Incredibly true! The impact these two platform alone have had on music has come significant leaps and bounds from when the iPod was invented. The subscriptions, inclusions, and marketing alone have made these platforms a social media for music and artists.

since the person she loved died for her she now wants to die?

there is different versions all over the world which is cool and it is still the most popular ballad

I agree with this because even if you go through the same thing as someone else, you will both have different experiences. This also means you will have true, real life experiences AI won't have, and its important that no matter what you say or do, it's the authentic you. Not AI.

I agree with this, because I believe a lot of the times when a student uses AI to write something for them, it comes at a desperate time. Like when they think that their writing isn't good enough, so they ChatGPT it. The most important thing about writing nowadays in my opinion is the fact that it is actually your thoughts and feelings. Not AI's.

I agree with this because even in my personal life, when people around me are using AI, I hear the term, "good enough" pretty often. Now I can see how AI has lowered our expectation creativity wise.

I also agree with this. I think that between AI and the human mind they are very different. When having AI give you all the answers, you have no power to create your own thoughts. Misusing AI is basically like giving a machine the rights to your mind and thoughts.

I strongly agree with this piece in the text because I feel that nowadays people using AI to cheat is way more common then ever before. Now using AI the right way would be to actually ask it to just give you ideas. This also ties into the wisdom aspect of using AI.

I argee with this if people use AI for essay it not really going to help them think and make critical thinking for themselfs. AI will give a lot of information but it grabs information and put it all together and that is not going to help people develop there own personal thinking.

I agree with this because human have more ability that they can imagine. Computer doesn’t have what ability’s a human has.

art can be an individual or group practice, I believe its not very hard to define, because everyone has their tastes in life, and therefore everything is art. I believe there is no such thing as good or bad art, as long as your art is intentional and true to yourself or what you reflect back onto the world. emotions are used with art making the real and raw feelings stand out. the only type of art I do not agree with is AI art, as its not real and true, its not done with any efforts or talents that are meaningful to anyone.

a man dies from love and then after he died the women realized she loved him too and then dies from guilt and sadness

In my former work experience, in a small town I was asked to assist with pregnancy and family care center. Along with having a far right agenda the place was offering counseling without legitimate certification. At the time I did a bit of research and found this article on "Crisis Pregnancy Centers". After I refused to assist with the "clinic" or support it in any way, images of aborted fetus were left in my home mailbox as well as others on the same street. Afterwards a college told me that same clinic had black listed another person in the same line of work as me, for being openly gay.

Just going to leave this link here.

(https://www.arcc-cdac.ca/media/crisis-pregnancy-centres/CPC-regulation-in-Canada-and-5-western-countries.pdf)

Research more studies on these topics

Beginning with the images of the "odours," Eliot encases the viewer into a vision of an ethereal woman, whose beauty is compared to that of Venus and Cleopatra from the stories referenced. However, in comparison to the "gentle gales ... [dispersing] Native perfumes" (Milton, 158) of Eden, this setting is full of "strange synthetic perfumes". In Eden, not only is everything in it's original/purest state, but there's a continual sense of "gentle" interactions and beings. Here, we have the rude "synthetic perfumes" of the modern world lurking in on the (possibly) private/intimate setting. Not only that, but the drowning of the senses makes the scene feel even more overwhelming, and trapped. Overall, this small section presents various stories which each further pervert the "naked majestie" (Milton, 290) that God bestowed to Adam and Eve when he created them. In Baudelaire's story of the martyred woman, her body is described "sinuous / As a snake poised to strike" (Baudelaire, 1), invoking a slightly less pure sexuality, as well as the form Satan takes to persuade Eve to commit the original sin. Similarly in the Aeneid, cupid is requested by Venus/Aphrodite to woo Dido by putting with in her "a hidden fire and [beguiling] her with [his] poison." (Aeneid, 1). This fire is rampant, and even poisons Dido to throw herself onto the pyre and sword, taking her own life. Thus in both cases the more recent/post-creation story tales involve the corruption of the human body (most notably those of women) and love. This is continued by Eliot in the most agressive sense out of all, with the drowning of the reader in these synthetic drug-like smells.

In "A Game of Chess," line 172, Eliot closes the section with "Good night, ladies, good night, sweet ladies, good night, good night," which is an exact quote from Ophelia's exit line. Eliot isn't summarizing or echoing; he is stating exactly what is said by Ophelia, and in both texts, they function as an exit line. This idea led me to question why Ophelia and why her exact words. Ophelia's anger stems from the death of the men around her, and Eliot's Part II is full of women in similar situations. Ophelia's madness manifests as fragmented sentences, which is the same texture Eliot uses for the women in this section. He uses Ophelia as a template for this section and then directly quotes her to reveal the similarities between the women.

The word "other" indicates to me a different version of the same type of object, which in this case is a story. The other "stumps of time" are fragments of earlier stories that have survived into the present, but they are "withered" and simply "stumps," implying that they are not complete or retold with pride but rather allowed to remain damaged and decaying. When read alongside the stories of Dido and the woman in Baudelaire's A Martyred Woman, Dido's initially luxurious romance with Aeneas eventually ends with her death on a couch associated with their relationship, symbolizing abandonment and the inevitability of an end. Baudelaire draws a parallel by placing a woman against a backdrop of "perfume flasks, of sequined fabrics / And voluptuous furniture, / Of marble statues, pictures, and perfumed dresses." However, he describes the woman herself with language such as "inert, complacent flesh," "ghastly head," and "cold teeth," creating a disturbing contrast between material beauty and bodily destruction. Eliot's use of the word "withered" references all of these things: early stories and traditions are but decorations mounted on the walls that appear beautiful at first glance but have no real cohesiveness or hope for restoration/change.

These are the last words that Ophelia says in the play Hamlet. She ends up going crazy due to her grief over her father, her lover, and the lack of autonomy that she once had. Ophelia, like all the women referenced thus far in the poem, goes insane/dies not on her own but to the detriment of the male figures in her life. Like Sibyl, she went insane because of her failure to manipulate Hamlet. Nobody could understand what she was saying. The repetition of "Hurry up please its time" in full caps shows that the female protagonist in this section of the poem is experiencing similar levels of distress.

This passage may make reference to Virgil's Aeneid, and specifically the role of Venus in the story. Venus is herself a goddess, queen amongst the mortals. As the goddess of love, she has the power to intervene in the affairs of mortals. It is her guidance that helps bring together Dido and Aeneas in the first place. However, she also recognizes that she has been superseded by Jupiter, himself king of the gods. Although his wife Juno was complicit in allowing the love affair to develop, she has developed it in such a way that when it splits per the will of Jupiter, it will likely be messy. Hence, Venus explains to Cupid, who in this case peeps out, to "Phoenician Dido now holds him, staying him with soft words, and I dread what may be the outcome of Juno’s hospitality; at such a turning point of fortune she will not be idle." She then commands him to ensure her love lasts forever. It is that eternal love that ultimately drives Dido to suicide in his absence. Eliot continues his emphasis on tragic love stories, a theme he has expounded on with the stories of Tristan and Isolde and the Austrian Archdukes. Surrounded by destruction and sadness, romantic stories seem out of place. Furthermore, it may be a commentary on the nature of power. Venus, herself a goddess, is superseded by yet another god, and in particular, a male one. Whether or not Eliot is himself a feminist in pointing this out, he nevertheless notes the ever evolving nature of power and fact that but for God, someone is always above you.

Rather than the focus on the natural world and conditions of urban living like Burial of the Dead, A Game of Chess highlights wealth, gender imbalances, and love–as represented by cupid. The reference to a game of chess can be connected to the content of the section in two ways: the calculated existence of men and women as separate entities, or the cold and calculated nature of romantic relationships. Eliot begins by describing a scene of immense wealth and ascribing the ostentatious display to a female figure by using the pronoun her in, “The glitter of her jewels rose to meet it”. The woman who is seemingly overcome by wealth is then contrasted with the presence of the nightingale, an animal representation of the violence inflicted upon women. The nightingale, small, animal, and unable to speak, represents the subjugated state of the female race. Oppressed and incapable of responding to the tyranny of men, the woman is silenced and left to an empty, lavish lifestyle.

https://reddit.com/r/typewriters/comments/1wmszod/whats_this_key_for_on_my_groma_kolibri_luxus/

• m/n (moneda nacional - aka use national currency like the Argentine peso rather than other currencies)
• o/s as oro sellado (sealed gold or gold currency); might also have been used for orden de servicio or orden de salida as service order or dispatch order respectively

still to this day people celebrate how amazing of a person robin hood was and what he has done

so people don't know exactly WHO he was. theres just many different stories

robin hood has been famous for a very long time

She's talking about how she chose to go with Hades

why did the guy turn back, he was supposed to not look at Eurydice until they escaped the underworld.

