mean, I would LOVE it. But some might not.

This is a reach of an argument, but whatever.

I may not be a scholar, but I do not see how this is anything but a political act.

Creaticvty is great! I have several creative outlets. Maybe CREATE a system that renders the one you don't like obsolete! That's the only way this would work. Agrarian society rendered Hunter-Gathering obsolete, Industrial Revolution did the same to the Agrarian lifestyle, and Democracy did the same to Monarchy.

Is it without clear boundaries though? Their might be gray area, and aspects of overlap (every language has phonetics) but we can easily say what a language is not. Italian is not Dutch, or Cambodian. It is, in fact, mostly Grunts, "OH!'s, and hand gestures.

Yes. Decentralized, individually chosen, as it should be. Not scaled.

This is a great argument for translanguaging. I am all for tailoring around input to produce optimal output.

However, all four advantages assume the classroom has only two majority languages (Welsh/English). They fall apart when you have five or ten home languages.

The authors themselves say the field is split. Red flag! If even the experts can't agree on what translanguaging actually is, how, precisely, is a school district supposed to turn it into policy?

This sentence made something click in my brain: We cannot solve this debate until we differentiate between Rights vs Responsibilities. RIGHTS are also called "negative rights" which means you have autonomy to make your own decisions without infringing on the autonomy of others. Technically, if your 'right' requires someone else to do something, it is not called a right, because if they do not consent, you can only extract their labor through force. Freedom of Speech, assembly, religion do not require input from other people. They require others to leave you alone to make your own decisions.

This debate should be framed as a "Moral Responsibility." It is the moral thing to do to lift up those who are disenfranchised, but it seems like some are pushing for insitutional codification, which is putting a "moral responsibility" into the category of "rights" and it is just too complex to properly administer.

Calm down, Shakespeare.

This means one language, and the ideology is that linguistic entities should be preserved by treating them as different entities without overlap.

I like this 'input/output' framing. It captures what I believe should be the main debate, which is how to produce the best output with any input (wherever the student may be in their journey.) But now we are questioning whether the output is a colonialist construct that should be morally disassembled.

It MIGHT be, but it is, like all things, many many things in one. And it is not just defined by potentially the worst framing of it.

Exactly, as I said before, done on the fly when navigating two conflicting essential components of an important dilemma.

I'm trying to avoid a 'country club bourgeoisie' tone and this verbiage is not helping.

I believe that most aspects of society function best when there is bending of rules for in-the-moment functionality. Such as a parent telling their child "don't tell your father, we can keep this between us." to show support. Bending the parental agreement? Yes. But the devil is in the details in minor situations. But breaking the social contract at scale between parents leads to chaos. This feels like an appropriate metaphor.

Interesting, I thought it was supposed to protect the minority language? On we read!

Why stop at bilingual? Why not Pentalingual?

Boy, the 'globalist trans agenda' has infected everything! Just kidding.

A modern connection: This is Taylor Swift's song “Mad Woman”, which debuted as the twelfth track on her seventh studio album, Folklore, released on August 18, 2020. Written during the COVID-19 pandemic, the song addresses the criticism and societal backlash that women often face when expressing anger. I include this song as an annotation because it resonates strongly with Xantippe. Centuries of being villainized, described as “shrewd” or “crazy,” mirror the way society punished women for emotions that were deemed inappropriate. Swift’s lyrics capture this societal double standard: "And there's nothin' like a mad woman What a shame she went mad No one likes a mad woman You made her like that" These lines directly reflect the way Xantippe’s anger is treated, not as a natural or justified response, but as evidence of moral or personal failing. Swift continues: "And you'll poke that bear 'til her claws come out And you find something to wrap your noose around" This imagery parallels the way Xantippe is provoked and restricted by the expectations of her husband and society, until she finally lashes out, a physical and emotional release mirrored in Levy’s poem. Later lines, such as: "The master of spin has a couple of well-placed friends They'll tell you you're insane" highlight how women’s reputations and emotions are manipulated and controlled by societal judgment, reinforcing the same marginalization that Levy talks about. By including “Mad Woman”, we can see a direct line from Xantippe’s historical and literary treatment to modern discussions about women, anger, and the consequences of breaking imposed emotional boundaries.[]https://youtu.be/6DP4q_1EgQQ?si=e7ol3EKrAAWfHwSR

At this point in the poem, Xantippe has lost the mask she worked so hard to hold on to. Socrates has angered her, and in a sudden fit of rage, her body responds with a faster heartbeat as she flings the wine onto the floor. Xantippe seems to rarely allow herself to feel or express anger; she might often feel slighted or sad, but these emotions are usually restrained, like a quietly glowing ember. Here, however, a “sudden flame” erupts: the heat rises, and she finally releases it. The description of the red wine spilling onto the white marble serves as a powerful metaphor for the loss of innocence or purity. What was once clean and controlled is now marked and transformed. Xantippe crosses the threshold changed, “hair unbound, white garment stained to redness," no longer the restrained, composed figure she once was. Levy’s depiction of this moment reflects her interest in women’s emotional and intellectual repression. Just as Xantippe’s fury has been contained by societal expectations in ancient Athens, Victorian women like Levy faced pressures to restrain their feelings and intellect. By giving Xantippe a dramatic, physical release of her anger, Levy depicts the costs of suppression and illuminates the intense, hidden emotional lives of women. The poem becomes not only a historical reflection on Xantippe’s experience but also a nuanced critique of the constraints placed on women in Levy’s world by showing how powerful and transformative the acknowledgment of one’s own emotions can be.

Through Socrates’ choice of words in this passage, he comes across as arrogantly dismissive. Using sarcasm, he comments, what on the surface sounds like a compliment: “I thank thee for the wisdom which thy lips / Have thus let fall among us,” as actually belittling. The phrase “have thus let fall among us” portrays her input as something not freely given or valued; it was blurted out and unsolicited. His snide questions: “From what high source, from what philosophies / Didst cull the sapient notion of thy words?” imply that even if her words were worthy, they could not possibly have come from her own mind. Socrates shows no genuine respect for Xantippe, acknowledging her only in the limited capacity allowed to women in domestic spaces. Amy Levy’s choice of including this passage reflects her broader interest in exposing the ways women’s intelligence and emotional lives were devalued. By featuring Socrates’ dismissive tone, Levy demonstrates the societal conditions of Victorian women like herself, who were often denied intellectual recognition and confined to narrowly defined roles. Just as Xantippe is belittled despite her perception, Victorian women faced systemic obstacles to being taken seriously. This makes Levy’s dramatic monologue a commentary on the ongoing marginalization of women’s minds.

While looks and beauty were very important in the ancient Grecian times, Socrates broke the mold when it came to the beauty standards of his day. As noted to his appearance in the Stanford Encyclopedia of Philosophy, Socrates "had wide-set, bulging eyes that darted sideways and enabled him, like a crab, to see not only what was straight ahead, but what was beside him as well; a flat, upturned nose with flaring nostrils; and large fleshy lips like an ass." Despite the hair trends of his area, he grew his hair out and refused to stay clean or change his clothes. Amy Levy includes these details to accentuate the contrast between societal expectations and individual worth. Men like Socrates could be physically unconventional or even “ugly” and still admired for their intellect, yet there was no equivalent space for women to be recognized for their minds. A woman’s value was tied to her beauty and social conformity. By presenting Socrates’ unconventionality alongside the phrase “half with awe,” Levy highlights the tension between superficial judgment and true merit, a tension mirrored in "Xantippe: A Fragment", where women’s intellectual and emotional lives were historically ignored or dismissed.

When Xantippe describes her soul as one that “yearned for knowledge,” she describes a desire that classical society discouraged in women. This yearning reflects Amy Levy’s own intellectual ambitions and her struggle to access education in a world that restricted women’s academic opportunities. Levy pushed against these limitations of her time. She became the second Jewish woman ever admitted to Cambridge University and the first Jewish woman to enroll at Newnham College, one of the women’s colleges founded to expand access to higher learning. Levy’s personal experiences with gender barriers enhance her portrayal of Xantippe’s longing. By giving a classical woman, the same thirst for intellectual life that Levy felt as a Victorian woman, the poem creates a bridge between eras. Xantippe’s desire becomes not merely personal but representative of a long history of women whose intellectual aspirations were dismissed or deemed inappropriate. Through this moment of self-revelation, Levy highlights the emotional cost of systemic exclusion and places knowledge-seeking as both a private desire and an act of resistance.

This passage reflects experiences that are both historically and personally grounded by connecting the lives of women in Ancient Greece and Victorian England. As a Victorian woman, Levy would have understood the deep emotional pressures created by a society that limited women’s education, rights, and opportunities simply because of their gender. Many women writers of the time, including Charlotte Brontë (writing as Currer Bell) and Mary Ann Evans (writing as George Eliot), adopted male pen names just to have their work published. This is just one example of the systemic barriers women faced. The line “and I was sinning in those very thoughts—” adds an intense sense of shame and internalized guilt, drawing attention to how natural curiosity and intellectual exploration could be framed as morally wrong for a woman. By illuminating this tension, Levy exposes the emotional cost of societal restrictions, showing how both historical and contemporary pressures could make women feel as though their own minds were prohibited.

