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 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.

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.

Amazing that MCP is not funtamental.

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.

• 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.

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.

grand livre comptable

Eigenvectors*

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.

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.

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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.

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

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Summary:

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.

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

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

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.

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".

This article talks about the harm that Jimmy Kimmel's Halloween Candy Prank brings to children. It states that children's abilities to understand humor based on their ages, so when these pranks happen to children who are younger than 10 years old, it might be traumatic experiences for children because we can see the painful reactions from children. I think the Candy prank is definitely harmful to children, but parents can tell children that it is a prank and show them the candies right after the prank, so calling it a traumatic experience is way too exaggerated. It could be harmful, but it can also be the lesson of teaching children the sense of humor. I think parents decide if they should play the prank based on children's personalities and their ages.

This article talks about the relationship between trauma and shame. It talks about how shame can be a healthy form of healthy development. It talks about how after 2 years old children start to feel guilt, shame, and fear.

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.

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

YAASSSSS KING ARTHURRRR!!

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.

Translated to non-gibberish lingo: I can't chose what I desire. If this wasn't the case, it would create an endless loop of having ability to chose everything, an infinite amount of time back, and then having an ability to chose what to chose, and what are objects of desire in the first place, etc.

Yes, we must assume. But this is playing pretend. We also assume that patterns exist and can be measured, and that doing science makes sense. These are the foundations of a study.

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 .

Author response:

Reviewer #1:

We appreciate the reviewer’s positive assessment of TSvelo and their helpful technical comments. In the revised manuscript, we will:

(1) Provide a clearer discussion of TF–target annotations, their limitations, and potential integration of additional databases.

(2) Clarify the rationale for example-gene selection (e.g., in Fig. 2d).

(3) Re-evaluate and temper the interpretation regarding ANXA4 and early-stage cell-cycle transitions.

(4) Add appropriate references supporting neuronal inside-out migration.

(5) Include additional analysis comparing TF-based transcription rate estimation with ATAC-based estimates from MultiVelo.

(6) Clarify how lineages were determined in Fig. 6g and incorporate barcode-based validation where applicable.

(7) Correct all typographical errors noted.

Reviewer #2:

We appreciate the reviewer’s careful examination of modeling, benchmarking, and interpretation. To address these concerns, we will:

(1) Expand the methodological justification for initial-state selection, add simulations with ground truth, and evaluate U-to-S delay more broadly across genes.

(2) Clarify matrix formulations and ensure consistency in notation (e.g., Eq. 8).

(3) Assess robustness to prior-knowledge graphs and evaluate alternatives beyond ENCODE/ChEA.

(4) Add methodological details on parameter search.

(5) Improve benchmarking on pancreatic endocrine datasets by including clear definitions of velocity pseudotime, ARI for cell-type separation, quantitative evaluation of phase-portrait fits, and appropriate interpretation of consistency metrics for multi-lineage systems.

(6) Reframe claims about “accurate” or “correct” predictions where evidence is qualitative and strengthen quantitative support where possible.

(8) Clarify lineage segmentation and merging when applying PAGA-guided multi-lineage modeling.

Reviewer #3:

We thank the reviewer for highlighting the need for more rigorous benchmarking and conceptual clarity. In response, we will:

(1) Conduct an expanded simulation study incorporating different data-generating models.

(2) Revise all strong claims to more cautious, evidence-based language.

(3) Add a concise table summarizing conceptual and computational differences among RNA-velocity frameworks.

(4) More clearly articulate the conceptual novelty of TSvelo relative to existing approaches.

(5) Include runtime and memory benchmarks across representative datasets.

(6) Explore additional methods in conceptual comparisons and benchmarking analyses.We appreciate the reviewers’ thoughtful input and agree that the suggested analyses and clarifications will significantly improve the rigor and clarity of the manuscript. We will incorporate all recommended revisions in the resubmission and provide a full, detailed, point-by-point response at that time.

I am so glad that Schumann included these lines because Lu's dilemma really resonates with me. Her willingness to articulate doubt complicates the narrative of her as a devout Buddhist. It showcases the struggle one feels while trying to maintain reason and rationale alongside their faith. I truly empathise with her.

These lines suggest that Lu's transformation wasn’t superficial but doctrinally grounded. I find myself wondering why this specific text had such an impact like was it the karmic logic, the moral urgency, or the clarity of Yinguang’s system? It challenges my tendency to separate “intellectual reading” from “life practice.

This line reveals the intellectual context Lu was pushing against at that time. I’m struck by how entrenched these Orientalist hierarchies were like Pure Land is dismissed as “corrupt” just because it doesn’t match Western expectations of textual purity. It helps me appreciate and understand why Lu felt the need to intervene internationally.

This really stood out to me because it reframes Lu not just as a religious thinker but as someone consciously using Buddhist ethics to intervene in global political debates. I hadn’t thought of vegetarianism as something Chinese Buddhists mobilized outwards toward the world rather than just internally within China.

this

this

hoi

hi odin6343

timbre affects consonance

https://www.biorxiv.org/content/10.1101/2025.09.12.675104v1.full

https://www.biorxiv.org/content/10.1101/2025.09.12.675104v1.full

Possible primary source?

However, while some may say that statute blah sought to undermine Welsh identity (introduction) notes how a survey from the time suggested that the English system was replacing Welsh laws already. As such, we may argue that such tactics were used not only to supdue the Welsh, but to enable a more unified administration system to () this new colony p.34

Interestingly, () notes a contemporary survey which noted how English laws had already begun to replace Welsh ones, the move moreso one of administrational ease than (blah), neverthless, (blah) notes the continutation of Welsh customs (p.34)

A key cause for rebellion prior to the conquest, upon the Llewyllen ap Gruffudd's defeat, the Statute of Rhuddlan defined Wales as (), statute (blah) providing evidence for blah, the Edwardian conquest undermining Welsh identity through replacing the Welsh institution of blah with a Norman one

Mais POURQUOI ?? :D :D :D

The missing option: they were never read at all.

add two paragraphs of your thoughts, it can't just be a mere reporting of what happened and just presenting all sides. THIS IS YOUR OPINION.

transparency of what?

no solutions proposed???!

move this part of the paragraph to the next one so it explains how greenwashing works as a credible expert tells her POV on the problem.

what is that another survey, then?

Unite the thought into a straightforward actionable opinion.

But

add more practical paragraphs after the fifth paragraph so the masses can join the cause

the context paragraph is too long. IT should be directly saying the source of the news + the news + immediate opinion.

Then drop the statistics on the second paragrapn.

REMOVE the current quotation para Then convert the fourth paragraph into a direct quotation.

ARE WE SURE to repeat the quote on the pull quote?

let's replace it with "job" to get "job sessions" instead of just "backup sessions"

You also need a premium license.

if she cited any reasons for Greenwashing, it would be much better.

kinda okay, pero at least sa quote, let's include why she is linking it.

remove

state the why after the first sentence.

Drop "the"

delayed.

are we sure that she is a PH bank exec? They are bank regulators, not an executive of a bank.

BSP

I recommend adding a number here for MORE IMPACT since this news is centered around numbers of illiteracy

“3 out of 4 students…” 75% of PH students"

for - green bean harvester

UNE PARTIE INTERESSE

for - tepary bean - researcher - South Africa

eLife Assessment

This valuable study investigates the role of P-bodies in yeast proliferation and mRNA regulation within the phyllosphere, proposing that P-body assembly contributes to methanol metabolism and stress adaptation. The findings are of interest to researchers studying post-transcriptional gene regulation and microbial ecology in plants. However, the evidence is incomplete, as most experiments were performed under artificial conditions, relied on limited genetic validation, and were supported primarily by qualitative or low-resolution imaging.

Reviewer #1 (Public review):

Summary:

Stemming from the previous research on the adaptation of methylotrophic microbes in the phyllosphere environment, this paper tested a novel hypothesis on the molecular and cellular mechanisms by which yeast uses biomolecular condensates as unique niches for the regulation of methanol-induced mRNAs. While a few in vivo experiments were conducted in the phyllosphere, more assays were carried out on plates to mimic various stress conditions, diminishing the reliability of the conclusions in supporting the main hypothesis.

Strengths:

This study addressed an interesting and important biological question. Some of the experiments were conducted methodically and carefully. The visualization of both the biomolecular condensates and the mRNAs was helpful in addressing the questions. The results are expected to be useful in paving the way for the future study to directly test its main hypothesis. The results of this study could also have a general implication for the adaptation of a huge population of microbes in the enormous space of the phyllosphere on Earth.

Weaknesses:

The results were often over- and misinterpreted. Given mthat any hypotheses were tested indirectly on plates, the correlative results could only be used to carefully suggest the likelihood of the hypotheses. For example, a single edc3 mutant was used to represent a P-body-defective strain, although it is well known that EDC3 is a critical component in mRNA decapping; hence, the mutant should display a pleiotropic phenotype, rather than a mere reduced P-body phenotype. Using a similar reductionist approach, the study went on to employ a series of plate assays to argue that the conditions were mimicking the phyllosphere, which could be misleading under these circumstances. Furthermore, the low percentage of the colocalization between P-bodies and mimRNA granules and the similar results from negative control mRNAs do not convincingly support the idea that mimRNAs are sequestered between two biomolecular condensates, and P-bodies could serve as regulatory hubs. Given that the abundance of mimRNA granules was positively correlated with the transcript abundance of mimRNAs, and P-body abundance did not change too much under methanol induction, the results could not support an active mimRNA sequestration mechanism from mimRNA granules to P-bodies with a proportional increase of the overlap between the two condensates. More direct experiments conducted in the phyllosphere using multiple P-body defective yeast strains should strengthen the manuscript, assuming all the results turned out to be supportive.

Reviewer #2 (Public review):

Summary:

This article aims to elucidate the potential roles of P-bodies in yeast adaptation to complex environmental conditions, such as the plant leaf phyllosphere. The authors demonstrated that yeast mutants defective in one of the P-body-localized proteins failed to grow in the Arabidopsis thaliana phyllosphere. They conducted detailed imaging analyses, focusing particularly on the co-localization of P-bodies and mRNAs (DAS1) related to the methanol metabolism pathway under various environmental conditions. The study newly revealed that these mRNAs form dot-like structures that occasionally co-localize with a P-body marker. Furthermore, the authors showed that the number of P-body-labeled dots increases under stress conditions, such as H₂O₂ treatment, and that mRNA dots are more frequently localized to P-body-like structures. Based on these detailed observations, the authors hypothesize that P-bodies function to protect mRNAs from degradation, particularly under stress conditions.

Strengths:

I think the authors' attempt to elucidate the potential roles of P-bodies in yeast under stress conditions is novel, and the imaging data are overall very nice.

Weaknesses:

I believe the authors could make additional efforts to more clearly demonstrate that P-bodies are indeed required for yeast proliferation in the phyllosphere, as described below, since this represents the most novel aspect of the study.

Reviewer #3 (Public review):

Summary:

The authors use fluorescent microscopy and fluorescent markers to investigate the requirement of P-bodies during growth on methanol, a common substrate available on plant leaves, by using a yeast edc3 mutant defective in P-body formation. Growth on methanol upregulates the transcription of methanol metabolic genes, which accumulate in granular structures, as observed by microscopy. Co-localization of P-bodies and granules was quantified and described as dynamically enhanced during oxidative stress. Ultimately, the authors suggest a model where methanol induces the accumulation of methanol-induced mRNAs in cytosolic granules, which dynamically interact with P-bodies, especially during oxidative stress, to protect the mRNAs from degradation. However, this model is not strongly supported by the provided data, as the quantification of the co-localization between different markers (of organelles and between P-body and granules) is not well presented or described in the text.