She is a small soul compared to others that are suffering in the after life and she says that it must break before her soul is gone and never to be seen again.

important as rollouts continue to get longer, i.e. long-horizon tasks

I would research this topic since I have seen how mental health challenges have affected people around me. Learning more about the topic could also help me understand how to support others and myself better and to use resources that are available to me

I've had issues myself with those issues

Some personal experience that I've had to larger social issues is in the military; there were always problems with mental health, especially during deployment times.

Reading about more personal curiosities about life's problems can help you learn because its personalized to you so it might make you want to learn more about that topic

Didn't know these where originally films.

absence of ILA and its affect on signing a release

didnt actualy know what snow was

wrote about what she read in stories not personal experances

only read british and american stories in childhood

I feel like the best research is the ones that solve the real issues in our world

You can learn by asking your own questions to topics

Curiosity helps with asking your own questions that you have personal interests in.

concept

This is use of ethos because it shows the writer is very familiar with the horror genre by using different film examples.

This is pathos as it uses the emotions of fear and disgust. Readers are scared of snakes, darkness, and tight spaces. Readers too likely are grossed out by having to read about eating worms

Horror movies make us feel good about our own lives by showing use the pure evil of characters. By stripping away real-world morals and showing a clear line between good and bad, the genre reassures us of our own morality.

King argues that horror movies give us a safe outlet for our suppressed negative and violent thoughts. In everyday society, we're expected to suppress these "anti-civilization" urges, so watching something scary acts as a way to let off steam without actually harming ourselves or anyone else.

this part and under is boring. jsut wall of text.. make it ool interactive animated etc?

does this page sell? or show correct reason people should pay? etc?

why aabout? choose a price?

see the audit button shoudl bring here not the blocks below so peopel can choos numebr of contacts to clan and the calualtor should go all the way to 999 999 liek calcualtor on the website on reviewmemails.com/pricing

The hell's this sentence.

This can change for everyone. People are taught different things and how we need to be cautious of who's around us and what we are saying because sometimes people can say things that offend others without meaning too.

This reminds me of growing up and how adults tell children it's just common sense but in our early years we're just trying to figure things out. As we grow up "common sense" is just what our parents or teachers taught us and that could be different for everyone.

Back to the future with Ted and Doug

keeping the links out side the file

She wants a change on the moral of the industrial training for black people because white workers have better training

n

Ladies of the Georgia Educational League:

The telegram which you sent to Governor Northern to read to his audience, informing the people of the North of your willingness to undertake the moral training of the colored children of Georgia, merits more than a pass ing notice. It is the first time, we believe, in the history of the South where a body of representative Southern white women have shown such interest in the moral welfare of the children of their former slaves as to be willing to undertake to make them more worthy the duties and responsibilities of citizenship. True, there have been individual cases where courageous women have felt their moral responsibility, and have nobly met it, but one of the saddest things about the sad condition of affairs in the South has been the utter indifference which Southern women, who were guarded with unheard of fidelity during the war, have manifested to the mental and moral welfare of the children of their faithful slaves, who, in the lanuage of Henry Grady, placed a black mass of loyalty between them and dishonor. This was a rare opportunity for you to have shown your gratitude to your slaves and your interest in their future welfare.

The children would have grown up in utter ignorance had not the North sent thousands of her noblest daughters to the South on this mission of heroic love and mercy; and it is worthy of remark of those fair daughters of the North, that, often eating with Negroes, and hi the ear lier days sleeping in their humble cabins, and always surrounded by thousands of them, there is not one re corded instance where one has been the victim of violence or insult. If because of the bitterness of your feelings, of your deep poverty at the close of the war, conditions were such that you could not do this work yourselves, you might have give a Christian's welcome to the women who came a thousand miles to do the work, that, in all gratitude and obligation belonged to you, — but instead, these women were often persecuted, always they have been ruthlessly ostracised, even until this day; often they were lonely, often longed for a word of sympathy, often craved association with their own race, but for thirty years they have been treated by the Christian white women of the South, — simply because they were doing your work, — the work committed to you by your Saviour, when he said, "Inasmuch as you did it to one of the least of these my brethren, you did it unto me," — with a contempt that would serve to justify a suspicion that instead of being the most cultured women, the purest,

Josephine quotes on the newspaper "Womanhood is the gentler aspect of life", "and should not be prostituted to the baser motives of society. What is to become of the race if woman disregards the sacredness of motherhood and sisterhood? Thee genera; federation must rest its head upon the breast of the mother of tears or else go out into the shadows of the night and die

This statement gave me a lot to think about while I was reading. It makes me think about how AI is going to change cross-cultural communication. But then I made me consider all of the ways that this is could be good as well as all of the ways it could be bad. You touched on both of these things, especially addressing the assignment you give in one of your course. I think that the AI translation assignment it a great way to make your students aware of how AI can be inaccurate

en principe sans trait d’union ; et équivoque : « du surnaturel » ou « qui dépasse le naturalisme »

tour familier

à travers

Foucart

bout de souffle

Ponctuer après la citation et non avant (ici comme partout).

: «

impropre : « techniciste »

supprimer « de »

« tient-elle à » ou « ne tient-elle qu’à »

its called maximum capacity, According to Hund's first rule, electrons will fill each empty degenerate orbital with parallel spins before any orbital gets a second electron.

so its also like two elements joining like Table Salt NaCl: Sodium and chlorine

I really like this and he remains completely correct. The political devolution we feel now can directly be attested to the basic failure of civics and the regular discourse of civil duties or functions. We are, notably, citizens of the country.

He's right beyond his years and the fight to include people within the Constitution starts with the very document itself.

Still extremely relevant. Speaks to the divide that is still felt today between people. 175 years ago is not as long as people may make it seem, it is only the difference of three people.

Well-argued, especially coming from someone who did believe in a Divine God. The hypocrisy is glaring and clear to any being with eyes.

Extremely aware of his audience and how to sell the very real idea of how horrible slavery is and how pervasive it is to the American image. Two things to this audience are holy, and attaching the stain of slavery to it after detailing horrific acts so vividly creates a vile feeling in the whole.

Rhetorical, strong, sturdy.

Man. He spares no blows that are deserved.

Still relevant to today. Wow.

There it is, he understands both the true righteousness of his words and his ability to craft them. Awe-inspiring, powerful, and knowing.

I do not and will not know where he found the ability to express himself so beautifully on stands he himself found to be so foreign and cruel. I know it was for the cause of emancipation, and yet being able to speak to the duality of beauty within American ideals and yet find the realities brought with it reprehensible is a complex task.

Resolving what the very first words of his speech were; excluding himself from the Fourth of July- and for good reason.

Rhetorical and powerful statement guiding the conversation along.

I can only imagine the weight of the present on his mind. I too am frustrated with my present day and I relate to this, but to the caliber of his words? I don't think I ever can, which makes me all the more left in awe. How muster the strength?

Genuinely well-said, surmising his position as a person born in bondage while being able to recognize the revolutionary actions of great minds before him. Clearly setting the argument that the revolution was only half-complete, which is true.

Summarizing his thoughts on the Colonies and their shaping of national identity by directly acknowledging the weight of the Declaration of Independence.

Brilliant.

Setting up the stakes; upcoming argument that the feeling is much the same but higher for the slave.

Damn.

Representing his feeling well- poetic, simple, effective.

The emotional stakes of speaking on the 4th of July

"“My wife must move for Cassio to her mistress” means Iago wants Desdemona (his wife) to make a move on behalf of Cassio toward her “mistress” — here, “mistress” is a term of endearment for Desdemona herself, not a different woman"

I hate how happy Iago is that he messed with Cassio so badly.