Xantippe describes a “troubled sleep” filled with memories of “weary days," “stormy days,” "gladsome days" and "sunny days." All of which showcase a wide emotional spectrum that reflects the turbulence and instability of her inner life. As described in the Oxford English Dictionary, “weary” is defined as “having the feeling of loss of strength, languor, and need for rest, produced by continued exertion (physical or mental), endurance of severe pain, or wakefulness; tired, fatigued.” This definition highlights the depth of exhaustion Xantippe experiences, not just physical tiredness, but a profound emotional and psychological fatigue shaped by years of disappointment and suppression. The coupling of “weary” with “stormy” suggests that her life has been a mixture of long-term exhaustion and moments of upheaval. By layering images of fatigue and unrest, Levy conveys an emotional range that establishes the introspective tone of the dramatic monologue. This invites readers to witness Xantippe’s internal thought process, something historical accounts often have denied her. This emotional landscape also functions as a mirror ball to Levy's own inner life by reflecting in fragmented but vivid ways the themes that appear throughout her body of work. In the poems collected in "A Minor Poet and Other Verse," there is a theme of loss and burdens of being human. Her poem, "Sonnet" exemplifies this introspective shift by capturing the same sense of inner weariness, longing, and psychological strain that is felt through Xantippe. By reading "Xantippe: A Fragment" alongside her other poems, we can see how Levy's writing reflects different angles of the same emotional core, all of which emphasize the private struggles that women were expected to keep hidden.

Although “Xantippe: A Fragment” was published in 1880, nine years before Levy’s death in 1889, the poem already reveals the emotional turmoil that resulted in her long-standing, though undiagnosed, clinical depression. In these lines, Amy Levy gives a haunting voice to a figure who feels emotionally drained, as if her life’s flame were dimming. The imagery of a “lamp of life” burning low, mixed with the weariness of waking, resonates with Levy’s own recurring bouts of melancholic depression. As a young Jewish woman navigating the male-dominated intellectual circles of Victorian England, Levy often felt like an outsider, both socially and spiritually. According to the Jewish Women’s Archive (2021), a friend and confidant, Richard Garnett, described her as having "constitutional melancholy." By channeling that profound exhaustion through Xantippe, she not only critiques the silencing of women, but also reveals personal anxieties about her own worth, agency, and artistic survival.

2025-11-25 Tue 0:24

https://bafybeibepgtwo4ptltedy4eo3muao5krtbmsk35ncm6clyi3mownjweoc4.ipfs.dweb.link/next/0.0.2/index%20%2816%29.html

Since when did Trump care about libraries?

créances

Boeing is a company that uses tech to directly further colonialism in Palestine today and historically on campus. One example is the GBU-39/B precision strike system that Boeing sells to the IDF who uses it to colonize Palestine. And back in 1939, here on UW campus, the Kirsten Wind Tunnel tested the B-29. This is the plane that would go on to drop the atomic bombs on Hiroshima and Nagasaki. Modern tech companies like Microsoft and Amazon sell their cloud, AI, web, and other services to oppressive governments around the world that use their tech to do harm

This is just one more reason why many people are confused about copyright. Not only do the laws vary from country to country, but they can also be different based on regions within a country. How is anyone (who is not a copyright lawyer) supposed to keep track of this?

This change has definitely affected a wide audience. The stark jumps that companies make are seen all over the place, mainly with major criticism. An example I can think of is companies changing logos which the public usually hates so they are forced to live with the negativity or change it back such as the Cracker Barrel logo. I’ve noticed these subtle changes with the Youtube UI where they change the font or symbols for different buttons. I usually dislike it for about a day and am forced to live with it until they inevitably change it again.

Carol Cohn fala sobre a necessidade de controle e previsibilidade no discurso de defesa. O tom de Shelby não é apenas de crítica técnica; é de angústia (distressed).

Evidência no Texto: Ele chama o evento de "extremely dangerous situation" e diz estar "very concerned and distressed" (muito preocupado e angustiado).

Análise: A "domesticação" do perigo falhou. Em 1995, o perigo era domesticado pela piada ("exciting watch"). Em 1998, a realidade da proliferação rompeu a bolha da linguagem tecnoestratégica segura. O "cenário sombrio" (previsto no memorando de 95) se concretizou porque a inteligência estava ocupada demais subestimando o vigiado.

I would say that there is absolutely a breaking point as far as someones privileges to be amongst the public but to call it a point of no return is somewhat cruel. I believe that justice or reconciliation should be based on rehabilitation. There should always be a return offered if the problem is solved truly. This is only in the legal sense, in a personal sense no one owes you a second chance in that way accept maybe your parents.

I appreciate how it doesn’t shy away from the limits of reconciliation. By comparing the Nuremberg Trials with the South African Truth and Reconciliation Commission, it highlights how not all wrongdoing can be “fixed” in the same way — and sometimes punishment, not forgiveness, is what justice requires.

Often on social media when an influencer gets cancelled, they make an apology video. Whether or not the Internet forgives them depends on the sincerity of the influencer. In 2020, it was popular for influencers to do a "ukulele apology" and apologize in song form. This was often perceived as cringey and instead led to more hate and public shaming.

Many Nazis knew this very well and escaped across the globe. For some reason, South America was a popular place to go. Or maybe it was just a lot more difficult to identify them in South America causing Nazi refugees to remain undiscovered a long time. Its kind of odd to me that South America is somewhat popular to move to. In mid 1900's Japan, Brazil of all places was a common place to move to.

I have an observation experience where one of the students interrupted the learning of others. He kept calling out and making the other students laugh.

I think that things like this could be really beneficial especially in a choral classroom or lesson. This could be helpful in a lot of ways including seeing how a teacher that is around the student more often than a music teacher would get the student to learn the best.

How might these types of grouping for self-contained classrooms affect the students learning?

I have come to learn that this statement is true. My original thought is that inclusion classrooms are better because it allows students with differences and dissabilities to interact with their peers. However I observed a music class in a self contained classroom and now I realize how self-contained classrooms are needed and how it could get in the way of learning for students without differences or dissabilities. And on the other side I've observed classrooms where some students who are in inclusion classrooms can disrupt thier classmates from learning. I think that if they were in a self-contained classroom that would be better for both parties.

I agree with music educators collaborating with special education faculty to better reach students with differences and disabilities. I see this a lot in my current placement the special education faculty work with the teachers on how to help these students specifically. I feel like doing this is beneficial for everyone in the class.

Jimmy Kimmel on his show did a Halloween prank that resulted in the prank going viral and a lot of parents replicating the prank on their own children at home. Some people argue that the effects are really negative and traumatizing to the child but the author notes calling this type of prank a trauma betrays the true definition of a psychological trauma because there is no correcting of the wrong that the parent has done in the actual thing and in this prank the whole idea is you reveal to them at the end that all their candy is actually still there.

Based on my experiences i agree with this author. I pranked my little sister when she was 9 and she really felt for it and was extremely hurt

This is a parenting source that discusses how shame is a helpful resource in teaching your child the difference between "right" and "wrong" however this tool is only effective if you consul the child for what they did and notify them that the action is wrong not them as a person. Then shame turns into guilt which makes it even more helpful in teaching right and wrong.

Many people can't even notice the downside of a thing while they're laughing and enjoying. As this prank is an example, the audience who laugh at it can't notice what's wrong with this show, and probably won't exploit the potential negative influence it brought to the kids. In this case, we need someone to rethink this show from another perspective, even though they would be criticized, since they were standing on the opposite side of most of the audience.

While reading this article, I thought of those moments when people said "just kidding", but for children, it was not just a joke. Adults might find pranks amusing, but the fear and humiliation children feel at that moment are one hundred percent real. Especially when they are recorded by cameras, posted online, and shown to strangers as a joke, that sense of powerlessness may linger in their hearts for a long time. Children cannot understand that "this is entertainment", they only think that if even their parents can laugh at them, then who else can they trust? This made me realize that laughter and hurt are sometimes separated by only a very thin line, and we often cross it when children are at their most vulnerable.

I find its framework interesting — they don’t just dismiss shaming out of hand, but carefully analyze when and how it might be morally justified. Their conditions (like proportionality, necessity, respect for privacy, non-abusiveness, and reintegration) seem really well suited to thinking about social media shaming.

The Wikipedia page on the sociological concept of “face” explains how people try to maintain a positive social image in front of others. (pretend to) What stood out to me is how deeply this idea connects to online behavior too. On social media, people are constantly managing their “face” by organizing posts, deleting things that seem embarrassing, or apologizing when something they say gets taken the wrong way. It made me realize that a lot of the pressure we feel online, like worrying about how others see us comes from this same idea of protecting our social image.

I think this is an interesting case of public shaming because, in his apology thread, he explains some of the reasoning behind why he said what he said. He mentions how someone he knew told him he would bring a book to the bar to pick up girls, a performative act that doesn't illustrate who that person really is and shows how the book becomes a prop in that instance. I think, while the original tweet obviously is exaggerated and oversimplified, there are cases where someone reading a book in a bar, to pick up girls for example, would be seen as scummy by a large majority of people.

This website talked about the definition of trauma, and also introduced the way to heal the shame. The strategies that are provided on the website were also useful for most of people.

This source discusses how shame is connected to trauma and how, when specifically it is posted to social media, it can make people feel worthless. Online shaming can create real damage psychologically, especially when millions of people see it and comment on it, it can cause severe psychological harm.

The reasoning why we might be laughing at the children is due to seeing ourself as being more intelligence, and as such the seemingly lower intelligence behavior of the children seems humorous due to its apparent stupidity. This parallels why people laugh of footage of someone getting hurt in a silly or seemingly avoidable way. By laughing, it expresses a sense of superiority over the person in the footage, however this does not encapsulate the whole story behind the incidents. The introduction of the internet and subsequently short form content and videos makes this even more prevalent, with important context being cut out in a way that makes the original video humorous.