Considering that there is only a small EDC3-dependent overlap between P-bodies and mimRNA granules, the claim that P-bodies regulate mimRNAs is not fully justified. Rather, EDC3 could also be involved in mimRNA granule formation, independent of P-bodies.

Strengths:

(1) The authors could show convincingly that P-bodies (using a P-body-deficient edc3-KO strain) are important for colonizing the plant phyllosphere and for the regulation of methanol-induced mRNAs (mimRNA).

(2) The visualization of mimRNA granules and P-bodies using fluorescent markers is interesting and was validated by alternative methods, such as FISH staining.

(3) The dynamic formation of mimRNA granules and P-bodies was demonstrated during growth on leaves and in artificial medium during oxidative stress. The mimRNA granules showed a similar dynamic as the abundances of several mimRNAs and their corresponding proteins.

(4) A role of EDC3 in the formation of mimRNA granules was demonstrated. However, the link between P-bodies and mimRNA granules was not clearly shown.

Weaknesses:

(1) The study largely relies on fluorescent microscopy and co-localization measurements. However, the subcellular resolution is not very high; it is unclear how dot-like structures were measured and, importantly, how co-localization was quantified.

(2) The text does not clarify to what degree P-bodies and mimRNA granules are different structures. Based on the images, the size of P-bodies and granules seems to be vastly different, making it unclear whether these structures are fused or separate, even if their markers are reported to overlap.

(3) The evidence that mimRNA granules contain ribosome-free and ribosome-associated RNA is only based on inhibitors and microscopy, without providing further evidence measuring granule content by isolation and sequencing approaches.

(4) Similarly, the co-localization with other organelle markers is not supported by quantitative data.

eLife Assessment

This fundamental study presents experimental evidence on how geomagnetic and visual cues are integrated in a nocturnally migrating insect. The evidence supporting the conclusions is compelling. The work will be of broad interest to researchers studying animal migration and navigation.

Reviewer #1 (Public review):

Summary

The manuscript by Ma et al. provides robust and novel evidence that the noctuid moth Spodoptera frugiperda (Fall Armyworm) possesses a complex compass mechanism for seasonal migration that integrates visual horizon cues with Earth's magnetic field (likely its horizontal component). This is an important and timely study: apart from the Bogong moth, no other nocturnal Lepidoptera has yet been shown to rely on such a dual-compass system. The research therefore expands our understanding of magnetic orientation in insects with both theoretical (evolution and sensory biology) and applied (agricultural pest management, a new model of magnetoreception) significance.

The study uses state-of-the-art methods and presents convincing behavioural evidence for a multimodal compass. It also establishes the Fall Armyworm as a tractable new insect model for exploring the sensory mechanisms of magnetoreception, given the experimental challenges of working with migratory birds. Overall, the experiments are well-designed, the analyses are appropriate, and the conclusions are generally well supported by the data.

Strengths

(1) Novelty and significance: First strong demonstration of a magnetic-visual compass in a globally relevant migratory moth species, extending previous findings from the Bogong moth and opening new research avenues in comparative magnetoreception.

(2) Methodological robustness: Use of validated and sophisticated behavioural paradigms and magnetic manipulations consistent with best practices in the field. The use of 5-minute bins to study the dynamic nature of the magnetic compass which is anchored to a visual cue but updated with a latency of several minutes, is an important finding and a new methodological aspect in insect orientation studies.

(3) Clarity of experimental logic: The cue-conflict and visual cue manipulations are conceptually sound and capable of addressing clear mechanistic questions.

(4) Ecological and applied relevance: Results have implications for understanding migration in an invasive agricultural pest with an expanding global range.

(5) Potential model system: Provides a new, experimentally accessible species for dissecting the sensory and neural bases of magnetic orientation.

Weaknesses

While the study is strong overall, several recommendations should be addressed to improve clarity, contextualisation, and reproducibility:

(1) Structure and presentation of results

Requires reordering the visual-cue experiments to move from simpler (no cues) to more complex (cue-conflict) conditions, improving narrative logic and accessibility for non-specialists.

(2) Ecological interpretation

(a) The authors should discuss how their highly simplified, static cue setup translates to natural migratory conditions where landmarks are dynamic, transient or absent.

(b) Further consideration is required regarding how the compass might function when landmarks shift position, are obscured, or are replaced by celestial cues. Also, more consolidated (one section) and concrete suggestions for future experiments are needed, with transient, multiple, or more naturalistic visual cues to address this.

(3) Methodological details and reproducibility

(a) It would be better to move critical information (e.g., electromagnetic noise measurements) from the supplementary material into the main Methods.

(b) Specifying luminance levels and spectral composition at the moth's eye is required for all visual treatments.

(c) Details are needed on the sex ratio/reproductive status of tested moths, and a map of the experimental site and migratory routes (spring vs. fall) should be included.

(d) Expanding on activity-level analyses is required, replacing "fatigue" with "reduced flight activity," and clarifying if such analyses were performed.

(4) Figures and data presentation

(a) The font sizes on circular plots should be increased; compass labels (magnetic North), sample sizes, and p-values should be included.

(b) More clarity is required on what "no visual cue" conditions entail, and schematics or photos should be provided.

(c) The figure legends should be adjusted for readability and consistency (e.g., replace "magnetic South" with magnetic North, and for box plots better to use asterisks for significance, report confidence intervals).

(5) Conceptual framing and discussion

(a) Generalisations across species should be toned down, given the small number of systems tested by overlapping author groups.

(b) It requires highlighting that, unlike some vertebrates, moths require both magnetic and visual cues for orientation.

(c) It should be emphasised that this study addresses direction finding rather than full navigation.

(d) Future Directions should be integrated and consolidated into one coherent subsection proposing realistic next steps (e.g., more complex visual environments, temporal adaptation to cue-field relationships).

(e) The limitations should be better discussed, due to the artificiality of the visual cue earlier in the Discussion.

(6) Technical and open-science points

• Appropriate circular statistics should be used instead of t-tests for angular data shown in the supplementary material.

• Details should be provided on light intensities, power supplies, and improvements to the apparatus.

• The derivation of individual r-values should be clarified.

• Share R code openly (e.g., GitHub).

• Some highly relevant - yet missing - recent and relevant citations should be added, and some less relevant ones removed.

Reviewer #2 (Public review):

Summary:

This work provided experimental evidence on how geomagnetic and visual cues are integrated, and visual cues are indispensable for magnetic orientation in the nocturnal fall armyworm.

Strengths:

Although it has been demonstrated previously that the Australian Bogon moth could integrate global stellar cues with the geomagnetic field for long-distance navigation, the study presented in this manuscript is still fundamentally important to the field of magnetoreception and sensory biology. It clearly shows that the integration of geomagnetic and visual cues may represent a conserved navigational mechanism broadly employed across migratory insects. I find the research very important, and the results are presented very well.

Weaknesses:

The authors developed an indoor experimental system to study the influence of magnetic fields and visual cues on insect orientation, which is certainly a valuable approach for this field. However, the ecological relevance of the visual cue may be limited or unclear based on the current version. The visual cues were provided "by a black isosceles triangle (10 cm high, 10 cm 513 base) made from black wallpaper and fixed to the horizon at the bottom of the arena". It is difficult to conceive how such a stimulus (intended to represent a landmark like a mountain) could provide directional information for LONG-DISTANCE navigation in nocturnal fall armyworms, particularly given that these insects would have no prior memory of this specific landmark. It might be a good idea to make a more detailed explanation of this question.

eLife Assessment

This important work introduces a family of interpretable Gaussian process models that allows us to learn and model sequence-function relationships in biomolecules. These models are applied to three recent empirical fitness landscapes, providing convincing evidence of their predictive power. The findings should be of interest to the community working on the sequence-function relationship, on epistasis, and on fitness landscapes.

Reviewer #1 (Public review):

Summary:

Zhou and colleagues introduce a series of generalized Gaussian process models for genotype-phenotype mapping. The goal was to develop models that were more powerful than standard linear models, while retaining explanatory power as opposed to neural network approaches. The novelty stems from choices of prior distributions (and I suppose fitted posteriors) that model epistasis based on some form of site/allele-specific modifier effect and genotype distance. The authors then apply their models to three empirical datasets, the GB1 antibody-binding dataset, the human 5' splice set dataset, and a yeast meiotic cross dataset, and find substantially improved variance explained while retaining strong explanatory power when compared to linear models.

Strengths:

The main strength of the manuscript lies in the development of the modeling approaches, as well as the evidence from the empirical dataset that the variance explained is improved.

Weaknesses:

The main weakness of the paper is that none of the models were tested on an in silico dataset where the ground truth is known. Therefore, it is unclear if their model actually retains any explanatory power.

Impact:

Genotype-phenotype mapping is a central point of genetics. However, the function is complex and unknown. Simple linear models can uncover some functional link between genes and their effects, but do so through severe oversimplification of the system. On the other hand, neural networks can, in principle, model the function perfectly, but it does so without easy interpretation. Gaussian regression is another approach that improves on linear regression, allowing better fitting of the data while allowing interpretation of the underlying alleles and their effects. This approach, now computable with state-of-the-art algorithms, will advance the field of genotype-to-phenotype associations.

Reviewer #2 (Public review):

This paper builds on prior work by some of the same authors on how to model fitness landscapes in the presence of epistasis. They have previously shown how simply writing general expansions of fitness in terms of one-body plus two-body plus three-body, etc., terms often fails to generalize to good predictions. They have also previously introduced a Gaussian process regression approach regarding how much epistasis there should be of each order.

This paper contains several main advances:

(1) They implement a more efficient form of the Gaussian process model fitting that uses GPUs and related algorithmic advances to enable better fitting of these models to datasets for larger sequences.

(2) They provide a software package implementing the above.

(3) They generalize the models to allow the extent of epistasis associated with changes in sequence to depend on specific sites, alleles, and mutations.

(4) They show modest improvements in prediction and substantial improvements in interpretability with the more generalized models above.

Overall, while this paper is quite technical, my assessment is that it represents a substantial conceptual and algorithmic advance for the above reasons, and I would recommend only modest revisions. The paper seems well-written and clear, given the inherent complexity of this topic.

Reviewer #3 (Public review):

Summary:

The authors propose three types of Gaussian process kernels that extend and generalize standard kernels used for sequence-function prediction tasks, giving rise to the connectedness, Jenga, and general product models. The associated hyperparameters are interpretable and represent epistatic effects of varying complexity. The proposed models significantly outperform the simpler baselines, including the additive model, pairwise interaction model, and Gaussian process with a geometric kernel, in terms of R^2.

Strengths:

(1) The demonstrated performance boost and improved scaling with increasing training data are compelling.

(2) The hyperparameter selection step using the marginal likelihood, as implemented by the authors, seems to yield a reasonable hyperparameter combination that lends itself to biologically plausible interpretations.

(3) The proposed kernels generalize existing kernels in domain-interpretable ways, and can correspond to cases that would not be "physical" in the original models (e.g., $\mu_p>1$ in the original connectedness model that allows modeling of anticorrelated phenotypes).

Weaknesses:

(1) While enabling uncertainty quantification is a key advantage of Gaussian processes, the authors do not present metrics specific to the predicted uncertainties; all metrics seem to concern the mean predictions only. It would be helpful to evaluate coverage metrics and maybe include an application of the uncertainties, such as in active learning or Bayesian optimization.