"The phrase means that Desdemona’s love for Othello is so strong that she can make him do whatever she wants — as easily as a person’s appetite can dictate what a god does. In other words, her desire and will can “rule” over him, and he will comply without resistance. The “appetite” is a metaphor for her strong desire or will, and “play the god” means to act as if she were a deity, commanding and shaping his actions."

This word choice is amazing for saying keep asking and he will eventually say yes. It really shows that when you push and push it can weigh on the other person in a bad way.

"In this context, Iago is recalling or imagining a moment when Othello and Desdemona were together, then separated by him (or by the circumstances). The “blow and thrust” could be a metaphor for the tension or struggle between them, or it could be a literal image of a fight. The “brief” moment and the “close together” image are contrasted with the act of parting, which Iago is using to set up a scene of manipulation"

I thought this was more saying that Iago was just a huge manipulator and continues to portray him as a terrible person over and over.

Othello's emotions are taking over his logical judgment of things, and he is struggling to make good conscious decsiosons.

" In Othello, “I am hurt to the death” is Othello’s way of expressing that he is both physically maimed and spiritually destroyed, marking the final, fatal moment of the play’s tragedy"

I undertand that he is hurt in a physical and emotional way, but I wonder how this would look on stage? How would this be expressed.

"Montano is saying that Othello may not be aware of Cassio’s bad behavior, or that his kindness and respect for Cassio’s apparent virtues make him choose not to focus on Cassio’s faults. In other words, Othello’s “good nature” could blind him to the negative traits Cassio shows"

I love this part of the scene. I think this scene in the National theatre play would be hilarious and I am excited to watch.

"So in Othello, “potations pottle‑deep” is Shakespeare’s vivid, period‑specific way of saying that Roderigo had been drinking heavily and repeatedly until the cups were empt"

What a fun choice of words to say someone is drunk and continuing to drink so heavily.

I think it's cool how Cassio is speaking so highly of Desdemona. Especially since Iago talks about her in such a sexual and inhumane way.

"“It is not yet ten o’clock in the morning.” I did not understand what this meant at all, but copilots direct translation helps me understand what they were tlaking abouit.

I love how Othello is speaking with affection and also talking about his future with his soon to be wedded wife. He keeps expressing his happiness with marrying Desdemona.

A préciser pour les lecteurs non spécialistes.

Je suggèrerais de supprimer le début de cette phrase

direct translaiton, "The Herald is saying that Othello, our noble and brave general, has received news that the Turkish fleet has been completely destroyed. He wants everyone to celebrate — some by dancing, some by making bonfires, and each person enjoying whatever sport or revelry they like. This is also to mark the celebration of Othello’s marriage. Othello has ordered that all offices be open and that everyone is free to feast from 5 o’clock until 11 o’clock. The proclamation ends with a blessing for Cyprus and Othello."

In my opinion this is mainly saying Othello is calling out for a celebration from the war and his marriage. I feel like my interpretation is similar to what AI said it was.

This is the first time in the play so far where they are actually happy without a joke being said.

robin was named the best archer

whoever can shoot a bow the best gets a "prize"

robin hood and his men went on their way

he killed someone and basically got away with it is what i'm understanding

he was tarveling

.

robin hood lived in england, henry the second was king, robin hood was an outlaw (someone that did not fit in)

When looking at the different travel values, I really admire and find Appreciation important. Whenever I travel to a different place I feel so happy to get the opportunity to go out and see all these places and to live in the moment.

I think that it was very interesting to learn about "travel values", especially because I have never heard about them before! Although I have never been out of the country, I go on many roadtrips so next time I do, I think that creating travel values will be very helpful for my adventures! I also find travel values very important because they can give you a new perspective of each place you visit!

More religious usage

Do you compare with CelFiE-ISH and other new read-level deconvolution model like Methylbert? CancerDetector is out of date.

If the console is stuck at the Browse[n]> prompt and missing the TRUE/FALSE outputs, you likely created an accidental breakpoint.

Remove the breakpoint: Find and click the red dot next to the line numbers in your script editor to clear it.

This complete section before discussing Subjective information should be placed in the Basic Concepts Section. The reader at this point should be ready to learn content related to the assessment process.

the english ballads have a bunch of different themes and for every one it is different

a lot of the music is still popular today

instruments are used to help tell the story

What purpose does the SWOT analysis serve?

One critique with this PBS website is the unrelated articles in the related tab. The related tabs purpose is to help you find other sources related to the topic. However, having different links creates a clouded menu that is inaccessible. PBS could improve accessibility by creating a clearer visual separation between the main content and additional or related information without becoming distracted or overwhelmed.

On this website using closed captions as operable is not it's only function. The closed caption on the video also supports perceivable accessibility because it is in a visual text format instead of just accessing information by listening. This feature is very important and has become one of the main strategies to navigate online websites for people with different learning and accessibility needs. For example, using closed captions helps people who are deaf or hard of hearing.

The full written transcript creates an accessible website under the perceivable principle by creating an alternate way to access the information presented in the video. This is essential for people who are deaf or hard of hearing. One of the negative effects of transcripts and information being physical or online is that it's challenging for people who are blind, visually impaired or for people wanting to review the content at their own pace. Since the PBS includes a conversation between Tamara Keith and Amy Walter about Trump and the media some viewers might need to return to an important part of the conversation. Having a physical resource instead of a visual makes searching for a specific part in the video more accessible. However, it is important to recognize that even websites we think are "accessible" are not accessible for everyone.

Using clear headings and organized sections supports the understandable principle of accessibility. The titles are in bigger font compared to the main text and they are bolded showing important information. Headings help users navigate information and makes it more accessible to find specific types of information. One of the ways that the headings are organized is by distinguishing the two speakers in the transcript Liz Landers and Amna Nawaz. Having accessible speaker identification is important because users in the transcript can tell who is speaking in the video even if you don't watch it.

On this website's homepage, the video playback controls follow the key web accessibility principle of making websites operable. The main goal of operability is making sure your users on your website have control of how they interact with the video. Key features of operability include: playing and pausing the video, volume, controlling speed, and captions. This is an important function of an accessible website because it allows users to process the information and follow along at their own pace. As well it is important to recognize the option of a transcript and full audio file.

That's a weird way to continue. If this was actually the script it wouldn't flow very well from the last line from section 9.

Weird way to say this

That's just weird English

And the last noise you see or get in your certificates because there's less emails that don't exist. They are also fake opening and might be skewering your decision-making.

That is what they say but people must interpret that as 99% in boxing not 99% delivered minus one percent bounce

Should we not put the validity number or whatever the average is for liability in boxing for people or is there a reason why you put 60% here?

We need to make it clear. The email marketing software providers are unable to give a real sperm rate. The largest portion of the list is un included in that spam rate we need to make that clear.

I want a good time to explain to people how different even marketing tools charge people with some of them will charge them even if the contact is unsubscribe and some will even allow you to add an email in the suppression list, but will remain active in the main list and some, if not all of them will charge you for duplicate contacts or multiple lists and then we also should explain that if people are in multiple lists and subscribe the subscribe from the list they don't subscribe from the account, so therefore people might have the impression they're unsubscribing and then they get an email from another list that they're in on your end and they get angry and complain and then you have no idea because of the complaints and spam rates aren't given yada yada

this is a bit too on the nose? we ned to be ore smooth or add things os it doensT' sound lieka sles pitch lol

and the other waya round right?

we can show calaultaor here no? or a cool version of it? ad some the other way aroudn right? like when trackign pizel doesnt ping or downoad or open

we keep saing apple 2021, but like gmail has been prefetching or micsosft uderfetching fro years its ot jsut apple they jsut made it public

we can say like you deliverablity tab isn't a deliverbaility tab, its jstu a delivery tba that shows you thigns that are miinsterpratred weirdly

and then google postmaster dns yahoo sender heub that sreal , toosl liek emailconsul show real deliverbaility and sender reptuation adn everm then ther are limitaitons.

weird to say that liek that lol

at some poitn shoudl probably say how logn it take osn average for a list to be more stale aan alive

or give you an email they dont' use as much on purpose, or they get too much spam, they change provduer ose their passwords, passaway,,,

the number one thing or the first thing to thinkg about or something )jsut so we don't compete with kennedy's message) we have to be more smooth

even if by how many emails, if lessc ontcts you send less... so you still save..

whre are the tst studies iwe can put in the screen as links or wtv

the on screen ... feel a lot of talkign coudl be translated as a animation in the background that goes with what someone says no? i mean i dont' knwo how we time it with nakimbe sayign things but jow cool owuld it be to animate things?

sotirng. maybe in more than on e places right? invoicing tools, inbox, crm a gogole doc. etc. etc

no need to reference ocld email ever. we can say like peopl ewho want to get your emails

try that

Zmenil som rok z 1982 na 1989, keďže pôvodné články už neboli na ACM dostupné.