This article explains how shame is deeply connected to trauma and can make people feel worthless, isolated, and unsafe. Reading this made me think about public shaming online — it’s not just “calling people out,” it can actually reopen or create real psychological damage, especially when it happens in front of millions. It makes me more cautious about joining online pile-ons, because we never know what someone is already carrying.

The summary and details of this link is : Shame has a function in normal child development, but shame arising from complex traumatic experiences is fundamentally different from that in ordinary situations. For adolescents who have experienced trauma, shame can quickly permeate their core identity. Therefore, to help traumatized adolescents change their behavior, it is essential to "work with their shame," that is, to acknowledge, understand, and respond to their shameful experiences.

This article explains the downside of what Jimmy Kimmel would call a "joke." The author looks deeper into the psychological effects of parents pretending to steal their kids' Halloween candy. She explains that the kids might experience lasting betrayal. Seeing their parents steal and lie can uproot their sense of morals and safety. The psychological effects on the child is not worth a five-second laugh on TV. This shows us that we must be careful about what "jokes" we promote on social media and television.

I found this quote to be of interest. "The norm violator's culpability increases with the extent to which she is responsible for the violation and not excused".

Relating back to what comes after being canceled, I agree it ranges from different things. I had a close friend of mine getting cancelled online which then transferred to in person. The bullying turned so bad that she was forced to quit her job, move schools, and create a new identity. She was 17 at the time. So through that I saw there were so many "consequences" that came from getting canceled.

It seems that only videos or posts with shaming and can make the audience laugh would grab more attention and have more exposure on the internet. I want to say that watching people criticize or shame someone is a natural behavior among humans, while the “Three Character Classic" suggests that people are kind by nature. Therefore, this remains the question that is our kindness been taught or it's just our natural beings

I find it interesting how normalized public shaming is to the point where there are entire segments on a talk show that make fun of children for reacting in a completely normal way to the information they are receiving. It shocks me how normal it is, especially when they are targeting kids who would obviously be sad if their parents ate all of their Halloween candy which they worked all night to collect.

Cancel culture is something that I feel like has emerged more in the past couple of years, especially with the rise of TikTok and other big social media platforms. While I don't think canceling somebody is bad, it must be for the right reasons. I think that many people get canceled for things that are not really a big deal, and therefore, they can lose everything in their lives. I can agree with cancel culture; however, when the person really did do something bad.

I think the most important factors in making an instance of public shaming bad is shaming that person's other characteristics. For example, if the individual is being publicly shamed for saying something inappropriate and then making fun of the way they talk or look or dress, then that can really affect the person in a harmful way.

Of course the reasoning the person is being shamed is important, but I argue the length that the person faces the shape, and the degree of shape that is bought onto the person is just as important. A period of shame may allow the person to realize the consequence of their actions the effect is has on other people, but prolonged shape might result in self depreciation and self hate, resulting in lowered self worth. Is this condition of lowered self worth, this might result in lashing out or other actions that might result in more harm.

I think public shaming becomes dangerous when it targets normal people who never asked for attention. It’s very different from holding powerful figures accountable. When someone goes viral by accident, they suddenly face millions of strangers judging their whole life based on one moment. That doesn’t feel like justice — it feels like entertainment. To me, the most important thing is whether the person has real power and whether the punishment is way bigger than the mistake.

This sentence reminds me of when I was a child and was scolded by adults. If I only heard, "How could you be so undisciplined!" that kind of hurt would linger in my heart for a long time. But if someone added a sentence after being angry, "I still love you very much, but you can't do this thing," the feeling would be completely different. It makes the child know that mistakes can be corrected, but they are not unlovable just because they made a mistake. Such comfort is actually teaching the child a safe self-perception: I can make mistakes, but I can also become better.

These two feeling played a big role for many children's childhood. The feeling of shame invisibly changed people's life. Sometimes, people will hide their opinion if they always feel shame. The feeling of guilty might be better, because this feeling let them want to fix and repair the harm of their action.

Yeah, I think it is right. For example, now I am applying master degree, HK give me the offer but it need me to deposit 130k. Sometime, I will think that, what if I cannot find a job after master, or what if I did not do well in job market. I will fell guilt, if I spend such amount money to do this choice, but what if I did not do well after I make this choice, I will fell guilt for my family.

Amazing that MCP is not funtamental.

• Google released Nano Banana Pro (gemini-3-pro-image-preview), a new AI image generation model.
• Nano Banana Pro excels in general intelligence, tool use, and creating complex scenes with less hallucination.
• It can use Google Search and Maps to gather data and reason visually through "thought images."
• Pushing infographic and map generation to new frontiers, enabling visually rich and factually accurate images.
• Can create detailed photorealistic images based on complex, multi-element prompts.
• Not reliable for electrical circuit designs yet, as it may produce erroneous circuit diagrams.
• Human intelligence still surpasses it in domain-specific tasks like accurate circuit design.
• Nano Banana Pro is seen as a game changer in practical, production-ready AI image generation.
• Tool use enables more factually accurate and data-driven generated images than previous models.
• Benchmarking AI image generation quality still needs development for production use assessment.
• The community is impressed with Nano Banana Pro's nuanced prompt following and image creation capabilities.

If Twitter had a retraction feature, I think the user should be able to choose whether to keep the original tweet visible with a retracted label or completely delete the content. It would also be helpful if the user could write a short explanation, like why they retracted it or what information was incorrect. When someone views a retracted tweet, I imagine it would look faded out with a note saying something like “The author has retracted this tweet,” instead of showing the full text. For retweets or quote tweets, I think they should also show the retraction label, so people won’t keep spreading the old message without knowing it was taken back. I’m not sure if there should be notifications, maybe only to people who already interacted with the tweet, so they know something changed.

grand livre comptable

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Manuscript number: RC-2025-03208

Corresponding author(s): Jared Nordman

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1. General Statements [optional]

All three reviewers of our manuscript were very positive about our work. The reviewers noted that our work represents a necessary advance that is timely, addresses important issues in the chromatin field, and will of broad interest to this community. Given the nature of our work and the positive reviews, we feel that this manuscript would best be suited for the Journal of Cell Biology.

2. Description of the planned revisions

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

Summary:

The authors investigate the function of the H3 chaperone NASP, which is known to bind directly to H3 and prevent degradation of soluble H3. What is unclear is where NASP functions in the cell (nucleus or cytoplasm), how NASP protects H3 from degradation (direct or indirect), and if NASP affects H3 dynamics (nuclear import or export). They use the powerful model system of Drosophila embryos because the soluble H3 pool is high due to maternal deposition and they make use of photoconvertable Dendra-tagged proteins, since these are maternally deposited and can be used to measure nuclear import/export rates.

Using these systems and tools, they conclude that NASP affects nuclear import, but only indirectly, because embryos from NASP mutant mothers start out with 50% of the maternally deposited H3. Because of the depleted H3 and reduced import rates, NASP deficient embryos also have reduced nucleoplasmic and chromatin-associated H3. Using a new Dendra-tagged NASP allele, the authors show that NASP and H3 have different nuclear import rates, indicating that NASP is not a chaperone that shuttles H3 into the nucleus. They test H3 levels in embryos that have no nuclei and conclude that NASP functions in the cytoplasm, and through protein aggregation assays they conclude that NASP prevents H3 aggregation.

Major comments:

The text was easy to read and logical. The data are well presented, methods are complete, and statistics are robust. The conclusions are largely reasonable. However, I am having trouble connecting the conclusions in text to the data presented in Figure 4.

First, I'm confused why the conclusion from Figure 4A is that NASP functions in the cytoplasm of the egg. Couldn't NASP be required in the ovary (in, say, nurse cell nuclei) to stimulate H3 expression and deposition into the egg? The results in 4A would look the same if the mothers deposit 50% of the normal H3 into the egg. Why is NASP functioning specifically in the cytoplasm when it is also so clearly imported into the nucleus? Maybe NASP functions wherever it is, and by preventing nuclear import, you force it to function in the cytoplasm. I do not have additional suggestions for experiments, but I think the authors need to be very clear about the different interpretations of these data and to discuss WHY they believe their conclusion is strongest.

The concern raised by the reviewer regarding NASP function during oogenesis has been addressed in a previous work published from our lab. Unfortunately, we did not do a good job conveying this work in the original version of this manuscript. We demonstrated that total H3 levels are unaffected when comparing WT and NASP mutant stage 14 egg chambers. This means that the amount of H3 deposited into the eggs does not change in the absence of NASP. To address the reviewer's comment, we will change the text to make the link to our previous work clear.

Second, an alternate conclusion from Figure 4D/E is that mothers are depositing less H3 protein into the egg, but the same total amount is being aggregated. This amount of aggregated protein remains constant in activated eggs, but additional H3 translation leads to more total H3? The authors mention that additional translation can compensate for reduced histone pools (line 416).

Similar to our response above, the total amount of H3 in wild type and NASP mutant stage 14 egg chambers is the same. Therefore, mothers are depositing equal amounts of H3 into the egg. We will make the necessary changes in the text to make this point clear.

As the function of NASP in the cytoplasm (when it clearly imports into the nucleus) and role in H3 aggregation are major conclusions of the work, the authors need to present alternative conclusions in the text or complete additional experiments to support the claims. Again, I do not have additional suggestions for experiments, but I think the authors need to be very clear about the different interpretations of these data and to discuss WHY they believe their conclusion is strongest.