(2) The more complex models, like the general product model, place a heavier burden on the hyperparameter selection step. Explicitly discussing the optimization routine used here would be helpful to potential users of the method and code.

eLife Assessment

This important study describes a novel Bayesian psychophysical approach that efficiently measures how well humans can discriminate between colors across the entire isoluminant plane. The evidence was considered compelling, as it included successful model validation against hold-out data and published datasets. This approach could prove to be of use to color vision scientists, as well as to those who use computational psychophysics and attempt to model perceptual stimulus fields with smooth variations over coordinate spaces.

Reviewer #1 (Public review):

Summary:

This paper presents an ambitious and technically impressive attempt to map how well humans can discriminate between colours across the entire isoluminant plane. The authors introduce a novel Wishart Process Psychophysical Model (WPPM) - a Bayesian method that estimates how visual noise varies across colour space. Using an adaptive sampling procedure, they then obtain a dense set of discrimination thresholds from relatively few trials, producing a smooth, continuous map of perceptual sensitivity. They validate their procedure by comparing actual and predicted thresholds at an independent set of sample points. The work is a valuable contribution to computational psychophysics and offers a promising framework for modelling other perceptual stimulus fields more generally.

Strengths:

The approach is elegant and well-described (I learned a lot!), and the data are of high quality. The writing throughout is clear, and the figures are clean (elegant in fact) and do a good job of explaining how the analysis was performed. The whole paper is tremendously thorough, and the technical appendices and attention to detail are impressive (for example, a huge amount of data about calibration, variability of the stim system over time, etc). This should be a touchstone for other papers that use calibrated colour stimuli.

Weaknesses:

Overall, the paper works as a general validation of the WPPM approach. Importantly, the authors validate the model for the particular stimuli that they use by testing model predictions against novel sample locations that were not part of the fitting procedure (Figure 2). The agreement is pretty good, and there is no overall bias (perhaps local bias?), but they do note a statistically-significant deviation in the shape of the threshold ellipses. The data also deviate significantly from historical measurements, and I think the paper would be considerably stronger with additional analyses to test the generality of its conclusions and to make clearer how they connect with classical colour vision research. In particular, three points could use some extra work:

(1) Smoothness prior.<br /> The WPPM assumes that perceptual noise changes smoothly across colour space, but the degree of smoothness (the eta parameter) must affect the results. I did not see an analysis of its effects - it seems to be fixed at 0.5 (line 650). The authors claim that because the confidence intervals of the MOCS and the model thresholds overlap (line 223), the smoothing is not a problem, but this might just be because the thresholds are noisy. A systematic analysis varying this parameter (or at least testing a few other values), and reporting both predictive accuracy and anisotropy magnitude, would clarify whether the model's smoothness assumption is permitting or suppressing genuine structure in the data. Is the gamma parameter also similarly important? In particular, does changing the underlying smoothness constraint alter the systematic deviation between the model and the MOCS thresholds? The authors have thought about this (of course! - line 224), but also note a discrepancy (line 238). I also wonder if it would be possible to do some analysis on the posterior, which might also show if there are some regions of color space where this matters more than others? The reason for doing this is, in part, motivated by the third point below - it's not clear how well the fits here agree with historical data.

(2) Comparison with simpler models. It would help to see whether the full WPPM is genuinely required. Clearly, the data (both here and from historical papers) require some sort of anisotropy in the fitting - the sensitivities decrease as the stimuli move away from the adaptation point. But it's >not< clear how much the fits benefit from the full parameterisation used here. Perhaps fits for a small hierarchy of simpler models - starting with isotropic Gaussian noise (as a sort of 'null baseline') and progressing to a few low-dimensional variants - would reveal how much predictive power is gained by adding spatially varying anisotropy. This would demonstrate that the model's complexity is justified by the data.

(3) Quantitative comparison to historical data. The paper currently compares its results to MacAdam, Krauskopf & Karl, and Danilova & Mollon only by visual inspection. It is hard to extract and scale actual data from historical papers, but from the quality of the plotting here, it looks like the authors have achieved this, and so quantitative comparisons are possible. The MacAdam data comparisons are pretty interesting - in particular, the orientations of the long axes of the threshold ellipses do not really seem to line up between the two datasets - and I thought that the orientation of those ellipses was a critical feature of the MacAdam data. Quantitative comparisons (perhaps overall correlations, which should be immune to scaling issues, axis-ratio, orientation, or RMS differences) would give concrete measures of the quality of the model. I know the authors spend a lot of time comparing to the CIE data, and this is great.... But re-expressing the fitted thresholds in CIE or DKL coordinates, and comparing them directly with classical datasets, would make the paper's claims of "agreement" much more convincing.

Overall, this is a creative and technically sophisticated paper that will be of broad interest to vision scientists. It is probably already a definitive methods paper showing how we can sample sensitivity accurately across colour space (and other visual stimulus spaces). But I think that until the comparison with historical datasets is made clear (and, for example, how the optimal smoothness parameters are estimated), it has slightly less to tell us about human colour vision. This might actually be fine - perhaps we just need the methods?

Related to this, I'd also note that the authors chose a very non-standard stimulus to perform these measurements with (a rendered 3D 'Greebley' blob). This does have the advantage of some sort of ecological validity. But it has the significant >disadvantage< that it is unlike all the other (much simpler) stimuli that have been used in the past - and this is likely to be one of the reasons why the current (fitted) data do not seem to sit in very good agreement with historical measurements.

Reviewer #2 (Public review):

Summary:

Hong et al. present a new method that uses a Wishart process to dramatically increase the efficiency of measuring visual sensitivity as a function of stimulus parameters for stimuli that vary in a multidimensional space. Importantly, they have validated their model against their own hold-out data and against 3 published datasets, as well as against colour spaces aimed at 'perceptual uniformity' by equating JNDs. Their model achieves high predictive success and could be usefully applied in colour vision science and psychophysics more generally, and to tackle analogous problems in neuroscience featuring smooth variation over coordinate spaces.

Strengths:

(1) This research makes a substantial contribution by providing a new method to very significantly increase the efficiency with which inferences about visual sensitivity can be drawn, so much so that it will open up new research avenues that were previously not feasible. Secondly, the methods are well thought out and unusually robust. The authors made a lot of effort to validate their model, but also to put their results in the context of existing results on colour discrimination, transforming their results to present them in the same colour spaces as used by previous authors to allow direct comparisons. Hold-out validation is a great way to test the model, and this has been done for an unusually large number of observers (by the standards of colour discrimination research). Thirdly, they make their code and materials freely available with the intention of supporting progress and innovation. These tools are likely to be widely used in vision science, and could of course be used to address analogous problems for other sensory modalities and beyond.

Weaknesses:

It would be nice to better understand what constraints the choice of basis functions puts on the space of possible solutions. More generally, could there be particular features of colour discrimination (e.g., rapid changes near the white point) that the model captures less well? The substantial individual differences evident in Figure S20 (comparison with Krauskopf and Gegenfurtner, 1992) are interesting in this context. Some observers show radial biases for the discrimination ellipses away from the white point, some show biases along the negative diagonal (with major axes oriented parallel to the blue-yellow axis), and others show a mixture of the two biases. Are these genuine individual differences, or could the model be performing less accurately in this desaturated region of colour space?

Reviewer #3 (Public review):

Summary:

This study presents a powerful and rigorous approach for characterizing stimulus discriminability throughout a sensory manifold, and is applied to the specific context of predicting color discrimination thresholds across the chromatic plane.

Strengths:

Color discrimination has played a fundamental role in studies of human color vision and for color applications, but as the authors note, it remains poorly characterized. The study leverages the assumption that thresholds should vary smoothly and systematically within the space, and validates this with their own tests and comparisons with previous studies.

Weaknesses:

The paper assumes that threshold variations are due to changes in the level of intrinsic noise at different stimulus levels. However, it's not clear to me why they could not also be explained by nonlinearities in the responses, with fixed noise. Indeed, most accounts of contrast coding (which the study is at least in part measuring because the presentation kept the adapt point close to the gray background chromaticity, and thus measured increment thresholds), assume a nonlinear contrast response function, which can at least as easily explain why the thresholds were higher for colors farther from the gray point. It would be very helpful if a section could be added that explains why noise differences rather than signal differences are assumed and how these could be distinguished. If they cannot, then it would be better to allow for both and refer to the variation in terms of S/N rather than N alone.

Related to this point, the authors note that the thresholds should depend on a number of additional factors, including the spatial and temporal properties and the state of adaptation. However, many of these again seem to be more likely to affect the signal than the noise.

An advantage of the approach is that it makes no assumptions about the underlying mechanisms. However, the choice to sample only within the equiluminant plane is itself a mechanistic assumption, and these could potentially be leveraged for deciding how to sample to improve the characterization and efficiency. For example, given what we know about early color coding, would it be more (or less) efficient to select samples based on a DKL space, etc?

eLife Assessment

This valuable study demonstrates that self-motion strongly affects neural responses to visual stimuli, comparing humans moving through a virtual environment to passive viewing. However, evidence that the modulation is due to prediction is incomplete as it stands, since participants may come to expect visual freezes over the course of the experiment. This study bridges human and rodent studies on the role of prediction in sensory processing, and is therefore expected to be of interest to a large community of neuroscientists.

Reviewer #1 (Public review):

In this paper, the authors wished to determine human visuomotor mismatch responses in EEG in a VR setting. Participants were required to walk around a virtual corridor, where a mismatch was created by halting the display for 0.5s. This occurred every 10-15 seconds. They observe an occipital mismatch signal at 180 ms. They determine the specificity of this signal to visuomotor mismatch by subsequently playing back the same recording passively. They also show qualitatively that the mismatch response is larger than one generated in a standard auditory oddball paradigm. They conclude that humans therefore exhibit visuomotor mismatch responses like mice, and that this may provide an especially powerful paradigm for studying prediction error more generally.

Asking about the role of visuomotor prediction in sensory processing is of fundamental importance to understanding perception and action control, but I wasn't entirely sure what to conclude from the present paradigm or findings. Visuomotor prediction did not appear to have been functionally isolated. I hope the comments below are helpful.

(1) First, isolating visuomotor prediction by contrasting against a condition where the same video stream is played back subsequently does not seem to isolate visuomotor prediction. This condition always comes second, and therefore, predictability (rather than specifically visuomotor predictability) differs. Participants can learn to expect these screen freezes every 10-15 s, even precisely where they are in the session, and this will reduce the prediction error across time. Therefore, the smaller response in the passive condition may be partly explained by such learning. It's impossible to fully remove this confound, because the authors currently play back the visual specifics from the visuomotor condition, but given that the visuomotor correspondences are otherwise pretty stable, they could have an additional control condition where someone else's visual trace is played back instead of their own, and order counterbalanced. Learning that the freezes occur every 10-15 s, or even precisely where they occur, therefore, could not explain condition differences. At a minimum, it would be nice to see the traces for the first and second half of each session to see the extent to which the mismatch response gets smaller. This won't control for learning about the specific separations of the freezes, but it's a step up from the current information.

(2) Second, the authors admirably modified their visual-only condition to remove nausea from 6 df of movement (3D position, pitch, yaw, and roll). However, despite the fact it's far from ideal to have nauseous participants, it would appear from the figures that these modifications may have changed the responses (despite some pairwise lack of significance with small N). Specifically, the trace in S3 (6DOF) and 2E look similar - i.e., comparing the visuomotor condition to the visual condition that matches. Mismatch at 4/5 microvolts in both. Do these significantly differ from each other?