Главное задача сейчас пройти по основному флоу. и только больше я сейчас не особо пояснию.

главное сделать проект для него менее абстрактым. главное, чтоб у Вити появилась картинка в голове по поводу этого проекта.

Not a narrative lede but effective. It probably suits this short profile better but cutting right into the story.

Very interesting work! The combined analysis of gene expression and genomic data at single cell resolution is a real strength. In three independent scRNA-seq datasets from HNSCC (including HPV+ HNSCC, HPV-negative HNSCC and normal tonsil epithelium) we (Smith et al EMBO J. 2025 (PMID 39548236)) observed peak A3A expression in differentiating keratinocytes and peak A3B expression in cycling keratinocytes but minimal expression of either gene in resting basal cells. We also provided evidence at the protein level (IHC in tumours) and in cultured keratinocytes (A3A, A3B mRNA expression and A3A RNA editing activity) that A3A is expressed in differentiating, rather than basal cells. From what I can see from your UMAP plots in Fig 1 and in Supplemental Fig 1 in your manuscript, the peak A3A expression and many of cells displaying greatest SBS2 enrichment appear to be 'stratified squamous epithelial cell' (if I am interpreting the key in Supp Fig 1 correctly), which I suspect is equivalent to what we called 'differentiating epithelial cells' (i.e. cells that express genes such as Involucrin and cytokeratin 10, which would not be expected to be present in basal cells). So I think your observations are consistent with ours but that we are using different terminology to describe the relevant epithelial cell populations. I'd be interested in your thoughts on this, and it might be worth looking at the additional scRNA-seq datasets that we analysed in our study to compare them with your dataset.

de mate waarin iemand vatbaar is voor de suggesties, ideeën of invloeden van anderen

Jyutping: hip3 zo6 English: to assist; to help; to support 例句 1. 本文旨在協助專業人士處理有產後情緒問題嘅家長。 This article aims to assist professionals in supporting parents with postpartum emotional difficulties. 2. 我哋會協助家長了解 BB 嘅需要。 We will help parents understand their baby's needs. 3. 專業人士可以協助家長建立更健康嘅親子關係。 Professionals can help parents build a healthier parent-child relationship. 💡 協助 比 幫 正式，例如 協助家長、協助專業人士、協助處理問題。

Jyutping: gau3 sing4 English: to constitute; to form; to make up; to create 例句 1. 產後抑鬱會對家長自身構成負面影響。 Postpartum depression can have a negative impact on parents themselves. 2. 長期壓力可能會對家庭關係構成負面影響。 Long-term stress may negatively affect family relationships. 3. 呢個問題可能會對 BB 嘅健康發展構成影響。 This issue may affect the baby's healthy development. 💡 常见搭配： - 構成威脅 — pose a threat - 構成影響 — have an impact - 構成負面影響 — have a negative impact - 構成風險 — pose a risk

Jyutping: faat3 zin2 English: development; to develop 例句 1. BB嘅健康發展好重要。 The baby's healthy development is very important. 2. 父母嘅回應會影響 BB 嘅情緒發展。 Parents' responses can affect the baby's emotional development. 3. 每個 BB 嘅發展速度都唔一樣。 Every baby's rate of development is different. 4. 良好嘅親子關係有助於 BB 健康發展。 A healthy parent-child relationship helps support the baby's healthy development.

• 健康發展 — healthy development
• 情緒發展 — emotional development
• 社交發展 — social development
• 認知發展 — cognitive development
• 語言發展 — language development

適切 Jyutping: sik1 cit3 English: appropriate; suitable; proper 例如： 例句 1. 如果家長有產後情緒問題，就需要適切嘅介入。 If a parent has postpartum emotional problems, appropriate intervention is needed. 2. 專業人士會根據家長嘅需要，提供適切嘅支援。 Professionals provide appropriate support based on the parents' needs. 3. 及早提供適切嘅幫助，可以減低問題對家庭嘅影響。 Providing appropriate help early can reduce the impact of the problem on the family.

Jyutping: zing1 zong6 English: symptom; symptoms 例句： 1. 產後抑鬱有咩常見徵狀？ What are the common symptoms of postpartum depression? 2. 如果發現自己有呢啲徵狀，可以盡早搵專業人士幫手。 If you notice these symptoms, you can seek professional help as early as possible. 3. 有啲家長可能會出現焦慮、失眠等徵狀。 Some parents may experience symptoms such as anxiety and insomnia. 💡 徵狀 is commonly used in medical/psychological contexts.

jing1 ji4- baby

cou1 zeon3 promote / facilitate / foster all work depending on the sentence: - 促进亲子关系 → foster a healthy parent-child relationship - 促进 BB 的健康发展 → promote the baby's healthy development - 促进家长与 BB 之间的联系 → facilitate the connection between parents and their baby

Around 10% of mothers and 5% of fathers in Hong Kong experience postpartum depression. Research has found a moderate positive correlation between mothers' and fathers' depressive symptoms after childbirth.

Roderigo’s inability to be convinced in scenes such as these could be an interesting point of study as the story develops him towards a much different character in terms of trust and distrust.

Here lies another instance of Iago utilizing discrimination to his own advantage when it suits him. He is slowly built as a character that is never fearful of making a decision in the moment that bends the situation to his will.

At war, Desdemona is fearful of the words that each one says unfortunately coming true. While in the scene, it seems like a simple lover’s gesture of minor concern. From the reader’s viewpoint, the fact that this story is a tragedy makes this line especially powerful as if every line such as this is slowly compiling their misfortune.

At first, Iago's plan was about Roderigo's money and Cassio's job. Now it becomes personal: he wants revenge "wife for wife", or, failing that, to make Othello sick with a jealousy that reason can't cure. His real goal is to destroy Othello's mind.

From this line, the soldiers are expectant of Othello and Desdemona’s arrival because it could restore morale to the army that was previously at the bay. Knowing that this story is a tragedy, one must question how unfortunate it must be for the soldiers to celebrate this relationship.

Iago uses a musical metaphor: Othello's marriage is like a perfectly tuned instrument, and he will loosen the pegs so the music falls apart. By adding "as honest as I am", he mocks his own reputation — "honest Iago" is the play's biggest lie.

I find this line to be quite out of place for the rest of the scene, almost as if it was a random thought within the scene as well. The random interjection about Othello’s marital status could outline how easily their mind has been taken off of the war, foreshadowing worse conflict to come.

This is foreshadowing: at his happiest moment, Othello links love and death, and he will indeed die because of this love. It echoes Solon's idea that no man can be called happy until he's dead — perfect happiness in tragedy never lasts.

This shows Iago as a mastermind: he sees himself as a spider trapping a big, blind fly, Cassio. He doesn't need anything big — just a small gesture of politeness he'll twist into proof. That's his method for the whole play.

Like Iago's "I am not what I am", Desdemona admits she hides behind a mask — but a defensive one: hers hides worry, his hides malice. It shows something important for the rest of the play: even the purest characters wear masks.

Iago plays the role of the blunt, joking soldier so no one can unmask him. His "jokes" show his misogyny: for him, women are fake and only think about sex. He'll use this same prejudice to convince Othello that Desdemona is unfaithful.

By ending the war, Shakespeare shifts to an inner, mental war between the characters, leaving them all on a small island far from Venice's order. Othello loses his role as a soldier, and the only enemy left is Iago.

This is my comment on this sentence

شیوه‌ها

eLife Assessment

This paper uses rigorous experimental methods to investigate the role of covert attention in decision-making. The authors identify aspects of the decision problem that shape the allocation of covert attention and show that covert attention attenuates the impact of overt gazes on choice. These findings are solid and methodologically and theoretically valuable for researchers working at the intersection of attention and decision-making.