A common issue raised by all three reviewers was to more convincingly demonstrate that assay that we have used to isolate protein aggregates does, in fact, isolate protein aggregates. To verify this, we will be performing the aggregate isolation assay using controls that are known to induce more protein aggregation. We will perform the aggregation assay with egg chambers or extracts that are exposed to heat shock or the aggregation-inducing chemicals Canavanine and Azetidine-2-carboxylic acid. The chemical treatment was a welcome suggestion from reviewer #3. These experiments will significantly strengthen any claims based on the outcome of the aggregation assay.

We will also make changes to the text and include other interpretations of our work as the reviewer has suggested.

Data presentation:

Overall, I suggest moving some of the supplemental figures to the main text, adding representative movie stills to show where the quantitative data originated, and moving the H3.3 data to the supplement. Not because it's not interesting, but because H3.3 and H3.2 are behaving the same.

Where possible, we will make changes to the figure display to improve the logic and flow of the manuscript

Fig 1:

It would strengthen the figure to include representative still images that led to the quantitative data, mostly so readers understand how the data were collected.

We will add representative stills to Figure 1 to help readers understand how the data is collected. We will also a representative H3-Dendra movie similar to the NASP supplemental movie.

The inclusion of a "simulated 50% H3" in panel C is confusing. Why?

We used a 50% reduction in H3 levels because that is reduction in H3 we measure in embryos laid by NASP-mutant mothers in our previous work. A reduction in H3 levels alone would be predicted to change the nuclear import rate of H3. Thus, having a quantitative model of H3 import kinetics was key in our understanding of NASP function in vivo. We will revise the text to make this clear.

I would also consider normalizing the data between A and B (and C and D) by dividing NASP/WT. This could be included in the supplement (OPTIONAL)

We can normalize the values and include the data in a supplemental figure.

Fig S1:

The data simulation S1G should be moved to the main text, since it is the primary reason the authors reject the hypothesis that NASP influences H3 import rates.

This is a good point. We will move S1G into the Figure 1.

Fig 2:

Once again, I think it would help to include a few representative images of the photoconverted Dendra2 in the main text.

We will add representative images of the photoconversion in Figure 2.

I struggled with A/B, I think due to not knowing how the data were normalized. When I realized that the WT and NASP data are not normalized to each other, but that the NASP values are likely starting less than the WT values, it made way more sense. I suggest switching the order of data presentation so that C-F are presented first to establish that there is less chromatin-bound H3 in the first place, and then present A/B to show no change in nuclear export of the H3 that is present, allowing the conclusion of both less soluble AND chromatin-bound H3.

The order of the presentation of the data was to test if NASP was acting as a nuclear receptor. Since Figure 1 compares the nuclear import, we wanted to address the nuclear export and provide a comprehensive analysis of the role of NASP in H3 nuclear dynamics before advancing on to other consequences of NASP depletion. We can add the graphs with the un-normalized values in the Supplemental Figure to show the actual difference in total intensity values.

Fig S2:

If M1-M3 indicate males, why are the ovaries also derived from males? I think this is just confusing labeling.

We will change the labelling.

Supplemental Movie S1:

Beautiful. Would help to add a time stamp (OPTIONAL).

Thank you! We will add the time stamp to the movie

Fig 3:

Panel C is the same as Fig S1A (not Fig 1A, as is said in the legend), though I appreciate the authors pointing it out in the legend. Also see line 276.

We appreciate the reviewer for pointing this out. We will make the change in the text to correct this.

Panel D is a little confusing, because presumably the "% decrease in import rate" cannot be positive (Y axis). This could be displayed as a scatter (not bar) as in Panels B/C (right) where the top of the Y axis is set to 0.

We understand the reviewer's concern that the decrease value cannot be positive. We can adjust the y-axis so that it caps off at 0.

Fig S3:

A: What do the different panels represent? I originally thought developmental time, but now I think just different representative images? Are these age-matched from time at egg lay?

The different panels show representative images. We can clarify that in the figure legend.

C: What does "embryos" mean? Same question for Fig 4A.

In this figure, embryos mean the exact number of embryos used to form the lysate for the western blot. We will clarify this in the figure legend.

Fig 4:

A: What does "embryos" mean? Number of embryos? Age in hours?

In this figure, embryos mean the exact number of embryos used to form the lysate for the western blot. We will clarify this in the figure legend.

C: Not sure the workflow figure panel is necessary, as I can't tell what each step does. This is better explained in methods. However I appreciated the short explanation in the text (lines 314-5).

The workflow panel helps to identify the samples labelled as input and aggregate for the western blot analysis. Since our input in the western blots does not refer to the total protein lysate, we feel it is helpful to point out exactly what stage at the protocol we are utilizing the sample for our analysis.

Minor comments:

The authors should describe the nature of the NASP alleles in the main text and present evidence of robust NASP depletion, potentially both in ovaries and in embryos. The antibody works well for westerns (Fig S2B). This is sort of demonstrated later in Figure 4A, but only in NAAP x twine activated eggs.

We appreciate the reviewer's comments about the NASP mutant allele. In our previous publication, we characterized the NASP mutant fly line and its effect on both stage 14 egg chambers and the embryos. We will emphasize the reference to our previous work in the text.

Lines 163, 251, 339: minor typos

Line 184: It would help to clarify- I'm assuming cytoplasmic concentration (or overall) rather than nuclear concentration. If nuclear, I'd expect the opposite relationship. This occurs again when discussing NASP (line 267). I suspect it's also not absolute concentration, but relative concentration difference between cytoplasm and nucleus. It would help clarify if the authors were more precise.

We appreciate the reviewer's point and will add the clarification in the text.

Line 189: Given that the "established integrative model" helps to reject the hypothesis that NASP is involved in H3 import, I think it's important to describe the model a little more, even though it's previously published.

We will add few sentences giving a brief description of the model to the text.

Line 203: "The measured rate of H3.2 export from the nucleus is negligible" clarify this is in WT situations and not a conclusion from this study.

We will add the clarification of this statement in the text.

Line 211: How can the authors be so sure that the decrease in WT is due to "the loss of non-chromatin bound nucleoplasmic H3.2-Dendra2?"

From the live imaging experiments, the H3.2-Dendra2 intensity in the nucleus reduces dramatically upon nuclear envelope breakdown with the only H3.2-Dendra2 intensity remaining being the chromatin bound H3.2. Excess H3.2 is imported into the nucleus and not all of it is incorporated into the chromatin. This is a unique feature of the embryo system that has been observed previously. We mention that the intensity reduction is due to the loss of non-chromatin bound nucleoplasmic H3.2.

Line 217: In the conclusion, the authors indicate that NASP indirectly affects soluble supply of H3 in the nucleoplasm. I do believe they've shown that the import rate effect is indirect, but I don't know why they conclude that the effect of NASP on the soluble nucleoplasmic H3 supply is indirect. Similarly, the conclusion is indirect on line 239. Yet, the authors have not shown it's not direct, just assumed since NASP results in 50% decrease to deposited maternal histones.

We appreciate the feedback on the conclusions of Figure 2 from the reviewer. Our conclusions are primarily based on the effect of H3 levels in the absence of NASP in the early embryos. To establish direct causal effects, it would be important to recover the phenotypes by complementation experiments and providing molecular interactions to cause the effects. In this study we have not established those specific details to make conclusions of direct effects. We will change the text to make this more clear.

Line 292: What is the nature of the NASP "mutant?" Is it a null? Similarly, what kind of "mutant" is the twine allele? Line 295.

We will include descriptions of the NASP and twine mutants in the text.

Line 316: Why did the authors use stage 14 egg chambers here when they previously used embryos? This becomes more clear later shortly, when the authors examine activated eggs, but it's confusing in text.

The reason to use stage 14 egg chambers was to establish NASP function during oogenesis. We will modify the text to emphasize the reason behind using stage 14 egg chambers.

Lines 343-348: It's unclear if the authors are drawing extended conclusions here or if they are drawing from prior literature (if so, citations would be required). For example, why during oogenesis/embryogenesis are aggregation and degradation developmentally separated?

This conclusion is based primarily based on the findings from this study (Figure 4) and out previous published work. We will modify the text for more clarity.

Lines 386-7: I do not understand why the authors conclude that H3 aggregation and degradation are "developmentally uncoupled" and why, in the absence of NASP, "H3 aggregation precedes degradation."

This is based data in Figure 4 combined with our previous working showing that the total level of H3 in not changed in NASP-mutant stage 14 egg chambers. Aggregates seem to be more persistent in the stage 14 egg chambers (oogenesis) and they get cleared out upon egg activation (entry into embryogenesis). This provides evidence for aggregation occurring prior to degradation and these two events occurring in different developmental stages. We will change the text to make this more clear.

Line 395: Why suddenly propose that NASP also functions in the nucleus to prevent aggregation, when earlier the authors suggest it functions only in the cytoplasm?

We will make the necessary edits to ensure that the results don't suggest a role of NASP exclusive to the cytoplasm. Our findings highlight a cytoplasmic function of NASP, however, we do not want to rule out that this same function couldn't occur in the nucleus.

Lines 409-413: The authors claim that histone deficiency likely does not cause the embryonic arrest seen in embryos from NASP mutant mothers. This is because H3 is reduced by 50% yet some embryos arrest long before they've depleted this supply. However, the authors also showed that H3 import rates are affected in these embryos due to lower H3 concentration. Since the early embryo cycles are so rapid, reduced H3 import rates could lead to early arrest, even though available H3 remains in the cytoplasm.