(3) It generally seems that if the authors wish to suggest that this paradigm can be used to study prediction error responses, they need to have controlled for the actions performed and the visual events. This logic is outlined in Press, Thomas, and Yon (2023), Neurosci Biobehav Rev, and Press, Kok, and Yon (2020) Trends Cogn Sci ('learning to perceive and perceiving to learn'). For example, always requiring Ps to walk and always concurrently playing similar visual events, but modifying the extent to which the visual events can be anticipated based on action. Otherwise, it seems more accurately described as a paradigm to study the influence of action on perception, which will be generated by a number of intertwined underlying mechanisms.

More minor points:

(1) I was also wondering whether the authors may consider the findings in frontal electrodes more closely. Within the statistical tests of the frontal electrodes against 0, as displayed in Figure 3c, the insignificance of the effect of Fp2 seems attributable to the small included sample size of just 13 participants for this electrode, as listed in Table S1, in combination with a single outlier skewing the result. The small sample size stands out especially in comparison to the sample size at occipital electrodes, which is double and therefore enjoys far more statistical power. It looks like the selected time window is not perfectly aligned for determining a frontal effect, and also the distribution in 3B looks like responses are absent in more central electrodes but present in occipital and frontal ones. I realise the focus of analysis is on visual processing, but there are likely to be researchers who find the frontal effect just as interesting.

(2) It is claimed throughout the manuscript that the 'strongest predictor (of sensory input) - by consistency of coupling - is self-generated movement'. This claim is going to be hard to validate, and I wonder whether it might be received better by the community to be framed as an especially strong predictor rather than necessarily the strongest. If I hear an ambulance siren, this is an especially strong predictor of subsequent visual events. If I see a traffic light turn red, then yellow, I can be pretty certain what will happen next. Etc.

(3) The checkerboard inversion response at 48 ms is incredibly rapid. Can the authors comment more on what may drive this exceptionally fast response? It was my understanding that responses in this time window can only be isolated with human EEG by presenting spatially polarized events (cf. c1, e.g., Alilovic, Timmermans, Reteig, van Gaal, Slagter, 2019, Cerebral Cortex)

Reviewer #2 (Public review):

Summary:

This study investigates whether visuomotor mismatch responses can be detected in humans. By adapting paradigms from rodent studies, the authors report EEG evidence of mismatch responses during visuomotor conditions and compare them to visual-only stimulation and mismatch responses in other modalities.

Strengths:

(1) The authors use a creative experimental design to elicit visuomotor mismatch responses in humans.

(2) The study provides an initial dataset and analytical framework that could support future research on human visuomotor prediction errors.

Weaknesses:

(1) Methodological issues (e.g., volume conduction, channel selection, lack of control for eye movements) make it difficult to confidently attribute the observed mismatch responses to activity in visual cortical regions.

(2) A very large portion of the data was excluded due to motion artefacts, raising concerns about statistical power and representativeness. The criteria for trial inclusion and the number of accepted trials per participant appear arbitrary and not justified with reference to EEG reliability standards.

(3) The comparison across sensory modalities (e.g., auditory vs. visual mismatch responses) is conceptually interesting, but due to the choice of analyzing auditory mismatch responses over occipital channels, it has limited interpretability.

The authors successfully demonstrate that visuomotor mismatch paradigms can, in principle, be applied in human EEG. However, due to the issues outlined above, the current findings are relatively preliminary. If validated with improved methodology, this approach could significantly advance our understanding of predictive processing in the human visual system and provide a translational bridge between rodent and human work.

Reviewer #3 (Public review):

Summary:

Solyga, Zelechowski, and Keller present a concise report of an innovative study demonstrating clear visuomotor mismatch responses in ambulating humans, using a mobile EEG setup and virtual reality. Human subjects walked around a virtual corridor while EEGs were recorded. Occasionally, motion and visual flow were uncoupled, and this evoked a mismatch response that was strongest in occipitally placed electrodes and had a considerable signal-to-noise ratio. It was robust across participants and could not be explained by the visual stimulus alone.

Strengths:

This is an important extension of their prior work in mice, and represents an elegant translation of those previous findings to humans, where future work can inform theories of e.g., psychiatric diseases that are believed to involve disordered predictive processing. For the most part, the authors are appropriately circumspect in their interpretations and discussions of the implications. I found the discussion of the polarity differences they found in light of separate positive and negative prediction errors, intriguing.

Weaknesses:

The primary weaknesses rest in how the results are sold and interpreted.

Most notably, the interpretation of the results of the comparison of visuomotor mismatches to the passive auditory oddball induced mismatch responses is inappropriate, as suboptimal electrode choices, unclear matching of trial numbers, and other factors. To clarify, regarding the auditory oddball portion in Figure 5, the data quality is a concern for the auditory ERPs, and the choice of Occipital electrodes is a likely culprit. Typically, auditory evoked responses are maximal at Cz or FCz, although these contacts don't seem to be available with this setup. In general, caution is warranted in comparing ERP peaks between two different sensory modalities - especially if attention is directed elsewhere (to a silent movie) during one recording and not during the other. The authors discuss this as a purely "qualitative" comparison in the text, which is appreciated, and do acknowledge the limitations within the results section, but the figure title and, importantly, the abstract set a different tone. At least, for comparisons between auditory mismatch and visuomotor mismatch, trial numbers need to be equated, as ERP magnitude can be augmented by noise (which reduces with increased numbers of trials in the average). And more generally, the size of the mismatch event at the scalp does not scale one-to-one with the size at the level of the neural tissue. One can imagine a number of variables that impact scalp level magnitudes, which are orthogonal to actual cortex-level activation - the size, spread, and polarity variance of the activated source (which all would diminish amplitude at the scalp due to polyphasic summation/cancelation). The variance of phase to a stimulus across trials (cross trial phase locking) vs magnitude of underlying power - the former, in theory, relates to bottom-up activity and the latter can reflect feedback (which has more variability in time across trials; the distance of the scalp electrode from the activated tissue (which, for the auditory system, would be larger (FCz to superior temporal gyrus) than for the visual system (O1 to V1/2)). None of this precludes the inclusion of the auditory mismatch, which is a strength of the study, but interpretations about this supporting a supremacy of sensory-motor mismatch - regardless of validity - are not warranted. I would recommend changing the way this is presented in the abstract.

Otherwise, the data are of adequate quality to derive most of their conclusions.

The authors claim that the mismatch responses emanate from within the occipital cortex, but I would require denser scalp coverage or a demonstration of consistent impedances across electrodes and across subjects to make conclusions about the underlying cortical sources (especially given the latencies of their peaks). In EEG, the distribution of voltage on the scalp is, of course, related to but not directly reflective of the distribution of the underlying sources. The authors are mostly careful in their discussion of this, but I would strongly recommend changing the work choice of "in occipital cortex" to "over occipital cortex" or even "posteriorly distributed". Even with very dense electrode coverage and co-registration to MRIs for the generation of forward models that constrain solutions, source localization of EEG signals is very challenging and not a simple problem. Given the convoluted and interior nature of human V1, the ability to reliably detect early evoked responses (which show the mismatch in mouse models) at the scalp in ERP peaks is challenging - especially if one is collapsing ERPs across subjects. And - given the latency of the mismatch responses, I'd imagine that many distributed cortical regions contribute to the responses seen at the scalp.

I think that Figure 3C, but as a difference of visual mismatch vs halting flow alone (in the open loop) might be additionally informative, as it clarifies exactly where the pure "mismatch" or prediction error is represented.

As a suggestion, the authors are encouraged to analyse time-frequency power and phase locking for these mismatch responses, as is common in much of the literature (see Roach et al 2008, Schizophrenia Bulletin). This is not to say that doing so will yield insights into oscillations per se, but converting the data to the time-frequency domain provides another perspective that has some advantages. It fosters translations to rodent models, as ERP peaks do not map well between species, but e.g., delta-theta power does (see Lee et al 2018, Neuropsychopharmacology; Javitt et al 2018, Schizophrenia research; Gallimore et al 2023, Cereb Ctx). Further, ERP peaks can be influenced by the actual neuroanatomy of an individual (especially for quantifying V1 responses). Time frequency analyses may aid in interpreting the "early negative deflection with a peak latency of 48 ms " finding as well.

Finally, the sentence in the abstract that this paradigm " can trigger strong prediction error responses and consequently requires shorter recording 20 times would simplify experiments in a clinical setting" is a nice setup to the paper, but the very fact that one third of recordings had to be removed due to movement artifact, and that hairstyle modulates the recording SnR, is reason that this paradigm, using the reported equipment, may have limited clinical utility in its current form. Further, auditory oddball paradigms are of great clinical utility because they do not require explicit attention and can be recorded very quickly with no behavioral involvement of a hospitalized patient. This should be discussed, although it does not detract from the overall scientific importance of the study. The authors should reconsider putting this statement in the abstract.

eLife assessment

This meta-analysis provides a fundamental synthesis of evidence demonstrating that transcranial magnetic stimulation targeting the hippocampal-cortical network reliably enhances episodic memory performance across diverse study designs. The evidence is convincing, with rigorous methodology and consistent effects observed despite modest sample sizes and some heterogeneity in stimulation approaches. The work highlights the specificity of memory improvements to hippocampal-dependent memories and identifies key methodological factors-such as individualized targeting-that influence efficacy. Overall, this study offers a timely and integrative framework that will inform both basic memory research and the design of future clinical trials for cognitive enhancement.

Reviewer #1 (Public review):

Summary:

Goicoechea et al. conducted a timely and thorough meta-analysis on the potential for indirect hippocampal targeted transcranial magnetic stimulation (TMS) to improve episodic memory. The authors included additional factors of interest in their meta-analysis, which can be used to inform the next generation of studies using this intervention. Their analysis revealed critical factors for consideration: TMS should be applied pre-encoding, individualized spatial targeting improves efficacy, and improvement of recollection was stronger than recognition.

Strengths:

As mentioned previously, the meta-analysis is timely and summarizes an emerging set of studies (over the past decade since Wang et al., Science 2014). Those outside of the field may not be aware of the robustness of improvements in episodic memory from hippocampal targeted TMS. The authors were quite thorough in including additional factors that are important for the interpretation of these findings. These factors also address the differences in approach across studies. The evidence that individualized spatial targeting improves TMS efficacy is consistent with recent advances in TMS for major depressive disorder. The specificity of the cognitive improvements to recollection of episodic memory and not for other cognitive domains is consistent with hippocampal targeting. The authors also plan to post the complete dataset on an open-source repository, which enables additional analysis by other researchers.

Weaknesses:

The write-up is succinct and emphasizes the scientific decisions that underlie key differences in the various experimental designs. While the manuscript is written for a scientific audience, the authors are likely aware that findings like this will be of broad appeal to the field of neurology, where treatments for memory loss are desperately needed. For this reason, the authors could consider including a statement regarding an interpretation of this meta-analysis from a clinical standpoint. Statements such as 'safe and effective' imply a clinical indication, and yet the manuscript does not engage with clinical trials terminology such as blinding, parallel arm versus crossover design, and trial phase. While the authors might prefer not to engage with this terminology, it can be confusing when studies delivering intervention-like five days of consecutive TMS (e.g., Wang et al., 2014) are clustered with studies that delivered online rhythmic TMS, which tests target engagement (e.g., Hermiller et al., 2020). While the 'sessions' variable somewhat addresses the basic-science versus intervention-like approach, adding an explicit statement regarding this in the discussion might help the reader navigate the broad scope of approaches that are utilized in the meta-analysis.