Reviewer #1 (Public review):

Summary:

In this paper, the authors combine a well-controlled decision-making task with a secondary probe task in order to understand how covert attention is shaped and how it influences the ongoing decision process. The authors report that the likelihood of overtly attending an alternative is shaped both by its decision relevance and its decision value. Further, covert attentional allocation comes at the expense of overt attentional allocation and attenuates the impact that covert gazes have on choice. Importantly, covert attentional allocation is dissociable from pre-saccadic activity. This paper sheds new light on the role of covert attention in decision-making. From an experimental aspect, the authors make excellent use of behavioural and process-tracing methods, presenting an exemplary paradigm that could be valuable to researchers working in related fields.

Strengths:

(1) The paper utilises a very rich and clever experimental design, which allows for probing covert attention at the level of a single trial. Extending the paradigm to ternary choices was an excellent addition. The decision task is also well-controlled. Overall, the results are very clearly presented, and the paper is very well written.

(2) The paper addresses important yet overlooked questions in decision neuroscience: what influences covert attention during decision-making and what is the functional relevance of covert attentional shifts. It is impressive that the authors are able to tackle these questions using behavioural and eye-tracking data alone.

(3) The authors thoroughly check that the secondary probe task can indeed be used as a proxy for covert attention. Checking that covert attentional allocation is dissociable from pre-saccadic activity was an excellent control.

Weaknesses:

(1) It is not clear why the decision task is described as "value-guided" instead of "perceptual". Participants receive momentary reward on the basis of their overall accuracy, but this is common practice in perceptual tasks. A value-based analogue of this task would provide trial-by-trial reward as a function of the perceptual magnitude (decision value) of the chosen stimulus. On a related note, the secondary task was not incentivised. The authors should further justify this choice.

(2) The authors do a great job in describing the determinants of covert attention. However, the relevant analyses are rather "phenomenological". It would be useful to try and dig further into the data in order to understand at a deeper, causal level these determinants. I suggest doing so by utilising in full the rich behavioural and overt attention data this paradigm offers. Specifically, the authors show that the higher the value of an alternative, the higher the likelihood this alternative is covertly attended. Decision values, however, could impact aspects of saccadic behaviour that directly influence covert attentional allocation (saccadic speed, dwell times, locus of gaze). At a more global level, decision value and/ or value difference could affect sampling behaviour (frequency of switching) or decision times, which can manifest in probe accuracy. Overall: a) overt sampling behaviour is at present reduced to the presence of saccades; but saccadic behaviour could be decomposed into richer metrics, b) although the task is "free-response", decision times are not analysed. Metrics based on a) and b) could be tested as mediating factors, enabling the authors to better understand the causal determinants of covert attention.

(3) The authors claim that covert attention attenuates: a) the impact of the last fixation on choice, b) the "time advantage" of the alternative that was overtly attended for longer. To further qualify these claims as functional/causal, further analyses are required. A few suggestions follow below. On trials where the "unattended" probe letter was successfully reported, the last fixation may exhibit different characteristics, which could explain away its reduced impact. For example, it may differ in duration, or it might correspond to a "switching" (rather than "staying" on the same alternative) saccade. These potential covariates need to be further examined. Additionally, correctly reporting unattended probes could come at the expense of decision accuracy due to dual task demands. If that is the case, then the reported attenuation of the impact of the last fixation could just be due to noisier responses. Similar points can be raised about the "time advantage", especially given that decision times do not feature in any of the analyses. More difficult decisions may lead to higher probe accuracy but also to prolonged decision times. The authors quantify the "time advantage" using cumulative fixation time, but perhaps relative metrics (e.g., relative fixation time or cumulative fixation time normalised by decision time) are better suited for this analysis.

Reviewer #2 (Public review):

Summary:

The authors combine a value-based choice task with a gaze-contingent covert attention paradigm to investigate how covert attention relates to value-guided decisions. This is an important and timely question, as many models of decision-making implicitly equate gaze with attention despite extensive evidence that covert attention can be dissociated from eye position. I particularly appreciated the relatively naturalistic task design, which allows participants to freely explore the options while covert attention is sampled during the decision process. Overall, the manuscript is well written, the experiments are carefully executed, and the analyses are generally appropriate and clearly presented.

My main reservation concerns the strength of some conceptual claims. The data convincingly demonstrate that covert attention can be decoupled from gaze and that probe report is modulated by option value. However, I found the evidence less directly supportive of the stronger conclusions that covert attention dynamically competes with overt attention throughout the decision process and that covert attention itself causally influences subsequent choice. Many of the reported analyses are consistent with these interpretations but, in my view, do not uniquely support them.

Strengths:

The manuscript addresses an important and timely question at the intersection of visual attention and value-based decision making. The experimental paradigm is elegant and relatively naturalistic, allowing covert attention to be sampled during ongoing decision making while participants freely explore the choice display. The experiments are carefully executed, and the analyses clearly demonstrate that covert attention can be dissociated from gaze and is systematically modulated by option value. The control analyses addressing the contribution of presaccadic attention are an important addition that substantially strengthens the manuscript, even though I had questions regarding their interpretation.

Weaknesses:

(1) Interpreting the causal role of covert attention in guiding choice

The manuscript puts forward two central conclusions: first, that covert attention is shaped by decision-relevant factors such as option value; and second, that covert attention, in turn, influences subsequent choice behavior. I found the evidence for the first conclusion compelling. My reservations concern the second conclusion, namely whether the current data uniquely establish a causal influence of covert attention on choice. Throughout the manuscript, the authors conclude that covert attention exerts "downstream consequences for choice behavior" and that an estimate of covert attention "influences the final choice." However, I am not sure that the presented analyses fully disentangle a causal influence of covert attention from a common influence of option value on both covert attention and the eventual decision.

One aspect that illustrates this concern is the interpretation of the last-fixation bias. While the reported association between the final fixation and the chosen option is clear, it is less obvious that the final fixation itself biases the subsequent choice. An equally plausible interpretation is that participants have largely committed to a decision and subsequently direct one final gaze shift toward the option they have already selected before executing the button press. In other words, the relationship may reflect choice influencing gaze rather than gaze influencing choice. Unless the temporal dynamics allow these alternatives to be distinguished, I would encourage the authors to use more neutral language (e.g., an association between last fixation and subsequent choice) or explicitly discuss this alternative interpretation.

More generally, I found myself looking for a more direct demonstration that covert attention biases choice toward the attended option. Most analyses ask whether successful peripheral probe report attenuates established gaze-related choice biases, such as the last-fixation or time-advantage bias. While these are interesting findings, they remain relatively indirect. A particularly compelling demonstration would be to examine situations in which option value cannot explain the relationship between covert attention and choice.

(2) Dynamic competition between covert attention and overt attention

I think one of the key strengths of this manuscript is its explicit attempt to dissociate covert attention from gaze-a distinction that is often overlooked in both empirical studies and computational models of value-based decision making. My reservation concerns the stronger claim that covert attention is dynamically reallocated during decision making and competes with overt attention. The present results clearly show that covert attention can be dissociated from gaze, but I found less direct evidence for the proposed dynamic competition. Throughout the experiments, probe report accuracy remains highest at the currently fixated option, whereas peripheral benefits, although reliable, are comparatively modest (e.g., Figures 2 and 3). This raises the question of how often, and under which circumstances, covert attention is actually reallocated away from the current fixation.

Because the authors possess precise eye-movement timing, I think the manuscript would be substantially strengthened by an analysis of attentional dynamics within individual fixations, to provide more direct support for the proposed dynamic interplay between covert and overt attention.

More generally, I found several interpretations somewhat stronger than the presented evidence. For example, the statement that increased covert attention to higher-valued peripheral options occurs "at the expense of" overt attention at the fixated location seems to imply a direct trade-off that is not explicitly demonstrated. Unless such temporal dynamics can be shown, I would encourage the authors to distinguish more clearly between demonstrating that covert attention can be dissociated from gaze and demonstrating that it dynamically competes with overt attention throughout the decision process.