We thank the reviewer for their suggestion. This conclusion is based on the findings from the previous study from our lab which showed that the majority of the embryos laid by NASP mutant females get arrested in the very early nuclear cycles (Reviewer #1 (Significance (Required)):

The significance of the work is conceptual, as NASP is known to function in H3 availability but the precise mechanism is elusive. This work represents a necessary advance, especially to show that NASP does not affect H3 import rates, nor does it chaperone H3 into the nucleus. However, the authors acknowledge that many questions remain. Foremost, why is NASP imported into the nucleus and what is its role there?

I believe this work will be of interest to those who focus on early animal development, but NASP may also represent a tool, as the authors conclude in their discussion, to reduce histone levels during development and examine nucleosome positioning. This may be of interest to those who work on chromatin accessibility and zygotic genome activation.

I am a genetics expert who works in Drosophila embryogenesis. I do not have the expertise to evaluate the aggregate methods presented in Figure 4.

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

Summary:

This manuscript focuses on the role of the histone chaperone NASP in Drosophila. NASP is a chaperone specific to histone H3 that is conserved in mammals. Many aspects of the molecular mechanisms by which NASP selectively binds histone H3 have been revealed through biochemical studies. However, key aspects of NASP's in vivo roles remain unclear, including where in the cell NASP functions, and how it prevents H3 degradation. Through live imaging in the early Drosophila embryo, which possesses large amounts of soluble H3 protein, Das et al determine that NASP does not control nuclear import or export of H3.2 or H3.3. Instead, they find through differential centrifugation analysis that NASP functions in the cytoplasm to prevent H3 aggregation and hence its subsequent degradation.

Major Comments:

The protein aggregation assays raise several questions. From a technical standpoint, it would be helpful to have a positive control to demonstrate that the assay is effective at detecting protein aggregates. Ie. a genotype that exhibits increased protein aggregation; this could be for a protein besides H3. A common issue raised by all three reviewers was to more convincingly demonstrate that assay that we have used to isolate protein aggregates does, in fact, isolate protein aggregates. To verify this, we will be performing the aggregate isolation assay using controls that are known to induce more protein aggregation. We will perform the aggregation assay with egg chambers or extracts that are exposed to heat shock or the aggregation-inducing chemicals Canavanine and Azetidine-2-carboxylic acid. The chemical treatment was a welcome suggestion from reviewer #3. These experiments will significantly strengthen any claims based on the outcome of the aggregation assay.

If NASP is not required to prevent H3 degradation in egg chambers, then why are H3 levels much lower in NASP input lanes relative to wild-type egg chambers in Fig 4D? We appreciate the reviewer's inputs regarding the reduced H3 levels in the NASP mutant egg chambers. We observe this reduction in H3 levels in the input because of the altered solubility of H3 which leads to the loss of H3 protein at different steps of the aggregate isolation assay. We will add a supplement figure showing H3 levels at different steps of the aggregate isolation assay. We do want to stress, however, that the total levels of H3 in stage 14 egg chambers does not change between WT and the NASP mutant.

A corollary to this is that the increased fraction of H3 in aggregates in NASP mutants seems to be entirely due to the reduction in total H3 levels rather than an increase in aggregated H3. If NASP's role is to prevent aggregation in the cytoplasm, and degradation has not yet begun in egg chambers, then why are aggregated H3 levels not increased in NASP mutants relative to wild-type egg chambers? If the same number of egg chambers were used, shouldn't the total amount of histone be the same in the absence of degradation?

In previously published work, we demonstrated that total H3 levels are unaffected when comparing WT and NASPmutant stage 14 egg chambers. This means that the amount of H3 deposited into the eggs does not change in the absence of NASP. To address the reviewer's comment, we will change the text to make the link to our previous work clear. As stated above, we will add a supplement figure showing H3 levels at different steps of the aggregate isolation assay.

The live imaging studies are well designed, executed, and quantified. They use an established genotype (H3.2-Dendra2) in wild-type and NASP maternal mutants to demonstrate that NASP is not directly involved in nuclear import of H3.2. Decreased import is likely due to reduced H3.2 levels in NASP mutants rather than reduced import rates per se. The same methodology was used to determine that loss of NASP did not affect H3.2 nuclear export. These findings eliminate H3.2 nuclear import/export regulation as possible roles for NASP, which had been previously proposed.

Thank you.

Live imaging also conclusively demonstrates that the levels of H3.2 in the nucleoplasm and in mitotic chromatin are significantly lower in NASP mutants than wild-type nuclei. Despite these lower histone levels, the nuclear cycle duration is only modestly lengthened. The live imagining of NASP-Dendra2 nuclear import conclusively demonstrate that NASP and H3.2 are unlikely to be imported into the nucleus as one complex.

Thank you.

Minor Comments:

Additional details on how the NASP-Dendra2 CRISPR allele was generated should be provided. In addition, additional details on how it was determined that this allele is functional should be provided (e.g. quantitative assays for fertility/embryo viability of NASP-Dendra2 females) We will make these additions to the text.

If statistical tests are used to determine significance, the type of test used should be reported in the figure legends throughout.

We will make the addition of the statistical tests to the figure legends.

The western blot shown in Figure 4A looks more like a 4-fold reduction in H3 levels in NASP mutants relative to wild-type embryos, rather than the quantified 2-fold reduction. Perhaps a more representative blot can be shown.

We have additional blots in the supplemental figure S3C. The quantification was performed after normalization to the total protein levels and we can highlight that in the figure legend.

Reviewer #2 (Significance (Required)):

As a fly chromatin biologist with colleagues that utilize mammalian experimental systems, I feel this manuscript will be of broad interest to the chromatin research community. Packaging of the genome into chromatin affects nearly every DNA-templated process, making the mechanisms by which histone proteins are expressed, chaperoned, and deposited into chromatin of high importance to the field. The study has multiple strengths, including high-quality quantitative imaging, use of a terrific experimental system (storage and deposition of soluble histones in early fly embryos). The study also answers outstanding questions in the field, specifically that NASP does not control nuclear import/export of histone H3. Instead, the authors propose that NASP functions to prevent protein aggregation. If this could be conclusively demonstrated, it would be valuable to the field. However, the protein aggregation studies need improvement. Technical demonstration that their differential centrifugation assay accurately detects aggregated proteins is needed. Further, NASP mutants do not exhibit increased H3 protein aggregation in the data presented. Instead, the increased fraction of aggregated H3 in NASP mutants seems to be due to a reduction in the overall levels of H3 protein, which is contrary to the model presented in this paper.

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

This manuscript by Das et al. entitled "NASP functions in the cytoplasm to prevent histone H3 aggregation during early embryogenesis", explores the role of the histone chaperone NASP in regulating histone H3 dynamics during early Drosophila embryogenesis. Using primarily live imaging approaches, the authors found that NASP is not directly involved in the import or export of H3. Moreover, the authors claimed that NASP prevents H3 aggregation rather than protects against degradation.

Major Comments:

Figure 1A-B: The plotted data appear to have substantial dispersion. Could the authors include individual data points or provide representative images to help the reader assess variability?

We chose to show unnormalized data in Figure 1 so readers could better compare the actual import values of H3 in the presence and absence of NASP. We felt it was a better representation of the true biological difference although raw data is more dispersive. We did also include normalized data in the supplement. Regardless, we will add representative stills to Figure 1 and include a H3-Dendra2 movie in the supplement to show the representative data.

Given that the authors conclude that the reduced nuclear import is due to lowered H3 levels in NASP-deficient embryos, would overexpression of H3 rescue this phenotype? This would directly test whether H3 levels, rather than import machinery per se, drive the effect.

We thank the reviewer for their valuable suggestion. We and others have tried to overexpress histones in the Drosophila early embryo without success. There must be an undefined feedback mechanism preventing histone overexpression in the germline. In fact, a recent paper has been deposited on bioRxiv (https://doi.org/10.1101/2024.12.23.630206) that suggest H4 protein could provide a feedback mechanism to prevent histone overexpression. While we would love to do this experiment, it is not technically feasible at this time.

Figure 2A-B: The authors present the Relative Intensity of H3-Dendra2, but this metric obscures absolute differences between Control and NASP knockout embryos. Please include Total Intensity plots to show the actual reduction in H3 levels.

We will add the total H3-Dendra2 intensity plots to the supplemental figure for the export curves.

Additionally, Western blot analysis of nucleoplasmic H3 from wild-type vs. NASP-deficient embryos would provide essential biochemical confirmation of H3 level reductions.

We will measure nuclear H3 levels by western from 0-2 hr embryos laid by WT and NASP mutant flies.

Figure 4: To support the conclusion that NASP prevents H3 aggregation, I recommend performing aggregation assays by adding compounds that induce unfolding (amino acid analogues that induce unfolding, like canavanine or Azetidine-2-carboxylic acid) or using aggregation-prone H3 mutants.

This is a very helpful suggestion! It is difficult to get chemicals into Drosophila eggs, but we will treat extracts directly with these chemicals. Additionally, we will use heat shocked eggs and extracts as an additional control.

Inclusion of CMA and proteasome inhibition experiments could also clarify whether degradation pathways are secondarily involved or compensatory in the absence of NASP.

The degradation pathway for H3 in the absence of NASP is unknown and a major focus of our future work is to define this pathway. Drosophila does not have a CMA pathway and therefore, we don't know how H3 aggregates are being sensed.