Reviewer #2 (Public review):

Summary:

In 2014, Wang et al. showed that noninvasive stimulation of a parietal site, connected functionally to the hippocampus, increased resting state connectivity throughout a canonical network associated with episodic memory. It also produced a memory boost, which correlated with the connectivity increase across subjects. Their discovery that an imaging biomarker could be used to target a network (rather than a single cortical site) in individual subjects and provide a scaling measure of target modulation should have revolutionized the noninvasive neuromodulation field. This meta-analysis by members of the same group covers memory effects from noninvasive stimulation of various nodes of the "hippocampal" network.

Strengths:

This is a very timely summary and meta-analysis of this very promising application of TMS. To the limited extent of my expertise in meta-analysis, the methodology seems rigorous, and the central finding, that high-frequency stimulation of nodes in the hippocampal network reproducibly improves event recall, is amply supported. This should provide impetus for larger clinical trials and further quantification of the optimal dose, duration of effect, etc.

Weaknesses:

My critical comments are mainly on the framing and argument:

(1) While the introduction centers on the role of the hippocampus in episodic memory and posits hippocampal neuromodulation by TMS as causative, the true mechanism may be more complex. Clean hippocampal lesions in primates cause focal loss of spatial and place memory, and I am aware of no specific evidence that the hippocampus does more than this in humans. Moreover, there is evidence that lateral parietal TMS also reaches neighboring temporal lobe regions, which contribute to episodic memory. The hippocampus may, therefore, be a reliable deep seed for connectivity-based targeting of the episodic memory network, but might not be the true or only functional target.

(2) The meta-analysis combines studies with confirmation of targeting and target-network engagement from fMRI and studies without independent evidence of having stimulated the putative target (e.g., Koch et al). That seems like a more important methodological distinction than merely the use of any individual targeting method. In my experience, atlas-based estimates are at least as accurate as eyeballing cortical areas in individuals. Hence, entering individual functional targeting as a factor might reveal an effect on efficacy.

(3) The funnel plot and Egger's regression for episodic memory outcomes suggested possible bias, and the average sample size of 23 is small, contributing to the likelihood of false positive results. It would be informative, therefore, to know how many or which studies had formal power estimates and what the predicted effect sizes were.

(4) In the Discussion, the authors might provide a comparison between the effect size for memory improvement found here with those reported for other brain-targeted interventions and behavioral strategies. It may also be worthwhile pointing out that HITS/memory is one of the very few, or perhaps the only, neuromodulatory effects on cognition that has been extensively reproduced and survived rigorous meta-analysis.

(5) The section of the Discussion on specificity compares HITS to transcranial electrical stimulation without specifying an anatomical target or intended outcome. A better contrast might be the enormous variety of cognitive and emotional effects claimed for TMS of the dorsolateral prefrontal cortex.

(6) With reference to why other nodes in the episodic memory network have not been tested, current flow modeling shows TMS of the medial prefrontal cortex is unlikely to be achievable without stronger stimulation of the convexity under the coil, in addition to being uncomfortable. The lateral temporal lobe has been stimulated without undue discomfort.

(7) Finally, a critical question hanging over the clinical applicability of HITS and other neuromodulation techniques is how well they will work on a damaged substrate. Functional and/or anatomical imaging might answer this question and help screen for likely responders. The authors' opinion on this would be informative.

Reviewer #3 (Public review):

Summary:

The manuscript by Goicoechea et al. assesses the influence of hippocampal-network targeted TMS to parietal cortex on episodic memory using a meta-analytic approach. This is an important contribution to the literature, as the number of studies using this approach to modulate memory/hippocampal function has clearly increased since the initial publication by Wang et al. 2014. This manuscript makes an important contribution to the literature. In general, the analysis is straightforward and the conclusions are well-supported by the results; I have mostly minor comments/concerns.

Strengths:

(1) A meta-analysis across published work is used to evaluate the influence of hippocampal-network-targeted TMS in parietal cortex on episodic memory. By pooling results across studies, the meta-analytic effects demonstrate an influence of TMS on memory across the diversity of many details in study design (specific tasks, stimuli, TMS protocols, study populations).

(2) Selectivity with regard to episodic memory vs. non-episodic memory tasks is evaluated directly in the meta-analysis.

(3) The investigation into supplemental factors as predictors of TMS's influence on memory was tested. This is helpful given the diversity of study designs in the literature. This analysis helps to shed light on which study designs, e.g., TMS protocols, etc., are most effective in memory modulation.

Weaknesses:

(1) My only significant concern is how studies are categorized in the 'Timing' factor (when stimulation is applied). Currently, protocols in which TMS is administered across days are categorized as 'pre-encoding' in the Timing factor. This has the potential to be misleading and may lead to inaccurate conclusions. When TMS is administered across multiple days, followed by memory encoding and retrieval (often on a subsequent day), it is not possible to attribute the influence of TMS to a specific memory phase (i.e., encoding or retrieval) per se. Thus, labeling multi-day TMS studies as 'pre-encoding' may be misleading to readers, as it may imply that the influence of TMS is due to modulation of encoding mechanisms per se, which cannot be concluded. For example, multi-day TMS protocols could be labeled as 'pre-retrieval' and be similarly accurate. This approach also pools results from TMS protocols with temporal specificity (i.e., those applied immediately during encoding and not on board during memory testing) and without temporal specificity (i.e., the case of multi-day TMS) regarding TMS timing. Given the variety of paradigms employed in the literature, and to maximize the utility/accuracy of this analysis, one suggestion is to modify the categories within the Timing factor, e.g., using labels like 'Temporally-Specific' and 'Temporally Non-specific'. The 'Temporally-Specific' category could be subdivided based on the specific memory process affected: 'encoding', 'retrieval', or 'consolidation' (if possible). I think this would improve the accuracy of the approach and help to reach more meaningful conclusions, given the variety of protocols employed in the literature.

(2) As the scope of the meta-analysis is limited to TMS applied to parietal or superior occipital cortex, it is important to highlight this in the Introduction/Abstract. The 'HITS' terminology suggests a general approach that would not necessarily be restricted to parietal/nearby cortical sites.

Minor:

(1) To reduce the number of study factors tested, data reduction was performed via Lasso regression to remove factors that were not unique predictors of the influence of TMS on memory. This approach is reasonable; however, one limitation is that factors strongly correlated with others (and predict less unique variance) will be dropped. This may result in a misrepresentation, i.e., if readers interpret factors left out of this analysis as not being strongly related to the influence of TMS on memory. I do see and appreciate the paragraph in the Discussion which appropriately addresses this issue. However, it may be worth also considering an alternative analysis approach, if the authors have not already done so, which explicitly captures the correlation structure in the data (i.e., shown in Figure S2) using a tool like PCA or an appropriate factor analysis. Then, this shared covariance amongst factors can be tested as predictors of the influence of TMS - e.g., by testing whether component scores for dominant PCs are indeed predictive of the influence of TMS. This complementary approach would capture rather than obfuscate the extent to which different factors are correlated and assess their joint (rather than independent) influence on memory, potentially resulting in more descriptive conclusions. For example, TMS intensity and protocol may jointly influence memory.

(2) Given the specific focus on TMS applied to parietal cortex to modulate hippocampal and related network function, it would be fruitful if the authors could consider adding discussion/speculation regarding whether this approach may be effectively broadened using other stimulation methods (e.g., tACS, tDCS), how it may compare to other non-invasive brain stimulation methods with depth penetration to target hippocampal function directly (transcranial temporal interference, or transcranial focused ultrasound), and/or how or whether other stimulation sites may or may not be effective.

(3) Studies were only included in the meta-analysis if they contained objective episodic memory tests. How were studies handled that included both objective and subjective memory, or other non-episodic memory measures? For example, Yazar et al. 2014 showed no influence of TMS on objective recall, but an impairment in subjective confidence. I assume confidence was not included in the meta-analysis. Similarly, Webler et al. 2024 report results from both the mnemonic similarity task (presumably included) and a fear conditioning paradigm (presumably excluded). Please clarify in the methods how these distinctions were handled.

(4) The analysis comparing memory to non-memory measures is important, showing the specificity of stimulation. Did the authors consider further categorizing the non-memory tasks into distinct domains (i.e., language, working memory, etc.)? If possible, this could provide a finer detail regarding the selectivity of influences on memory vs. other aspects of cognition. It is likely that other aspects of cognition dependent on hippocampal function may be modulated as well, i.e., tasks with high relational/associative processing demands.

(5) In the analysis of the Intensity factor, how were studies using Active (rather than resting) MT categorized? Only resting MT is mentioned in Table S1. This is important as the original theta-burst TMS protocol from Huang et al. 2005 determines intensity based on Active Motor Threshold.

(6) Is there a reason why the study by Koen et al. 2018 (Cognitive Neuroscience) was not included? TMS was performed during encoding to the left AG, and objective memory was assessed, so it would seemingly meet the inclusion criterion.

(7) It would be helpful to briefly differentiate the current meta-analysis from that performed by Yeh & Rose (How can transcranial magnetic stimulation be used to modulate episodic memory?: A systematic review and meta-analysis, 2019, Frontiers in Psychology) (other than being more current).

(8) For transparency and to facilitate further understanding of the literature and potential data re-use, it would be great if the authors consider sharing a supplementary table or file that describes how individual studies/memory measures were categorized under the factors listed in Table S1.

This is well-said.

eLife Assessment

This useful study provides a well-constructed computational investigation of how intermittent theta-burst stimulation (iTBS) influences synaptic plasticity within the corticothalamic circuit, improving our mechanistic understanding of how stimulation parameters interact with intrinsic brain oscillations. The authors build a corticothalamic population model that generates individual alpha rhythms with a calcium-dependent metaplasticity rule, and provide solid evidence that aligning stimulation frequencies to brain-intrinsic oscillatory subharmonics enhances plasticity effects. This insight could open a route toward personalized, more effective stimulation protocols.

Reviewer #1 (Public review):

Summary:

The authors show that the lower frequency (~5Hz) stimulation of the intermittent theta-burst stimulation (iTBS) via repetitive transcranial magnetic stimulation (rTMS) serves as a more effective stimulation paradigm than the high-frequency protocols (HF-rTMS, ~10Hz) with enhancing plasticity effects via long-term potentiation (LTP) and depression (LTD) mechanisms. They show that the 5 Hz patterned pulse structure of the iTBS is an exact subharmonic of the 10 Hz high-frequency rTMS, creating a connection between the two paradigms and acting upon the same underlying synchrony mechanism of the dominant alpha-rhythm of the corticothalamic circuit.

First, the authors create a corticothalamic neural population model consisting of 4 populations: cortical excitatory pyramidal and inhibitory interneuron, and thalamic excitatory relay and inhibitory reticular populations. Second, the authors include a calcium-dependent plasticity model, in which calcium-related NMDAR-dependent synaptic changes are implemented using a BCM metaplasticity rule. The rTMS-induced fluctuations in intracellular calcium concentrations determine the synaptic plasticity effects.

Strengths:

The model (corticothalamic neural population with calcium-dependent plasticity, with TBS input for rTMS) is thoroughly built and analyzed.