(3) Dissociating covert from presaccadic attention

The control analyses aimed at dissociating covert from presaccadic attention are an important strength of the manuscript. However, I found the rationale underlying these analyses difficult to follow. The key conclusion-that value modulates probe report even when covert attention is not presaccadic-rests on the distinction illustrated in Figure 6, yet it remained unclear to me exactly how this distinction isolates presaccadic from non-presaccadic covert attention. My understanding is that the authors compare probe performance at previously fixated locations depending on whether those locations are subsequently re-fixated. If this interpretation is correct, a more explicit explanation of why this operationalization isolates presaccadic attention would help readers evaluate this conclusion. Conceptually, I found it more intuitive to think about a dissociation based on probe performance at locations that are subsequently fixated versus not subsequently fixated following the first fixation, when the currently fixated option, the upcoming saccade target, and a third unfixated option are distinct. Such situations would seem to provide the clearest opportunity to separate attentional enhancement associated with the upcoming saccade target from covert attention directed elsewhere.

I also found the interpretation of the time-to-saccade control stronger than the evidence directly supports. The authors argue that a purely presaccadic account predicts a monotonic decline in probe accuracy as a function of time-to-saccade. This assumption seems stronger than supported by the presaccadic attention literature, which primarily characterizes attentional enhancement during approximately the final 100-200 ms before saccade onset. Within this temporal window, the present data actually appear consistent with such an increase (Figure S6). By contrast, I am not aware of theoretical or empirical work predicting a monotonic presaccadic attentional shift extending hundreds of milliseconds, or even a second, before saccade onset.

The observed U-shaped relationship is nevertheless inconsistent with a simple presaccadic-only account, as probe performance remains elevated even when the upcoming saccade is relatively distant. I therefore agree that the findings suggest additional attentional processes. However, I do not think they uniquely establish that probe performance indexes covert attention independently of presaccadic planning.

Author response:

We thank the reviewers for their thoughtful and constructive feedback on our work.

We have attempted to synthesize the main suggestions across both reviewers into four main areas, which we plan to address in a revised version of the manuscript.

(1) The relationship between covert attention, overt attention, and choice

While the current analyses address elements of relationship between covert attention, overt attention, and choice, the reviewers highlighted an opportunity to make full use of our rich gaze data to examine more explicitly how these processes are related to one another over the course of a decision. [Reviewer #1 Point 2, Point 3 i; Reviewer #2 Point 2 para 2]

(2) The role of decision difficulty and decision time

The reviewers point out that decision difficulty may i) account for the relationship between covert attention (probe response correctness) and choice; and ii) influence decision times, which are currently not analyzed; and that these considerations may affect how we interpret the relationship between covert attention and choice. [Reviewer #1 Points 2, 3 ii and iii; Reviewer #2 Point 1 paras 1 and 3]

(3) Evidence for competition between covert and overt attention

The reviewers requested more direct evidence for our claim that covert attention to higher-valued peripheral options occurs "at the expense of" overt attention [Reviewer #2 Point 2 paras 1 and 3, Point 3 para 3]

(4) Figure 6: Presaccadic vs. non-presaccadic attention

Reviewer 2 found the rationale behind the analysis distinguishing presaccadic from non-presaccadic covert attention difficult to follow and suggested and alternative to make this comparison more explicit [Reviewer #2 Point 3].

In response to these suggestions, we will include additional analyses in the revised manuscript which more directly examine the relationship between covert attention, overt attention, and choice. We will also introduce new analyses which more simply and directly depict: why decision difficulty cannot wholly account for the link between covert attention and choice; a competition/trade-off between overt and covert attention; and that value modulation existed at non-presaccadic options. In addition to these four main areas, we will clarify several elements of the task design and analysis which may not have been sufficiently clear in the manuscript.

Here, we offer a provisional response to each of the points above.

(2) The relationship between covert attention, overt attention, and choice

We agree that the relationship between covert attention, overt attention, and choice could be examined in more detail using the available eye-tracking data. In the revised manuscript, we plan to include further analysis looking at fixation and sampling measures before the decision is made.

For example, to investigate the relationship between covert attention and choice (as reviewer 1 suggested), we plan to include an analysis which asks about how decision accuracy changes as a function of decision difficulty (relative option value) when the unfixated probe is correctly reported, versus when it is not. This will straightforwardly test for the influence of probe report on choice, controlling for the influence of decision difficulty.

More generally, we will revise the manuscript to distinguish more carefully between associations demonstrated by the present data and stronger causal interpretations. To address this, we will either attempt more direct statistical tests of causality, or use more neutral language where the causal directionality between fixation, covert attention, and choice cannot be uniquely established by the present design.

An important clarification on the dynamics of the task is relevant to several of the reviewers’ comments concerning the last-fixation analyses. Probe performance was generally measured earlier than the final fixation and choice – as it was always at the first to third option visit by the participant (or only first to second option visit in Experiment 1). Only 10.4% of probes occurred during the final option visit, and on average, participants made 2.8 further option visits after probe presentation. We can see that our original presentation of these results may have been difficult to follow. We will make this clarification in the revised manuscript, particularly where we refer to “last-fixated” or “last-unfixated” options. These terms refer retrospectively to which option was ultimately visited or not visited last before choice, rather than to the fixation occurring at the time the probe was presented.

(2) The role of decision difficulty and decision time

We agree with the reviewers that decision difficulty and in turn decision time should be considered more carefully.

We will make explicit that relative option value was already controlled for in the existing analyses relating to covert attention and choice. Specifically, for the last-fixation analysis, relative value difference was included as a control variable in the regression. For the time-advantage analysis, we use a corrected dependent variable which is intended to remove possible influences of relative value (i.e., decision difficulty) on the fixation durations, following what has been done in previous work (e.g., Eum et al., 2023; Krajbich et al., 2010; Krajbich & Rangel, 2011).

We additionally plan to include new analyses which more directly examine the role of decision difficulty and decision time. In particular, we will test whether the relationship between peripheral probe performance and choice remains after accounting for decision difficulty, and whether decision time differs as a function of probe performance.

We would also like to take the opportunity to clarify that “time advantage” was operationalised as a relative measure: the difference in cumulative dwell time between the two relevant options, rather than the absolute cumulative dwell time on a single option. This definition will be stated more clearly in the main text and figure caption.

Finally, to address the possibility that peripheral probe report errors reflect a general dual-task cost or increased response noise, we will examine decision accuracy as a function of decision difficulty separately for trials in which the peripheral probe was reported correctly versus incorrectly. This will help determine whether successful peripheral probe report is associated with poorer decision performance.

However, we should note that a closer examination of Figure 4 offers clues to this question; it suggests that correctly reporting the probe at the last-unfixated item makes people more accurate, rather than happening at the expense of decision accuracy. To explain this:

When objectively Option 1 is worse than Option 2 (i.e., to the left of x-axis center), choice for a last-fixated Option 1 (solid line) is unfairly boosted; but if the last-unfixated probe was correctly reported, then the choice proportion is correctly closer to 0%.

When objectively Option 1 is better than Option 2 (i.e., to the right of x-axis center), choice for a last-unfixated Option 1 (dotted line) is unfairly discounted; but if the last-unfixated probe was correctly reported, then the choice proportion is correctly closer to 100%.

That is, the bias-attenuation effect of correctly reporting the probe at the last-unfixated item seems to operate on 1) reducing choice boosting of the objectively-worse fixated option (left of x-axis center, solid line is further down on right panels than left panels); and 2) increasing choice boosting of the objectively-better unfixated option (right of x-axis center, dotted line is further up on right panels than left panels).

(3) Evidence for competition between covert and overt attention

To address more directly the question of whether covert attention occurs at the expense of overt attention, we will add an analysis testing whether successful report of a probe at an unfixated option negatively predicts report of the simultaneous probe at the fixated option. A negative relationship would provide a more direct test of the predicted trade-off between attentional allocation to the fixated and peripheral locations. We will revise the strength of the corresponding language in the manuscript according to what this analysis supports.

(4) Figure 6: Presaccadic vs. non-presaccadic attention

We agree that the rationale underlying the current Figure 6 analysis is too implicit in the current manuscript.

To clarify the underlying rationale: The key logic of the analysis was to ask whether value modulation of probe performance is present at an unfixated option even when that option is not the target of the upcoming saccade. In the original analysis, we only compared attention at peripheral options that had previously been viewed: we compared attention at peripheral options that had previously been viewed and were subsequently fixated next, with those that had previously been viewed but were not subsequently fixated next. The requirement that the option had previously been viewed arose because, in the gaze-contingent “hidden” condition, the value information of an unvisited peripheral option would not yet have been available to the participant, and therefore there would be no possibility of any value modulation effects existing at that location.