Minor Comments:

(1) The Introduction would benefit from mentioning the two NASP isoforms that exist in mammals (sNASP and tNASP), as this evolutionary context may inform interpretation of the Drosophila results.

We will make the edits in the text to include that Drosophila NASP is the sole homolog of sNASP and that tNASP ortholog is not found in Drosophila.

(2) Could the authors comment on the status of histone H4 in their experimental system? Given the observed cytoplasmic pool of H3, is it likely to exist as a monomer? If this H3 pool is monomeric, does that suggest an early failure in H3-H4 dimerization, and could this contribute to its aggregation propensity?

In our previous work we noted that NASP binds more preferentially to H3 and the levels of H3 we much more reduced upon NASP depletion than H4. We pointed out in this publication that our data was consistent with H3 stores being monomeric in the Drosophila embryo. We don't' have a H4-Dendra2 line to test. In the future, however, this is something we are very keen to look at.

Reviewer #3 (Significance (Required)):

This work addresses a timely and important question in the field of chromatin biology and developmental epigenetics. The focus on histone homeostasis during embryogenesis and the cytoplasmic role of NASP adds a novel perspective. The live imaging experiments are a clear strength, providing valuable spatiotemporal insights. However, I believe that the manuscript would benefit significantly from additional biochemical validation to support and clarify some of the mechanistic claims.

3. Description of the revisions that have already been incorporated in the transferred manuscript

• *

4. Description of analyses that authors prefer not to carry out

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

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Referee #3

Evidence, reproducibility and clarity

This manuscript by Das et al. entitled "NASP functions in the cytoplasm to prevent histone H3 aggregation during early embryogenesis", explores the role of the histone chaperone NASP in regulating histone H3 dynamics during early Drosophila embryogenesis. Using primarily live imaging approaches, the authors found that NASP is not directly involved in the import or export of H3. Moreover, the authors claimed that NASP prevents H3 aggregation rather than protects against degradation.

Major Comments:

Figure 1A-B: The plotted data appear to have substantial dispersion. Could the authors include individual data points or provide representative images to help the reader assess variability? Given that the authors conclude that the reduced nuclear import is due to lowered H3 levels in NASP-deficient embryos, would overexpression of H3 rescue this phenotype? This would directly test whether H3 levels, rather than import machinery per se, drive the effect.

Figure 2A-B: The authors present the Relative Intensity of H3-Dendra2, but this metric obscures absolute differences between Control and NASP knockout embryos. Please include Total Intensity plots to show the actual reduction in H3 levels. Additionally, Western blot analysis of nucleoplasmic H3 from wild-type vs. NASP-deficient embryos would provide essential biochemical confirmation of H3 level reductions.

Figure 4: To support the conclusion that NASP prevents H3 aggregation, I recommend performing aggregation assays by adding compounds that induce unfolding (amino acid analogues that induce unfolding, like canavanine or Azetidine-2-carboxylic acid) or using aggregation-prone H3 mutants. Inclusion of CMA and proteasome inhibition experiments could also clarify whether degradation pathways are secondarily involved or compensatory in the absence of NASP.

Minor Comments:

(1) The Introduction would benefit from mentioning the two NASP isoforms that exist in mammals (sNASP and tNASP), as this evolutionary context may inform interpretation of the Drosophila results.

(2) Could the authors comment on the status of histone H4 in their experimental system? Given the observed cytoplasmic pool of H3, is it likely to exist as a monomer? If this H3 pool is monomeric, does that suggest an early failure in H3-H4 dimerization, and could this contribute to its aggregation propensity?

Significance

This work addresses a timely and important question in the field of chromatin biology and developmental epigenetics. The focus on histone homeostasis during embryogenesis and the cytoplasmic role of NASP adds a novel perspective. The live imaging experiments are a clear strength, providing valuable spatiotemporal insights. However, I believe that the manuscript would benefit significantly from additional biochemical validation to support and clarify some of the mechanistic claims.

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Referee #2

Evidence, reproducibility and clarity

Summary:

This manuscript focuses on the role of the histone chaperone NASP in Drosophila. NASP is a chaperone specific to histone H3 that is conserved in mammals. Many aspects of the molecular mechanisms by which NASP selectively binds histone H3 have been revealed through biochemical studies. However, key aspects of NASP's in vivo roles remain unclear, including where in the cell NASP functions, and how it prevents H3 degradation. Through live imaging in the early Drosophila embryo, which possesses large amounts of soluble H3 protein, Das et al determine that NASP does not control nuclear import or export of H3.2 or H3.3. Instead, they find through differential centrifugation analysis that NASP functions in the cytoplasm to prevent H3 aggregation and hence its subsequent degradation.

Major Comments:

1. The protein aggregation assays raise several questions.

a. From a technical standpoint, it would be helpful to have a positive control to demonstrate that the assay is effective at detecting protein aggregates. Ie. a genotype that exhibits increased protein aggregation; this could be for a protein besides H3.

b. If NASP is not required to prevent H3 degradation in egg chambers, then why are H3 levels much lower in NASP input lanes relative to wild-type egg chambers in Fig 4D?

c. A corollary to this is that the increased fraction of H3 in aggregates in NASP mutants seems to be entirely due to the reduction in total H3 levels rather than an increase in aggregated H3. If NASP's role is to prevent aggregation in the cytoplasm, and degradation has not yet begun in egg chambers, then why are aggregated H3 levels not increased in NASP mutants relative to wild-type egg chambers? If the same number of egg chambers were used, shouldn't the total amount of histone be the same in the absence of degradation? 2. The live imaging studies are well designed, executed, and quantified. They use an established genotype (H3.2-Dendra2) in wild-type and NASP maternal mutants to demonstrate that NASP is not directly involved in nuclear import of H3.2. Decreased import is likely due to reduced H3.2 levels in NASP mutants rather than reduced import rates per se. The same methodology was used to determine that loss of NASP did not affect H3.2 nuclear export. These findings eliminate H3.2 nuclear import/export regulation as possible roles for NASP, which had been previously proposed. 3. Live imaging also conclusively demonstrates that the levels of H3.2 in the nucleoplasm and in mitotic chromatin are significantly lower in NASP mutants than wild-type nuclei. Despite these lower histone levels, the nuclear cycle duration is only modestly lengthened. 4. The live imagining of NASP-Dendra2 nuclear import conclusively demonstrate that NASP and H3.2 are unlikely to be imported into the nucleus as one complex.

Minor Comments:

1. Additional details on how the NASP-Dendra2 CRISPR allele was generated should be provided. In addition, additional details on how it was determined that this allele is functional should be provided (e.g. quantitative assays for fertility/embryo viability of NASP-Dendra2 females)
2. If statistical tests are used to determine significance, the type of test used should be reported in the figure legends throughout.
3. The western blot shown in Figure 4A looks more like a 4-fold reduction in H3 levels in NASP mutants relative to wild-type embryos, rather than the quantified 2-fold reduction. Perhaps a more representative blot can be shown.

Significance

As a fly chromatin biologist with colleagues that utilize mammalian experimental systems, I feel this manuscript will be of broad interest to the chromatin research community. Packaging of the genome into chromatin affects nearly every DNA-templated process, making the mechanisms by which histone proteins are expressed, chaperoned, and deposited into chromatin of high importance to the field. The study has multiple strengths, including high-quality quantitative imaging, use of a terrific experimental system (storage and deposition of soluble histones in early fly embryos). The study also answers outstanding questions in the field, specifically that NASP does not control nuclear import/export of histone H3. Instead, the authors propose that NASP functions to prevent protein aggregation. If this could be conclusively demonstrated, it would be valuable to the field. However, the protein aggregation studies need improvement. Technical demonstration that their differential centrifugation assay accurately detects aggregated proteins is needed. Further, NASP mutants do not exhibit increased H3 protein aggregation in the data presented. Instead, the increased fraction of aggregated H3 in NASP mutants seems to be due to a reduction in the overall levels of H3 protein, which is contrary to the model presented in this paper.

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Referee #1

Evidence, reproducibility and clarity

Summary:

The authors investigate the function of the H3 chaperone NASP, which is known to bind directly to H3 and prevent degradation of soluble H3. What is unclear is where NASP functions in the cell (nucleus or cytoplasm), how NASP protects H3 from degradation (direct or indirect), and if NASP affects H3 dynamics (nuclear import or export). They use the powerful model system of Drosophila embryos because the soluble H3 pool is high due to maternal deposition and they make use of photoconvertable Dendra-tagged proteins, since these are maternally deposited and can be used to measure nuclear import/export rates.

Using these systems and tools, they conclude that NASP affects nuclear import, but only indirectly, because embryos from NASP mutant mothers start out with 50% of the maternally deposited H3. Because of the depleted H3 and reduced import rates, NASP deficient embryos also have reduced nucleoplasmic and chromatin-associated H3. Using a new Dendra-tagged NASP allele, the authors show that NASP and H3 have different nuclear import rates, indicating that NASP is not a chaperone that shuttles H3 into the nucleus. They test H3 levels in embryos that have no nuclei and conclude that NASP functions in the cytoplasm, and through protein aggregation assays they conclude that NASP prevents H3 aggregation.

Major comments:

The text was easy to read and logical. The data are well presented, methods are complete, and statistics are robust. The conclusions are largely reasonable. However, I am having trouble connecting the conclusions in text to the data presented in Figure 4.