The conclusions seem sound and justified. The authors justifiably link stimulation parameters (especially the alpha subharmonics iTBS frequency) with fluctuations in calcium concentration and their effects on LTP and LTD in relevant parts of the corticothalamic circuit populations leading to a dampening of corticothalamic loop gains and enhancement of intrathalamic gains with an overall circuit-wide feedforward inhibition (= inhibitory activity is enhanced via excitatory inputs onto inhibitory neurons) and a resulting suppression of the activity power. In other words: alpha-resonant iTBS protocols achieve broadband power suppression via selective modulation of corticothalamic FFI.

(1) The model is well-described, with the model equations in the main text and the parameters in well-formatted tables.

(2) The relationship between iTBS timing and the phase of rhythms is well explained conceptually.

(3) Metaplasticity and feedforward inhibition regulation as a driver for the efficacy of iTBS are well explored in the paper.

(4) Efficacy of TBS, being based on mimicry of endogenous theta patterns, seems well supported by this simulation.

(5) Recovery between periods of calcium influx as an explanation for why intermittency produces LTP effects where continuous stimulation fails is a good justification for calcium-based metaplasticity, as well as for the role of specific pulse rate.

(6) Circuit resonance conclusion is interesting as a modulating factor; the paper supports this hypothesis well.

(7) The analysis of corticothalamic dampening and intrathalamic enhancement in the 3D XYZ loop gain space is a strong aspect of the paper.

Weaknesses:

(1) Overall, the paper is difficult to follow narratively - the motivation (formulated as a specific research question) for each section can be a bit unclear. The paper could benefit from a minor rewrite at the start of each section to justify each section's reasoning. The Discussion is too long and should be shortened and limited to the main points.

(2) While the paper refers to modelling and data in discussion, there is no direct comparison of the simulations in the figures to data or other models, so it's difficult to evaluate directly how well the modelling fits either the existing model space or data from this region. Where exactly the model/plasticity parameters from Table 5 and the NFTsim library come from is not easy to find. The authors should make the link from those parameters to experimental data clearer. For example, which clinical or experimental data are their simulations of the resting-state broadband power suppression based on?

(3) The figures should be modified to make them more understandable and readable.

(4) The claim in the abstract that the paper introduces "a novel paradigm for individualizing iTBS treatments" is too strong and sounds like overselling. The paper is not the first computational modelling of TBS - as acknowledged also by the authors when citing previous mean-field plasiticity modelling articles. Btw. the authors could briefly mention and include also references also to biophysically more detailed multi-scale approaches such as https://doi.org/10.1016/j.brs.2021.09.004 and https://doi.org/10.1101/2024.07.03.601851 and https://doi.org/10.1016/j.brs.2018.03.010

(5) The modelling assumes the same CaDP model/mechanism for all excitatory synapses/afferents. How well is this supported by experimental evidence? Have all excitatory synaptic connections in the cortico-thalamic circuit been shown to express CaDP and metaplasticity? If not, these limitations (or predictions of the model) should be mentioned. Why were LTP calcium volumes never induced within thalamic relay-afferent connections se and sr? What about inhibitory synapses in the circuit model? Were they plastic or fixed?

(6) Minor point: Metaplasticity is modelled as an activity-dependent shift in NMDAR conductance, which is supported by some evidence, but there are other metaplasticity mechanisms. Altering NMDA-synapse affects also directly synaptic AMPA/NMDA weight and ratio (which has not been modelled in the paper). Would the model still work using other - more phenomenological implementation of the sliding threshold - e.g. based on shifting calcium-dependent LTP/LTD windows or thresholds (for a phenomenological model of spike/voltage-based STDP-BCM rules, see https://doi.org/10.1007/s10827-006-0002-x and https://doi.org/10.1371/journal.pcbi.1004588) - maybe using a metaplasticity extension of Graupner and Brunel CaDP model. A brief discussion of these issues might be added to the manuscript - but this is just a suggestion.

(7) Short-term plasticity (depression/facilitation) of synapses is neglected in the model. This limitation should be mentioned because adding short-term synaptic dynamics might affect strongly circuite model dynamics.

Reviewer #2 (Public review):

Transcranial magnetic stimulation is used in several medical conditions to alter brain activity, probably by induction of synaptic plasticity. The authors pursue the idea to personalise parameters of the stimulation protocol by adapting the stimulation frequency to an individual's brain rhythm. The authors test this approach in a population model connecting the cortex with deeper brain areas, the thalamocortical loop, which includes calcium-dependent plasticity for the connections within and between brain regions. While the authors relate literature-based experimental findings with their results, their results are so far not supported by experimental work.

The authors successfully highlight in their model that personalization of rTMS stimulation frequency to the brain intrinsic frequency has the potential to improve stimulation impact, and they relate this to specific changes in the network. Their arguments that this resonance improves efficacy are intuitive, and their finding that inhibition and excitation are selectively modulated is a good starting point for analysing the underlying mechanism.

As rTMS is used in clinical contexts, and the idea of aligning intrinsic and stimulation frequency is relatively easy to implement, the paper is conceptually of interest for the rTMS community, despite its weak points on the mechanistic explanation. The authors made the simulation code publicly available, which is a useful resource for further studies on the effects of metaplasticity. The same stimulation parameters have been tested in experiments, and a reanalysis of the experimental results following the idea of this paper could be influential for clinical optimisation of stimulation protocols.

A strength of the paper is that it takes into account also deeper brain areas, and their interaction with the cortex. The paper carefully measures system changes in response to different frequency differences between thalamocortical loop and stimulation. By explicitly modelling changes to connections, the authors do start dissect the mechanism underlying the observed effect. Unfortunately, the dissection of the mechanistic underpinning in the current version of the manuscript does not yet fully exploits the possibility of a computational model. Here are a couple of points related to this critique:

(1) The study reports that connections between thalamus and cortex as well as within the thalamus change, but the model is not used to separate the influence of both.

(2) The paper reports that a resonance between stimulation and brain increases stimulation effectiveness. This conclusion is solely based on the observation of strong reactions in the network to subharmonics of the brain's frequency, and lacks further support such as alternative measures of resonance, or an analysis of the role of the phase difference between stimulation and brain oscillation, which is likely changed by the stimulation. For example, for harmonic oscillators, resonance leads to a 90 degree phase difference between driving force and system response, and for rTMS, phase locking has been shown to be relevant.

(3) The authors claim that over-engagement of plasticity for HF-rTMS makes their intermittent protocol more effective. Yet, the study lacks a direct comparison between stimulation protocols that shows over-engagement of plasticity for the HF-protocol. The study also does not explore which time-scale of the plasticity mechanism rules the optimal stimulation protocol. Moreover, the study reports that only few number of pulses per burst show a good effect. This should depend on how strongly a single pulse changes the calcium volume, but this relation was not explored in the model.

(4) The authors report on the frequency spectrum of the cortical excitatory population, with the argument that the power of this population is most closely related to EEG measurements. A report of the other neuronal populations is missing, which might be informative on what is going on in the network.

Statistics:

(1) The authors do not state whether they test for assumptions of the multiple regression analysis, such as whether errors have equal variance or that residuals are normally distributed.

(2) For the statistical analysis, the authors ignore about half of their model simulations for which the change in the power was negligible. It is not clear to me which statistical analysis is meant; whether the figures show all model simulations, whether regression lines where evaluated ignoring them, and whether the multiple regression analysis used only half of the data points.

Reviewer #3 (Public review):

Summary:

This article presented a novel computer model to address an important question in the field of brain stimulation, using the magnetic stimulation iTBS protocol as an example, how stimulation parameters, frequency in particular, interfere with the intrinsic brain oscillations via plastic mechanisms. Brain oscillation is a critical feature of functional brains and its alteration signals the onset of many neuropsychiatric diseases or certain brain states. The authors suggested with their model that harmonic and subharmonic stimulations close to the individual alpha frequency achieved strong broadband power suppression.

Strengths:

The authors focused on the cortico-thalamic circuitry and managed to generate alpha oscillations in their four-population model. By adding the non-monotonic calcium-based BCM rule, they have also achieved both homeostasis and plasticity in response to magnetic stimulation. This work combined computer simulations and statistical analysis to demonstrate the changes in network architecture and network dynamics triggered by varied magnetic stimulation parameters. By delivering the iTBS protocol to the cortical excitatory population, the key findings are that harmonic and subharmonic stimulations close to the individual alpha frequency (IAF) achieved strong broadband power suppression. This resulted from increased synaptic weights of the corticothalamic feed-forward inhibitory projections, which were mediated by the calcium dynamics perturbed by iTBS magnetic stimulation. This finding endorsed the importance of applying customized stimulation to patients based on their IAFs and suggested the underlying mechanism at the circuitry level.

Weaknesses:

The drawbacks of this work are also obvious. Model validation and biological feasibility justification should be better addressed. The primary outcome of their model is the broadband power suppression and the optimal effects of (sub)harmonic stimulation frequency, but it lacks immediate empirical support in the literature. To the best of my knowledge, many alpha frequency tACS studies reported to increase but not suppress the power of certain brain oscillations. A review by Wang et al., 2024 (Frontiers in System Neuroscience) suggested hybrid changes to different brain oscillations by magnetic stimulation. Developing a model to fully capture such changes might be out of the scope of the present study and challenging in the entire field, but it undermines the quality of the present work if not extensively discussed and justified. Clarity and reproducibility of the work can be improved. Although it is intriguing to see how the calcium-dependent BCM plasticity mediates such changes, the writing of the methods part is not hard to follow. It was also not clear why only two populations were considered in the thalamus, how the entire network was connected, or how the LTP/LTD threshold alters with calcium dynamics. The figures were unfortunately prepared in a nested manner. The crowded layout and the tiny font sizes reduce the clarity. The third point comes to contextualization and comparison to existing models. It will strengthen the work if the authors could have compared their work to other TMS modeling work with plasticity rules, e.g, Anil et al., 2024. Besides, magnetic stimulation is unique in being supra-threshold and having focality compared to other brain stimulation modalities, e.g., tDCS and tACS, but they may share certain basic neural mechanisms if accounting for certain parameters, e.g., frequency. A solid literature review and discussion on this part may help the field better perceive the value and potential limitations of this work.

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eLife Assessment

This study is an important contribution to the field of viral sequencing, providing methods for more accurate characterization of viral genetic diversity using long-read sequencing and unique molecular identifiers (UMIs). Although it is a small pilot study, it shows promise as a convincing, validated methodology with broad applicability.

Reviewer #1 (Public review):

Tamao et al. aimed to quantify the diversity and mutation rate of the influenza (PR8 strain) in order to establish a high-resolution method for studying intra-host viral evolution . To achieve this, the authors combined RNA sequencing with single-molecule unique molecular identifiers (UMIs) to minimize errors introduced during technical processing. They proposed an in vitro infection model with a single viral particle to represent biological genetic diversity, alongside a control model using in vitro transcribed RNA for two viral genes, PB2 and HA.

Through this approach, the authors demonstrated that UMIs reduced technical errors by approximately tenfold. By analyzing four viral populations and comparing them to in vitro transcribed RNA controls, they estimated that ~98.1% of observed mutations originated from viral replication rather than technical artifacts. Their results further showed that most mutations were synonymous and introduced randomly. However, the distribution of mutations suggested selective pressures that favored certain variants. Additionally, comparison with closely related influenza strain (A/Alaska/1935) revealed two positively selected mutations, though these were absent in the strain responsible for the most recent pandemic (CA01).

Overall, the study is well-designed, and the interpretations are strongly supported by the data.