Although this analysis was intended to distinguish value modulation at likely presaccadic targets from value modulation at other peripheral locations, we now see that its rationale is not immediately transparent. In the revised manuscript, we therefore plan to replace or supplement this analysis with a simpler visualisation and analysis that addresses the same question more directly.

We will also revise text associated with the time-to-saccade analysis. Our use of the term “monotonic” was imprecise. The intended point was not that a purely presaccadic account predicts a strictly monotonic change across the full time-to-saccade interval, but rather that the observed U-shaped relationship is difficult to reconcile with a simple presaccadic-only account, because probe performance remains elevated even when the upcoming saccade is relatively distant. We will clarify this point and more carefully distinguish evidence inconsistent with a simple presaccadic-only explanation from the stronger claim (which we did not intend to make) that presaccadic contributions have been completely excluded.

(5) Additional clarifications

We agree with Reviewer 1 that the task can reasonably be described as perceptual rather than strictly value-based, but we believe that our central concerns regarding how covert and overt attention interact with multi-alternative decision-making still stand.

We will clarify why the secondary probe task was not incentivized. The probe task was intended to provide an incidental measure of covert attention while participants performed the primary decision task. Incentivizing probe performance could have encouraged participants to explicitly trade off performance on the two tasks or to alter their allocation of attention in response to the secondary task.

In summary, the revisions will combine clarification of several existing analyses with the addition of new analyses that more directly test the reviewers’ central concerns. In particular, we will more explicitly address whether the relationship between covert attention and choice remains after accounting for option value/decision difficulty; whether peripheral and fixated probe performance show evidence of an attentional trade-off between overt and covert attention; how decision time and richer measures of overt sampling relate to covert attention; and whether value modulation is present at peripheral locations that are not the target of the upcoming saccade. We will also moderate causal and mechanistic language where the current data do not uniquely establish directionality.

eLife Assessment

This study presents a useful approach to analyzing brain responses to transcranial focused ultrasound (tFUS), and potentially to neuromodulation-evoked activity more generally. By applying ideas from physics, it reveals changes in brain activity that measures of signal strength alone do not capture. The authors report a surprising non-monotonic relationship between irreversibility and stimulation dose, and a relationship between baseline irreversibility and susceptibility during stimulation. The evidence for the central interpretation remains incomplete, however: it is not yet clear that these changes reflect genuine reorganization of brain activity rather than artifacts of the calcium-recording method or an unaccounted-for side effect of the stimulation itself.

Reviewer #1 (Public review):

Summary:

The authors introduce ordinal EPR (Dsym) as a novel metric for characterizing tFUS-evoked calcium responses. The concept is interesting and could offer an innovative way of looking at neural responses to neuromodulation more broadly. Their main result, that EPR carries information beyond mean GCaMP amplitude, is compelling, but the manuscript would be strengthened by testing whether it holds against other conventional GCaMP metrics beyond mean amplitude (e.g. decay time). More importantly, the EPR metric requires further validation before its central claim, that it reflects genuine dynamical reorganization of the underlying circuit rather than artifacts of the measurement pipeline (e.g. GCaMP indicator kinetics), can be accepted. A rigorous surrogate/synthetic signal validation would be a very valuable, if not essential, addition to this paper.

Strengths:

This manuscript frames tFUS-evoked activity in a way that is uncommon in the field. Moving beyond amplitude-based readouts to ask how sonication reshapes the temporal organization of neural activity is a novel contribution to the ultrasound neuromodulation literature. The finding that EPR shows a distinct dose-response profile from calcium amplitude and retains dose-related structure after controlling for amplitude, is a promising demonstration that this framework can extract information not visible to conventional measures.

Methods are very detailed and well explained for reproducibility purposes.

Weaknesses:

Major weaknesses:

EPR may carry information beyond mean GCaMP amplitude- but is it carrying information beyond other elements of the GCaMP signal? For the on-target recovery window, the calcium signal has not yet returned to baseline (original data traces, Fig. 2C, Fig. 4A), so the trace still contains a residual decay transient at the time Dsym is computed. This decay rate reflects neuronal activity returning to baseline and will be dependent on things such as calcium indicator kinetics. Would a change in Dsym always accompany this kind of decay, regardless of whether this has anything to do with endogenous dynamics, irreversibility etc.? Would any gradual return to baseline/decay of this kind produce elevated Dsym on its own, independent of underlying stochastic fluctuations/endogenous dynamics? Is it not expected that a system that is not at steady state shows entropy production? Perhaps the decay rate is simply different by dose, which explains the findings? Could this be tested i.e. with synthetic data or by removing the decay? The fact that a change in EPR is measured when there is no significant GCaMP change indicates that the measure is not purely dominated by decay kinetics but it is not clear whether decay kinetics etc. could be confounding this result.

GCaMP's rise and decay kinetics are asymmetric (fast rise, slow decay). Since Dsym is a measure of asymmetry between forward and time-reversed ordinal statistics, could this kinetic asymmetry alone have any impact on "irreversibility", independent of underlying neural dynamics? Could this be tested with synthetic data? The authors note that the GCaMP signal is filtered through an indicator with slow kinetics but don't mention the asymmetry.

When different stimulation periods are used, what impact if any, does this have on computation of the EPR metric?

What impact does having a noisier/lower SNR calcium signal have on the EPR metric, if any?

Minor weaknesses:

The major advantage of TUS as a non-invasive brain stimulation technique is its capacity for spatially focused deep brain stimulation. However, it carries a significant auditory confound, which leads to activation of widespread (not spatially restricted) neuronal networks (Kop et al., 2024; Sato et al., 2018). The data used in this paper does not adequately control for this confound (i.e. deafened animals (Guo et al., 2023; Sato et al., 2018). Whilst this is not the responsibility of the authors of this manuscript, it should be mentioned in the discussion that any of the tFUS-calcium evoked responses could be due to indirect auditory stimulation rather than a direct pressure-mediated effect. The authors may just be measuring EPR in response to the auditory confound, which does not undermine the overall impact of this paper (as it is more focused on an approach to looking at this kind of data), but should be mentioned. I do think that presence of the auditory confound could impact interpretability of the results in the manuscript. Could the authors comment on this? Could auditory mediated arousal or state shift (perhaps activating brain regions not captured by the fiber) explain any of the presented EPR results or affect interpretation?

Guo, H., Salahshoor, H., Wu, D., Yoo, S., Sato, T., Tsao, D. Y., & Shapiro, M. G. (2023). Effects of focused ultrasound in a "clean" mouse model of ultrasonic neuromodulation. iScience, 26(12). https://doi.org/10.1016/j.isci.2023.108372<br /> Kop, B. R., Shamli Oghli, Y., Grippe, T. C., Nandi, T., Lefkes, J., Meijer, S. W., Farboud, S., Engels, M., Hamani, M., Null, M., Radetz, A., Hassan, U., Darmani, G., Chetverikov, A., den Ouden, H. E., Bergmann, T. O., Chen, R., & Verhagen, L. (2024). Auditory confounds can drive online effects of transcranial ultrasonic stimulation in humans. eLife, 12, RP88762. https://doi.org/10.7554/eLife.88762<br /> Sato, T., Shapiro, M. G., & Tsao, D. Y. (2018). Ultrasonic Neuromodulation Causes Widespread Cortical Activation via an Indirect Auditory Mechanism. Neuron, 98(5), 1031-1041.e5. https://doi.org/10.1016/j.neuron.2018.05.009

Reviewer #2 (Public review):

Summary:

The present paper studies the entropy production rate (EPR) before, during, and after thalamic sonication in freely moving mice from a previously published dataset. The motivation is that EPR contains information about the dynamics of neural activity that is not present in the average calcium amplitude. It was observed that the change in EPR reaches a maximum at an intermediate stimulation dosage, in contrast to the change in overall calcium amplitude, which increases monotonically with dose in the on-target condition. The paper further observes a statistically significant relation between the baseline EPR and the change in EPR during stimulation, but not for recovery, as well as a similar relation for the calcium response. The manuscript draws an analogy between the non-monotonic dependence and stochastic resonance.

Overall, these results present an interesting analysis of how acoustic stimulation affects the dynamics of neural activity. In my view, these tools would be quite useful for quantifying changes in neural activity under different perturbations.