First, I'm confused why the conclusion from Figure 4A is that NASP functions in the cytoplasm of the egg. Couldn't NASP be required in the ovary (in, say, nurse cell nuclei) to stimulate H3 expression and deposition into the egg? The results in 4A would look the same if the mothers deposit 50% of the normal H3 into the egg. Why is NASP functioning specifically in the cytoplasm when it is also so clearly imported into the nucleus? Maybe NASP functions wherever it is, and by preventing nuclear import, you force it to function in the cytoplasm. I do not have additional suggestions for experiments, but I think the authors need to be very clear about the different interpretations of these data and to discuss WHY they believe their conclusion is strongest.

Second, an alternate conclusion from Figure 4D/E is that mothers are depositing less H3 protein into the egg, but the same total amount is being aggregated. This amount of aggregated protein remains constant in activated eggs, but additional H3 translation leads to more total H3? The authors mention that additional translation can compensate for reduced histone pools (line 416).

As the function of NASP in the cytoplasm (when it clearly imports into the nucleus) and role in H3 aggregation are major conclusions of the work, the authors need to present alternative conclusions in the text or complete additional experiments to support the claims. Again, I do not have additional suggestions for experiments, but I think the authors need to be very clear about the different interpretations of these data and to discuss WHY they believe their conclusion is strongest.

Data presentation:

Overall, I suggest moving some of the supplemental figures to the main text, adding representative movie stills to show where the quantitative data originated, and moving the H3.3 data to the supplement. Not because it's not interesting, but because H3.3 and H3.2 are behaving the same.

Fig 1:

It would strengthen the figure to include representative still images that led to the quantitative data, mostly so readers understand how the data were collected. The inclusion of a "simulated 50% H3" in panel C is confusing. Why? I would also consider normalizing the data between A and B (and C and D) by dividing NASP/WT. This could be included in the supplement (OPTIONAL)

Fig S1:

The data simulation S1G should be moved to the main text, since it is the primary reason the authors reject the hypothesis that NASP influences H3 import rates.

Fig 2:

Once again, I think it would help to include a few representative images of the photoconverted Dendra2 in the main text. I struggled with A/B, I think due to not knowing how the data were normalized. When I realized that the WT and NASP data are not normalized to each other, but that the NASP values are likely starting less than the WT values, it made way more sense. I suggest switching the order of data presentation so that C-F are presented first to establish that there is less chromatin-bound H3 in the first place, and then present A/B to show no change in nuclear export of the H3 that is present, allowing the conclusion of both less soluble AND chromatin-bound H3.

Fig S2:

If M1-M3 indicate males, why are the ovaries also derived from males? I think this is just confusing labeling. Supplemental Movie S1: Beautiful. Would help to add a time stamp (OPTIONAL).

Fig 3:

Panel C is the same as Fig S1A (not Fig 1A, as is said in the legend), though I appreciate the authors pointing it out in the legend. Also see line 276. Panel D is a little confusing, because presumably the "% decrease in import rate" cannot be positive (Y axis). This could be displayed as a scatter (not bar) as in Panels B/C (right) where the top of the Y axis is set to 0.

Fig S3:

A: What do the different panels represent? I originally thought developmental time, but now I think just different representative images? Are these age-matched from time at egg lay? C: What does "embryos" mean? Same question for Fig 4A. Fig 4: A: What does "embryos" mean? Number of embryos? Age in hours? C: Not sure the workflow figure panel is necessary, as I can't tell what each step does. This is better explained in methods. However I appreciated the short explanation in the text (lines 314-5).

Minor comments:

The authors should describe the nature of the NASP alleles in the main text and present evidence of robust NASP depletion, potentially both in ovaries and in embryos. The antibody works well for westerns (Fig S2B). This is sort of demonstrated later in Figure 4A, but only in NAAP x twine activated eggs.

Lines 163, 251, 339: minor typos Line 184: It would help to clarify- I'm assuming cytoplasmic concentration (or overall) rather than nuclear concentration. If nuclear, I'd expect the opposite relationship. This occurs again when discussing NASP (line 267). I suspect it's also not absolute concentration, but relative concentration difference between cytoplasm and nucleus. It would help clarify if the authors were more precise. Line 189: Given that the "established integrative model" helps to reject the hypothesis that NASP is involved in H3 import, I think it's important to describe the model a little more, even though it's previously published. Line 203: "The measured rate of H3.2 export from the nucleus is negligible" clarify this is in WT situations and not a conclusion from this study. Line 201: How can the authors be so sure that the decrease in WT is due to "the loss of non-chromatin bound nucleoplasmid H3.2-Dendra2?" Line 217: In the conclusion, the authors indicate that NASP indirectly affects soluble supply of H3 in the nucleoplasm. I do believe they've shown that the import rate effect is indirect, but I don't know why they conclude that the effect of NASP on the soluble nucleoplasmic H3 supply is indirect. Similarly, the conclusion is indirect on line 239. Yet, the authors have not shown it's not direct, just assumed since NASP results in 50% decrease to deposited maternal histones. Line 292: What is the nature of the NASP "mutant?" Is it a null? Similarly, what kind of "mutant" is the twine allele? Line 295. Line 316: Why did the authors use stage 14 egg chambers here when they previously used embryos? This becomes more clear later shortly, when the authors examine activated eggs, but it's confusing in text. Lines 343-348: It's unclear if the authors are drawing extended conclusions here or if they are drawing from prior literature (if so, citations would be required). For example, why during oogenesis/embryogenesis are aggregation and degradation developmentally separated? Lines 386-7: I do not understand why the authors conclude that H3 aggregation and degradation are "developmentally uncoupled" and why, in the absence of NASP, "H3 aggregation precedes degradation." Line 395: Why suddenly propose that NASP also functions in the nucleus to prevent aggregation, when earlier the authors suggest it functions only in the cytoplasm? Lines 409-413: The authors claim that histone deficiency likely does not cause the embryonic arrest seen in embryos from NASP mutant mothers. This is because H3 is reduced by 50% yet some embryos arrest long before they've depleted this supply. However, the authors also showed that H3 import rates are affected in these embryos due to lower H3 concentration. Since the early embryo cycles are so rapid, reduced H3 import rates could lead to early arrest, even though available H3 remains in the cytoplasm.

Significance

The significance of the work is conceptual, as NASP is known to function in H3 availability but the precise mechanism is elusive. This work represents a necessary advance, especially to show that NASP does not affect H3 import rates, nor does it chaperone H3 into the nucleus. However, the authors acknowledge that many questions remain. Foremost, why is NASP imported into the nucleus and what is its role there?

I believe this work will be of interest to those who focus on early animal development, but NASP may also represent a tool, as the authors conclude in their discussion, to reduce histone levels during development and examine nucleosome positioning. This may be of interest to those who work on chromatin accessibility and zygotic genome activation.

I am a genetics expert who works in Drosophila embryogenesis. I do not have the expertise to evaluate the aggregate methods presented in Figure 4.

Определение сознания как необходимой операционки для работы дравйвверов - модулей сознания Еслибы работали хорошо и хорошо были связаны, то непрерывность истории вероятно не понадобилосбы...

А также определения сознания как историю расказываемую самому себе, и несколько вех смены самоопределений:<br /> детсво - значимый взрослый говорит какой я подросток - группа говорит мне какой я взрослый (сколько лет? ) сам формирует непротиворечивую историю про себя.

Кое-где читал, что история внутри нас появляется тогда, когда есть противоречие между частями нас. Таким образом, они нужны не столько нам, сколько как способ передать информацию о решении этого противоречия.

В связи с этим, если бы все части нас были интгерированы, то создавать историю о себе бы не требовалось.

Свою жизнь мы живем изнутри своей головы и в большей мере осведомлены о мотивах. Других же приоритетно мы судим по действиям. А не ищем намерения. Мудак или не мудак, лучше давай кредит кредит доверия

Это похоже на некоторые тезисы из буддизма. Интересно, для меня рациональность раньше была в основном через когнитивно-поведенческую терапию и теорию вероятностей.

Но кажется и пересечений с буддизмом довольно много.

Очень вымученый и тяжелый и крайне неудачный пример с кучей неинтуитивных натяжек в условиях, еще и про чувства...

Напомнить пример про принятие решений на основе и взвешивания вероятностей риска в деньгах и усилий

Почему не использовать слово условная вероятность

"вероятность, которая есть если верно утверждение Х".

Обратное от этого: априорная вероятность

Афтор походу обьясняет концепт истины. (которую можешь знать или нет но она есть) Но не понимаю почему автор выбрал такие жесткие абсолютные формулирвоки.

Сверхобобщение, гипертрофированные.

referencing the dynamic between men and women, possibly the idea of simping

acculturation

undermined welsh identity by making them fuedal subjects of the king, rather than welsh citizens

SLAY SLAY SLAY QUOTE! As such, statute I was a clear undermining of Welsh identity in the way that Wales was now an owned land, rather than a principality, it was no longer independent as was subject to the whims of the 'alien' english

here. could say underming welsh identity by trying to keep wales divided to stop them from rising up again

I wonder how he was able to convince so many different tribes to unite and choose him as their leader.

It’s interesting that this crusade was Christians attacking other Christians just because they believed differently.

After another major military failure in 1214 and over a decade of high taxes, fines, and seizures of baronial lands (the Robin Hood story is based on this period), John's subjects, the Norman-descended Barons, rebelled against his abuses and forced him to sign the Magna Carta in 1215.

I find it interesting that he grew up in Tunisia and the he learned several languages and skills as well.