The authors have addressed all the comments from the previous round of reviews. No further concerns.

Reviewer #2 (Public review):

Summary:

This manuscript presents a technically oriented application of UMI-based long-read sequencing to study intra-host diversity in influenza virus populations. The authors aim to minimize sequencing artifacts and improve the detection of rare variants, proposing that this approach may inform predictive models of viral evolution. While the methodology appears robust and successfully reduces sequencing error rates, key experimental and analytical details are missing, and the biological insight is modest. The study includes only four samples, with no independent biological replicates or controls, which limits the generalizability of the findings. Claims related to rare variant detection and evolutionary selection are not fully supported by the data presented.

Strengths:

The study addresses an important technical challenge in viral genomics by implementing a UMI-based long-read sequencing approach to reduce amplification and sequencing errors. The methodological focus is well presented, and the work contributes to improving the resolution of low-frequency variant detection in complex viral populations.

Weaknesses:

The application of UMI-based error correction to viral population sequencing has been established in previous studies (e.g., in HIV), and this manuscript does not introduce a substantial methodological or conceptual advance beyond its use in the context of influenza.

The study lacks independent biological replicates or additional viral systems that would strengthen the generalizability of the conclusions. Potential sources of technical error are not explored or explicitly controlled. Key methodological details are missing, including the number of PCR cycles, the input number of molecules, and UMI family size distributions. These are essential to support the claimed sensitivity of the method.

The assertion that variants at {greater than or equal to}0.1% frequency can be reliably detected is based on total read count rather than the number of unique input molecules. Without information on UMI diversity and family sizes, the detection limit cannot be reliably assessed.

Although genetic variation is described, the functional relevance of observed mutations in HA and NA is not addressed or discussed in the context of known antigenic or evolutionary features of influenza. The manuscript is largely focused on technical performance, with limited exploration of the biological implications or mechanistic insights into influenza virus evolution.

The experimental scale is small, with only four viral populations derived from single particles analyzed. This limited sample size restricts the ability to draw broader conclusions about quasispecies dynamics or evolutionary pressures.

Comments on revisions:

The revised manuscript provides additional methodological detail and clearer presentation, which improves transparency. However, the main limitations persist: the study remains small in scale, lacks independent validation, and relies on theoretical rather than empirical support for its claimed detection sensitivity. As a result, the work represents a modest technical advance rather than a substantive contribution to understanding influenza virus evolution.

Author response:

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

Reviewer #1 (Public review):

(1) The methods section is overly brief. Even if techniques are cited, more experimental details should be included. For example, since the study focuses heavily on methodology, details such as the number of PCR cycles in RT-PCR or the rationale for choosing HA and PB2 as representative in vitro transcripts should be provided.

We thank the reviewer for this important suggestion. We have now expanded the Methods section to include the number of PCR cycles used in RT-PCR (line 407) and have explained the rationale for choosing HA and PB2 as representative transcripts (line 388).

(2) Information on library preparation and sequencing metrics should be included. For example, the total number of reads, any filtering steps, and quality score distributions/cutoff for the analyzed reads.

We agree and have added detailed information on library preparation, filtering criteria, quality score thresholds, and sequencing statistics for each sample (line 422, Figure S2).

(3) In the Results section (line 115, "Quantification of error rate caused by RT"), the mutation rate attributed to viral replication is calculated. However, in line 138, it is unclear whether the reported value reflects PB2, HA, or both, and whether the comparison is based on the error rate of the same viral RNA or the mean of multiple values (as shown in Figure 3A). Please clarify whether this number applies universally to all influenza RNAs or provide the observed range.

We appreciate this point. We have clarified in the Results (line 140) that the reported value corresponds to PB2.

(4) Since the T7 polymerase introduced errors are only applied to the in vitro transcription control, how were these accounted for when comparing mutation rates between transcribed RNA and cell-culture-derived virus?

We agree that errors introduced by T7 RNA polymerase are present only in the in vitro–transcribed RNA control. However, even when taking this into account, the error rate detected in the in vitro transcripts remained substantially lower than that observed in the viral RNA extracted from replicated virus (line 140, Fig.3a). Thus, the difference cannot be explained by T7-derived errors, and our conclusion regarding the elevated mutation rate in cell-culture–derived viral populations remains valid.

(5) Figure 2 shows that a UMI group size of 4 has an error rate of zero, but this group size is not mentioned in the text. Please clarify.

We have revised the Results (line 98) to describe the UMI group size of 4.

Reviewer #2 (Public review):

(1) The application of UMI-based error correction to viral population sequencing has been established in previous studies (e.g., HIV), and this manuscript does not introduce a substantial methodological or conceptual advance beyond its use in the context of influenza.

We appreciate the reviewer’s comment and agree that UMI-based error correction has been applied previously to viral population sequencing, including HIV. However, to our knowledge, relatively few studies have quantitatively evaluated both the performance of this method and the resulting within-quasi-species mutation distributions in detail. In our manuscript, we not only validate the accuracy of UMIbased error correction in the context of influenza virus sequencing, but also quantitatively characterize the features of intra-quasi-species distributions, which provides new insights into the mutational landscape and evolutionary dynamics specific to influenza. We therefore believe that our work goes beyond a simple application of an established method.

(2) The study lacks independent biological replicates or additional viral systems that would strengthen the generalizability of the conclusions.

We agree with the reviewer that the lack of independent biological replicates and additional viral systems limits the generalizability of our findings. In this study, we intentionally focused on single-particle–derived populations of influenza virus to establish a proof-of-principle for our sequencing and analytical framework. While this design provided a clear demonstration of the method’s ability to capture mutation distributions at the single-particle level, we acknowledge that additional biological replicates and testing across diverse viral systems would be necessary to confirm the broader applicability of our observations. Importantly, even within this limited framework, our analysis enabled us to draw conclusions at the level of individual viral populations and to suggest the possibility of comparing their mutation distributions with known evolvability. This highlights the potential of our approach to bridge observations from single particles with broader patterns of viral evolution. In future work, we plan to expand the number of populations analyzed and include additional viral systems, which will allow us to more rigorously assess reproducibility and to establish systematic links between mutation accumulation at the single-particle level and evolutionary dynamics across viruses.

(3) Potential sources of technical error are not explored or explicitly controlled. Key methodological details are missing, including the number of PCR cycles, the input number of molecules, and UMI family size distributions.

We thank the reviewer for this important suggestion. We have now expanded the Methods section to include the number of PCR cycles used in RT-PCR (line 407). In addition, we have added information on the estimated number of input molecules. Regarding the UMI family size distributions, we have added the data as Figure S2 and referred to it in the revised manuscript.

Finally, with respect to potential sources of technical error, we note that this point is already addressed in the manuscript by direct comparison with in vitro transcribed RNA controls, which encompass errors introduced throughout the entire experimental process. This comparison demonstrates that the error-correction strategy employed here effectively reduces the impact of PCR or sequencing artifacts.

(4) The assertion that variants at ≥0.1% frequency can be reliably detected is based on total read count rather than the number of unique input molecules. Without information on UMI diversity and family sizes, the detection limit cannot be reliably assessed.

We thank the reviewer for raising this important issue. We agree that our original description was misleading, as the reliable detection limit should not be defined solely by total read count. In the revised version, we have added information on UMI distribution and family sizes (Figure S2), and we now state the detection limit in terms of consensus reads. Specifically, we define that variants can be reliably detected when ≥10,000 consensus reads are obtained with a group size of ≥3 (line 173). 

(5)  Although genetic variation is described, the functional relevance of observed mutations in HA and NA is not addressed or discussed.

We appreciate the reviewer’s suggestion. In our study, we did not apply drug or immune selection pressure; therefore, we did not expect to detect mutations that are already known to cause major antigenic changes in HA or NA, and we think it is difficult to discuss such functional implications in this context. However, as noted in discussion, we did identify drug resistance–associated mutations. This observation suggests that the quasi-species pool may provide functional variation, including resistance, even in the absence of explicit selective pressure. We have clarified this point in the text to better address the reviewer’s concern (line 330).

(6) The experimental scale is small, with only four viral populations derived from single particles analyzed. This limited sample size restricts the ability to draw broader conclusions.

We thank the reviewer for pointing out the limitation of analyzing only four viral populations derived from single particles. We fully acknowledge that the small sample size restricts the generalizability of our conclusions. Nevertheless, we would like to emphasize that even within this limited dataset, our results consistently revealed a slight but reproducible deviation of the mutation distribution from the Poisson expectation, as well as a weak correlation with inter-strain conservation. These recurring patterns highlight the robustness of our observations despite the sample size.

In future work, we plan to expand the number of viral populations analyzed and to monitor mutation distributions during serial passage under defined selective pressures. We believe that such expanded analyses will enable us to more reliably assess how mutations accumulate and to develop predictive frameworks for viral evolution.

Reviewer #1 (Recommendations for the authors):

(1)  Please mention Figure 1 and S2 in the text.

Done. We now explicitly reference Figures 1 and S2 (renamed to S1 according to appearance order) in the appropriate sections (lines 74, 124).

(2)  In Figure 4A, please specify which graph corresponds to PB2 and which to PB2-like sequences.

Corrected. Figure 4A legend now specify PB2 vs. PB2-like sequences.

(3)  Consider reducing redundancy in lines 74, 149, 170, 214, and 215.

We thank the reviewer for this stylistic suggestion. We have revised the text to reduce redundancy in these lines.

Reviewer #2 (Recommendations for the authors):

(1)  The manuscript states that "with 10,000 sequencing reads per gene ...variants at ≥0.1% frequency can be reliably detected." However, this interpretation conflates raw read counts with independent input molecules.

We have revised this statement throughout the text to clarify that sensitivity depends on the number of unique UMIs rather than raw read counts (line 173). To support this, we calculated the probability of detecting a true variant present at a frequency of 0.1% within a population. When sequencing ≥10,000 unique molecules, such a variant would be observed at least twice with a probability of approximately 99.95%. In contrast, the error rate of in vitro–transcribed RNA, reflecting errors introduced during the experimental process, was estimated to be on the order of 10⁻⁶ (line 140, Fig. 3a). Under this condition, the probability that the same artificial error would arise independently at the same position in two out of 10,000 molecules is <0.5%. Therefore, variants present at ≥0.1% can be reliably distinguished from technical artifacts and are confidently detected under our sequencing conditions.

(2) To support the claimed sensitivity, please provide for each gene and population: (a) UMI family size distributions, (b) number of PCR cycles and input molecule counts, and (c) recalculation of the detection limit based on unique molecules.

If possible, I encourage experimental validation of sensitivity claims, such as spike-in controls at known variant frequencies, dilution series, or technical replicates to demonstrate reproducibility at the 0.1% detection level.

We have added (a) histograms of UMI family size distributions for each gene and population (Figure S2), (b) detailed method RT-PCR protocol and estimated input counts (line 407), and (c) recalculated detection limits (line 173).

We appreciate the reviewer’s suggestion and fully recognize the value of spike-in experiments. However, given the observed mutation rate of T7-derived RNA and the sufficient sequencing depth in our dataset, it is evident that variants above the 0.1% threshold can be robustly detected without additional spike-in controls.

This is beautiful.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public review):

Summary:

The aim of this paper is to develop a simple method to quantify fluctuations in the partitioning of cellular elements. In particular, they propose a flow-cytometry based method coupled with a simple mathematical theory as an alternative to conventional imaging-based approaches.