So freedom as in J Raz's tripartite practical autonomy, NOT as in absolute uncoditional free will.

We need agency to play, apparently, but we don't have it, most likely!

Playthrough, or autoethnographic, or therapeutic self-writing reflections. It becomes true for you. It is your lived experience.

YAASSSSS KING ARTHURRRR!!

This hybrid framing challenges the idea that vegetarianism in China is simply a “return” to tradition. Instead, it seems like a negotiation of multiple moral vocabularies. It makes me wonder whether this hybridity stabilizes vegetarianism (by giving people many entry points) or destabilizes it (by diluting any single motivation).

This is so nostalgia-laced. It highlights to me how economic change reshapes the emotional meaning of food.

I find this compelling because it suggests that ethical exploration is happening outside traditional religious institutions. It makes me rethink how moral life is structured in contemporary China not necessarily through doctrine and orthodox practices, but through everyday consumption spaces

eLife Assessment

This study presents a valuable tool named TSvelo, a computational framework for RNA velocity inference that models transcriptional regulation and gene-specific splicing. The evidence supporting the claims of the authors is solid, although elaboration of the computational benchmark and datasets would have strengthened the study. The work will be of interest to computational scientists working in the field of RNA biology.

Reviewer #1 (Public review):

Summary:

In the paper, the authors propose a new RNA velocity method, TSvelo, which predicts the transcription rate linearly based on the expression of RNA levels of transcription factors. This framework is an extension of its recent work TFvelo by including unspliced reads and designing a coherent neuralODE framework. Improved performance was demonstrated in six diverse datasets.

Strengths:

Overall, this method introduces innovative solutions to link cell differentiation and gene regulation, with a balance between model complexity (neuralODE) and interpretability (raw gene space).

Weaknesses:

While it seems to provide convincing results, there are multiple technical concerns for the authors to clarify and double-check.

(1) The authors should clarify and discuss the TF-target map: here, the TF-target genes map is predefined by the TF binding's ChIP-seq data. This annotation is largely incomplete and mostly compiled from a set of bulk tissues. Therefore, for a certain population, the TF-target relation may change. This requires clarification and discussion, possibly exploring how to address this in the model. In addition, a regulon database could be added, e.g., DoRothEA?

(2) The authors should clarify how example genes are selected. This is particularly unclear in Figure 2d.

(3) The authors should clarify confidence in the statement in lines 179-180, that ANXA4 should initially decrease. This is particularly concerning, as TSvelo didn't capture the cell cycle transitions well during the initial part.

(4) A support reference should be added for the statement in line 260 that "neuron migrations are inside-out manner". There is no reference supporting this, and this statement is critical for the model assessment.

(5) The comparison to scMultiomics data is particularly interesting, as MultiVelo uses ATAC data to predict the transcription rate. It would be very insightful to add a direct comparison of the estimated transcription rate between using ATAC and directly using TFs' RNA expressions.

(6) In Figure 6g, it should be clarified how the lineage was determined. Did the authors use the LARRY barcodes, predicted cell fate, or any other methods? Here, the best way is probably using the LARRY barcodes for individual clones.

Reviewer #2 (Public review):

Summary:

Li et al. propose TSvelo, a computational framework for RNA velocity inference that models transcriptional regulation and gene-specific splicing using a neural ODE approach. The method is intended to improve trajectory reconstruction and capture dynamic gene expression changes in scRNA-seq data. However, the manuscript in its current form falls short in several critical areas, including rigorous validation, quantitative benchmarking, clarity of definitions, proper use of prior knowledge, and interpretive caution. Many of the authors' claims are not fully supported by the evidence.

Major comments:

(1) Modeling comments

(a) Lines 512-513: How does the U-to-S delay validate the accuracy of pseudotime? Using only a single gene as an example is not sufficient for "validation."

(b) Lines 512-518: The authors propose a strategy for selecting the initial state, but do not benchmark how accurate this selection procedure is, nor do they provide sufficient rationale. While some genes may indeed exhibit U-to-S delay during lineage differentiation, why does the highest U-to-S delay score indicate the correct initiation states? Please provide mathematical justification and demonstrate accuracy beyond using a single gene example. Maybe a simulation with ground truth could help here, too.

(c) Equation (8): The formulation looks to be incorrect. If $$W \in \mathbb{R}^{G\times G}$$ and $$W' - \Gamma' \in \mathbb{R}^{K\times K}$$, how can they be aligned within the same row? Please clarify.

(d) The use of prior knowledge graphs from ENCODE or ChEA to constrain regulation raises concerns. Much of the regulatory information in these databases comes from cell lines. How can such cell-line-based regulation be reliably applied to primary tissues, as is done throughout the manuscript? Additional experiments are needed to test the robustness of TSvelo with respect to prior knowledge.

(e) Lines 579-580: How is the grid search performed? More methodological details are required. If an existing method was used, please provide a citation.

(2) Application on pancreatic endocrine datasets

(a) Lines 140-141: What is the definition of the final pseudotime-fitted time t or velocity pseudotime?

(b) Lines 143-144: The use of the velocity consistency metric to benchmark methods in multi-lineage datasets is incorrect. In multi-lineage differentiation systems, cells (e.g., those in fate priming stages) may inherently show inconsistency in their velocity. Thus, it is difficult to distinguish inconsistency caused by estimation error from that arising from biological signals. Velocity consistency metrics are only appropriate in systems with unidirectional trajectories (e.g., cell cycling). The abnormally high consistency values here raise concerns about whether the estimated velocities meaningfully capture lineage differences.

(c) The improvement of TSvelo over other methods in terms of cross-boundary direction correctness looks marginal; a statistical test would help to assess its significance.

(d) Lines 177-178: Based on the figure, TSvelo does not appear to clearly distinguish cell types. A quantitative metric, such as Adjusted Rand Index (ARI), should be provided.

(e) Lines 179-183: The claim that traditional methods cannot capture dynamics in the unspliced-spliced phase portrait is vague. What specific aspect is not captured-the fitted values or something else? Evidence is lacking. Please provide a detailed explanation and quantitative metrics to support this claim.

(3) Application to gastrulation erythroid datasets

(a) Lines 191-194: The observation that velocity genes are enriched for erythropoiesis-related pathways is trivial, since the analysis is restricted to highly variable genes (HVGs) from an erythropoiesis dataset. This enrichment is expected and therefore not informative.

(b) Lines 227-228: It remains unclear how TSvelo "accurately captures the dynamics." What is the definition of dynamics in this context? Figure 3g shows unspliced/spliced vs. fitted time plots and phase portraits, but without a quantitative definition or measure, the claim of superiority cannot be supported. Visualization of a single gene is insufficient; a systematic and quantitative analysis is needed.

(4) Application to the mouse brain and other datasets

(a) Lines 280-281: The authors cannot claim that velocity streams are smoother in TSvelo than in Multivelo based solely on 2D visualization. Similarly, claiming that one model predicts the correct differentiation trajectory from a 2D projection is over-interpretation, as has been discussed in prior literature see PMID: 37885016.

(b) Lines 304-306: Beyond transcriptional signal estimation, how is regulation inferred solely from scRNA-seq data validated, especially compared with scATAC-seq data? Are there cases where transcriptome-based regulatory inference is supported by epigenomic evidence, thereby demonstrating TSvelo's GRN inference accuracy?

(c) The claim that TSvelo can model multi-lineage datasets hinges on its use of PAGA for lineage segmentation, followed by independent modeling of dynamics within each subset. However, the procedure for merging results across subsets remains unclear.

Reviewer #3 (Public review):

Despite the abundance of RNA velocity tools, there are still major limitations, and there is strong skepticism about the results these methods lead to. In this paper, the authors try to address some limitations of current RNA velocity approaches by proposing a unified framework to jointly infer transcriptional and splicing dynamics. The method is then benchmarked on 6 real datasets against the most popular RNA velocity tools.

While the approach has the potential to be of interest for the field, and may present improvements compared to existing approaches, there are some major limitations that should be addressed, particularly concerning the benchmark (see major comment 1).

Major comments:

(1) My main criticism concerns the benchmarking: real data lack a ground truth, and are absolutely not ideal for comparing methods, because one can only speculate what results appear to be more plausible.<br /> A solid and extensive simulation study, which covers various scenarios and possibly distinct data-generating models, is needed for comparing approaches. The authors should check, for example, the simulation studies in the BayVel approach (Section 4, BayVel: A Bayesian Framework for RNA Velocity Estimation in Single-Cell Transcriptomics). Clearly, all methods should be included in the simulation.

(2) Related to the above: since a ground truth is missing, the real data analyses need to be interpreted with caution. I recommend avoiding strong statements, such as "successfully captures the correct gene dynamics", or "accurately infer", in favour of milder statements supported by the data, such as "... aligns with the biological processes described" (as in page 12), or "results are compatible with current biological knowledge", etc...

(3) Many methods perform RNA velocity analyses. While there is a brief description, I think it'd be useful to have a schematic summary (e.g., via a Table) of the main conceptual, mathematical, and computational characteristics of each approach.

(4) Related to the above: I struggled to identify the main conceptual novelty of TSvelo, compared to existing approaches. I recommend explaining this aspect more extensively.

(5) A computational benchmark is missing; I'd appreciate seeing the runtime and memory cost of all methods in a couple of datasets.

(6) I think BayVel (mentioned above) should be added to the list of competing methods (both in the text and in the benchmarks). The package can be found here: https://github.com/elenasabbioni/BayVel_pkgJulia .