Strengths:

The approach they develop is simple to understand and its use with flow-cytometry measurements is clearly explained. Understanding how the fluctuations in the cytoplasm partition varies for different kinds of cells is particularly interesting.

Weaknesses:

The theory only considers fluctuations due to cellular division events. Fluctuations in cellular components are largely affected by various intrinsic and extrinsic sources of noise and only under particular conditions does partitioning noise become the dominant source of noise. In the revised version of the manuscript, they argue that in their setup, noise due to production and degradation processes are negligible but noise due to extrinsic sources such as those stemming from cell-cycle length variability may still be important. To investigate the robustness of their modelling approach to such noise, they simulated cells following a sizer-like division strategy, a scenario that maximizes the coupling between fluctuations in cell-division time and partitioning noise. They find that estimates remain within the pre-established experimental error margin.

We thank the Reviewer for her/his work in revising our manuscript.

Reviewer #2 (Public review):

Summary:

The authors present a combined experimental and theoretical workflow to study partitioning noise arising during cell division. Such quantifications usually require time-lapse experiments, which are limited in throughput. To bypass these limitations, the authors propose to use flow-cytometry measurements instead and analyse them using a theoretical model of partitioning noise. The problem considered by the authors is relevant and the idea to use statistical models in combination with flow cytometry to boost statistical power is elegant. The authors demonstrate their approach using experimental flow cytometry measurements and validate their results using time-lapse microscopy. The approach focuses on a particular case, where the dynamics of the labelled component depends predominantly on partitioning, while turnover of components is not taken into account. The description of the methods is significantly clearer than in the previous version of the manuscript.

We thank the Reviewer for her/his work in revising our manuscript. In the following, we address the remaining raised points.

I have only two comments left:

• In eq. (1) the notation has been changed/corrected, but the text immediately after it still refers to the old notation.

We have fixed the notation.

• Maybe I don't fully understand the reasoning provided by the authors, but it is still not entirely clear to me why microscopy-based estimates are expected to be larger. Fewer samples will increase the estimation uncertainty, but this can go either way in terms of the inferred variability.

We thank the Reviewer for giving us the opportunity to clarify this point. In the previous answer, we focused on the role of the gating strategy, highlighting how the limited statistics available with microscopy reduce the chances of a stronger selection of the events. The explanation for why the noise is biased toward increasing the estimation of division asymmetry relies on multiple aspects: First, due to the multiple sources of noise affecting fluorescence intensity, the experimental procedure, and the segmentation protocol, the measurements of the fluorescence intensity of single cells fluctuate. This variability adds to the inherent stochasticity of the partitioning process, thereby increasing the overall variance of the distribution.

To illustrate this effect, we simulated the microscopy data. We extracted a fraction f from a Gaussian distribution with mean µ = 𝑝 and standard deviation σ = σ_{𝑡𝑟𝑢𝑒} , i.e. 𝑁(𝑝, σ_{𝑡𝑟𝑢𝑒}). We then simulated different time frames by adding noise drawn from a Gaussian distribution with mean µ = 0 and standard deviation σ = σ_{𝑛𝑜𝑖𝑠𝑒} , i.e., 𝑁(0, σ_{𝑛𝑜𝑖𝑠𝑒}), to f. An equal process was applied to 1 − f. The added noise was resampled so that the two measurements remained independent. Figure 6 shows a sample dynamic where the empty gray circles represent the true fractions. We then fitted the two dynamics to a linear equation with a common slope and obtained an estimate of the partitioning noise.

By repeating this process a number of times consistent with the experiment, we measured the resulting standard deviation of the new partitioning distribution. Figure 7 shows the distribution of the measured standard deviation over multiple repetitions of the simulations. Each histogram is the variance of the partitioning distribution obtained from 100 simulations of the noisy (and non noisy) fluorescence dynamic. By comparing this with the distribution of the standard deviation of the non-noisy dynamics, it is possible to observe that, on average, the added noise leads to a greater estimated variance. The magnitude of this increase depends on the variance of the added noise, but it is always biased toward larger values.

This represents only one component of the effect. The shown distributions and simulations are intended solely to demonstrate the direction of the bias, and not to account for the exact difference between the flow cytometry and microscopy estimates. In the proposed case, where noise and true variance are equal, the resulting difference in division asymmetry is 1.3.

A second contribution arises from the segmentation protocol. As we stated, a major limitation of the microscopy-based approach is the need for manual image segmentation. This reduces the amount of available data and introduces potential errors. Even though different checks were applied, some situations are difficult to avoid. For example, when daughter cells are very close to each other, the borders may not be precisely recognized; cells may overlap; or speckles may remain undetected. In all these cases, it is easier to overestimate the fluorescence than to underestimate it, thereby increasing the chance of an extremal event.

Indeed, segmentation relies on both brightfield and fluorescence images. Errors in defining the cell outline are more likely when fluorescence is low, since borders, overlaps, and speckles are more evident against a darker background. This introduces an additional bias toward higher asymmetry, increasing the number of events in the tail of the partitioning distribution.

Both aspects described above could be mitigated by increasing the available statistics. In particular, by applying stricter selection criteria, such as imposing limits on fluorescence intensity fluctuations, the distribution should approach the expected one.

A similar issue does not arise in flow cytometry experiments. From the initial sorting procedure, which ensures a cleaner separation of peaks, to the morphological checks performed at each acquisition point, the availability of a large number of measured events reduces both measurement noise and segmentation errors.

A discussion on these aspects has been added in the revised version of the Supplementary Materials and in the Main Text.

Reviewer #2 (Public review):

The authors present a combined experimental and theoretical workflow to study partitioning noise arising during cell division. Such quantifications usually require time-lapse experiments, which are limited in throughput. To bypass these limitations, the authors propose to use flow-cytometry measurements instead and analyse them using a theoretical model of partitioning noise. The problem considered by the authors is relevant and the idea to use statistical models in combination with flow cytometry to boost statistical power is elegant. The authors demonstrate their approach using experimental flow cytometry measurements and validate their results using time-lapse microscopy. The approach focuses on a particular case, where the dynamics of the labelled component depends predominantly on partitioning, while turnover of components is not taken into account. The description of the methods is significantly clearer than in the previous version of the manuscript.

Reviewer #1 (Public review):

Summary:

The aim of this paper is to develop a simple method to quantify fluctuations in the partitioning of cellular elements. In particular, they propose a flow-cytometry based method coupled with a simple mathematical theory as an alternative to conventional imaging-based approaches.

Strengths:

The approach they develop is simple to understand, and its use with flow-cytometry measurements is clearly explained. Understanding how the fluctuations in the cytoplasm partition varies for different kinds of cells is particularly interesting.

Weaknesses:

The theory only considers fluctuations due to cellular division events. Fluctuations in cellular components are largely affected by various intrinsic and extrinsic sources of noise and only under particular conditions does partitioning noise become the dominant source of noise. In the revised version of the manuscript, they argue that in their setup, noise due to production and degradation processes are negligible but noise due to extrinsic sources such as those stemming from cell-cycle length variability may still be important. To investigate the robustness of their modelling approach to such noise, they simulated cells following a sizer-like division strategy, a scenario that maximizes the coupling between fluctuations in cell-division time and partitioning noise. They find that estimates remain within the pre-established experimental error margin.

Comments on previous version:

The authors have addressed all of my comments.

eLife Assessment

This study presents a useful method based on flow cytometry to study partitioning noise during cell division. The methods, data and analysis support the claims of the authors is convincing. This work will be of interest to cell biologists and biophysicists working on asymmetric partitioning during cell division.

Ik Altijd Praten Cause Praten Is Grappig, Niet? Intelligentie <br /> Aanleg Prestatie Creativiteit Persoonlijkheid Interessen Gedrag Neuropsychologisch

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Pas de " d' ".

Je trouve qu'il y a une erreur. Dans la formation sur HTML5 et CSS3 on nous apprend que notre code doit toujours avoir la structure suivante : header (composée de "nav"), main (composée de plusieurs "section") et de footer. Or, ici, on nous montre un "header" et un "main", et la réponse n'est que "main". Etrange … Il fallait écrire "quelles balises" afin qu'on puisse choisir les deux correspondantes.

Personally, I think the most important factor is abusiveness. Just like the enforcing norms say, "public shaming must aim at reintegration of the norm violator back into the community." When celebrities make mistakes, public shaming can work as the punishments to them, but the goal should be let them realize their mistakes and feel guilty. When public shaming becomes abusive and aims to permanently stigmatize these people, it would be an instance of bad public shaming.

I think public shaming can only be viewed as a helpful tool in terms of holding others accountable for their action. But there is a fine line of crossing the boundary of shaming. Because it can lead to negative outcomes and detriotate a persons life.

I don't think this is a good way of reflecting on life. It is not a "goal" really - happiness is an "industry". See "The Happiness Myth: Why What We Think Will Make Us Happy Never Does by Jennifer Michael Hecht.

I don't think the "choices" are varied enough to represent much of "life". The path is very very limited. For e.g. university is dominant as a stage in the game. Globally, only 12-15% of the population go to university. 85% of the world do not necessarily choose NOT to go (although it might be a conscious choice for some), most do not have the opportunity to go to university at all. The life path you set out, thus, excludes 85% of the world. The OER you are creating here is for global use, not for an elite 12-15% of the population.

It's also a fallacy that going to university results in employment ("stable job") vs. not going to university. The "choice" of going to university (if we are lucky enough to have such a choice) does not automatically open a door to "venture success". Clearly, 85% of the world who didn't go into higher education are doing something with their life - including being leaders in their fields.

Not sure about the assumption that we make "choices" in life. We don't choose to be born, get sick or die (just three of many examples...) So, there has to be a randomness in the game as well as decisiveness (free-will vs. determinism)...

Things that seem like choices ("have children") might not be (women have children against their will/not out of choice).

There are lots of ways in which the life paths are not diverse enough. e.g. jobs - assumption is that a job is a thing you "get", rather than being something you might create (e.g. being self employed is commonplace for artists).

The relationship/marriage component assumes that a) relationships transform into marriage b) being single is not an option

The space after the marriage decision gateway on both sides is "buy a house". 33% of the world's population do not own their own home. Why are they not represented in this game? Some countries have no culture of buying homes - e.g in Germany long-term renting is the norm. In the UK, only 20% of people under 35 can afford to get a mortgage, nearly all rent. etc....

It might be better to remove the points - they are arbitary (who says what will make who happy?) It gamifies this in ways that are unhelpful to the educational goal of reflecting on life choices/pathways we have no choice over....

Not WeChat - it is not open software (needs to be CC software) There are hundreds of open source e-dice - find one and add the link to it here (delete the WeChat suggestion).

To make this accessible to all readers, left-justify the text (do not 'centre' texts).

To make this accessible to all readers, left-justify the text (do not 'centre' texts).

To make this accessible to machine readers (for the blind), move the emoji to the end...

for - South African supplier - tepary bean

Might be better here to say find a manhole cover that is not in the middle of a road? The neighborhood doesn't HAVE to be familiar does it? That's a constraint we do not really need here.

Why does this have to be shared? To what end? The images are not connected (different covers, different places, entirely different systems of infrastructure, etc.) so offer no learning insights.

How is it shared in a way that makes it findable online? The user could post the image anywhere and it would never be found by another user... Again, here you have a node (image) being posted with no ties (hyperlinks, shared space for depositing images).

Again, looking at a single node does not allow anyone to understand the presence of a network. The node must be tied to another (manhole).