Strength:

The study is well motivated by the argument that quantifying neural response requires information about dynamics that is not contained in the average calcium amplitude. EPR or irreversibility has been shown to be a powerful tool in describing out-of-equilibrium biological processes. The paper successfully quantifies irreversibility using an ordinal surrogate of the entropy production rate. The non-monotonic dependence is an interesting result, which demonstrates (with caveats stated in Weakness) that EPR contains more information about neural response than the average calcium amplitude. Furthermore, the paper also clearly states limitations of the approach and performs a robustness check by varying the parameters used in the ordinal EPR estimation.

Weakness:

(1) My main concern, as the authors have touched upon in the Discussion, is that although irreversibility clearly provides a readout of the neuronal dynamics, it remains unclear what its biological significance is. While it is related to susceptibility, the mechanistic link remains weak. I suspect that this interpretability issue will limit the impact of this approach.

(2) I wonder how the measured irreversibility is affected by the asymmetric response of GCaMP, which typically rises quickly and decays slowly. It seems possible that this temporal asymmetry, combined with the average calcium response, already leads to a non-monotonic irreversibility curve. The paper should investigate this and other potential contributors to the temporal irreversibility in the readout.

(3) As shown in Fig. 2AB, the baseline EPR already varies considerably with acoustic intensity, by an amount comparable to the change in EPR after stimulation (Fig. 2E). Since the baseline window precedes stimulation, this variation cannot be caused by the sonication. It suggests either that the uncertainty in EPR quantification is larger than the error bars indicate, or that trials at different nominal intensities differ systematically in some other respect. It should be examined whether the non-monotonic trend is statistically significant in light of this baseline fluctuation.

Author response:

We thank the reviewers and the editor for providing a thorough and constructive review of the manuscript. Both reviewers indicated that the ordinal entropy production rate should be validated with a synthetic (surrogate) model of the calcium signal. We agree with this, and it will feature prominently in the revised version of the manuscript.

As described (see Author response image 1), we have already implemented a synthetic GCaMP model. In the simulations, the strength of the calcium intensity varies monotonically with the intensity of tFUS. Applying our D_sym measure to the synthetic data produces a monotonic dose-response for irreversibility. It does not reproduce the non-monotonic, moderate-dose peak that we found in our study. We report this preliminary finding here because it speaks directly to the reviewers’ central critique.

Below we provide details of the planned revision.

(1) A surrogate model of GCaMP’s response to tFUS does not reproduce the observed dose structure

We built a generative model of the measured calcium signal according to the underlying physics (Author response image 1A):

(1) A latent Poisson spike train whose rate during and after sonication is monotone in tFUS dose by construction, such that any non-monotonicity appearing downstream must follow from the measurement of irreversibility;

(2) Convolution with an indicator kernel having fast rise and slow decay, matching the kinetic asymmetry mentioned by the reviewers;

(3) A static saturating nonlinearity representing GCaMP6s saturation;

(4) Additive measurement noise applied after the nonlinearity, with standard deviation calibrated to the variance of the real data’s baseline periods;

(5) baseline z-scoring, windowing, and estimation of D_sym using the same parameters as the manuscript (m = 3, τ = 8, f_s = 32 Hz; 5 s baseline, 5 s stimulation, 10 s recovery).

We ran four separate versions of the simulation (with and without saturation, noise before and after the nonlinearity). Simulated GCaMP traces for the variant with saturation and post-nonlinearity noise are drawn in Author response image 1B.

Result. All four simulation variants produce ∆D_sym that increases monotonically with dose and is largest at the highest dose (Author response image 1C). During the recovery window, the difference between moderate (2.3 to 3.7 W/cm²) and maximum (7.4 W/cm²) dose is −0.297±0.066 for the full version with the nonlinearity, −0.222±0.070 without the saturating nonlinearity, and −0.006±0.003 for the two versions in which noise precedes the nonlinearity (mean ± SD across seeds). In contrast, the D_sym of the real data (reproduced for convenience in Author response image 1D) peaks at 3.7 W/cm².

Author response image 1.

A synthetic model of the calcium measurement chain does not reproduce the observed dose structure. (A) Generative model. A Poisson spike train whose rate is proportional to acoustic dose is convolved with a fast-rise, slow-decay indicator kernel, passed through a static saturating nonlinearity, corrupted by additive noise, and then analysed with the same windowing and ordinal estimator used in the manuscript. (B) Simulated traces at low, moderate, and high dose for the variant with saturation and post-nonlinearity noise. (C) Simulated change in irreversibility during recovery, for all four variants of the simulation. Every variant increases monotonically with dose. Markers denote the mean across 20 seeds (error bars show standard deviation). (D) Real data as shown in the manuscript: observed change in irreversibility during recovery for on-target sonication. Markers denote individual animals; boxes show the mean ± 1 SEM. Irreversibility peaks at moderate dose.

(1.1) Invariance of ordinal statistics

Ordinal pattern methods are invariant under any strictly monotonic pointwise transformation of the signal, because such a transformation preserves rank order. Two consequences follow, and we will state them in the Methods of the revision:

- The static component of the indicator nonlinearity, including saturation and the relationship between calcium concentration and fluorescence, cannot alter D_sym.

- The scaling of amplitude with dose does not affect D_sym.

Any potential confound of GCaMP kinetics on D_sym enters through the dynamics, which is what the simulations above address.

(1.2) Planned analyses

In the revision, we will add:

- A sweep over rise and decay constants, Hill coefficient, noise level, and spike statistics, reporting explicitly the region of parameter space, if any, in which a moderate-dose peak can be produced;

- Burstiness and adaptation in the latent source;

- Calibrating the amplitude of the simulated responses to the real data;

- Finite-sample bias and variability of D_sym as a function of window length, pattern count, and signal-to-noise ratio, addressing Reviewer 1’s questions about window duration and noise.

(2) Aspects of the data that argue against a confound of kinetics

Independent of simulation, certain results in the manuscript are not explainable from an effect of calcium indicator kinetics.

(2.1) Non-monotonic irreversibility is reported for off-target sonication – without an accompanying amplitude response. Off-target sonication produces no appreciable fluorescence response, meaning that there is no evoked transient whose kinetics could generate ordinal asymmetry, yet irreversibility still varies non-monotonically with dose. We will elaborate on the off-target EPR effects in the revision.

(2.2) The dose structure remains after adjustment for amplitude. Our nested model comparison shows that acoustic dose explains variance in D_sym beyond baseline irreversibility and the windowmatched change in fluorescence. This is inconsistent with the view of D_sym as a deterministic readout of the amplitude trajectory. In the revision, we will also include decay time constant as a covariate, as Reviewer 1 suggests.

(2.3) Prediction from the prestimulation window. Baseline D_sym is computed on a window that contains no stimulus and no evoked transient, and it predicts the subsequent calcium response after adjustment for baseline fluorescence and dose. The kinetics of a response that has not yet occurred cannot modulate the value of a quantity measured before it.

(3) Reviewer 2, point 3: variation in baseline EPR across nominal intensity

Reviewer 2 correctly observes that baseline irreversibility varies across nominal intensity by an amount comparable to the stimulation-induced change. This implies two candidate explanations, which we address separately.

Systematic differences between intensity groups. If trials at different nominal intensities were not evenly distributed through a recording session, then drifts in bleaching, arousal, or habituation would produce spurious baseline differences between intensity groups. We tested this directly in the 408 analysed CMT 2.5 Hz trials. Session position, defined as the index of a trial within its recording, does not differ across the six intensity levels: Kruskal-Wallis H(5) = 3.51, p = 0.62 on-target and H(5) = 1.70, p = 0.89 off-target, with mean session position varying only between 18.3 and 23.3 across intensities. Within individual recordings, the rank correlation between session position and intensity has a median of +0.03 and does not exceed 0.34 in magnitude in any of the 10 recordings. Acoustic intensity is therefore not confounded with position in the session, and we will report this in the revision.

Uncertainty larger than the plotted error bars. This is a fair concern and we will address it by reporting trial-resampling and pattern-count bootstrap confidence intervals for D_sym. We also note that our primary inference already includes baseline D_sym as a covariate in an ANCOVA specification.

(4) Reviewer 1, remaining points

Other conventional GCaMP metrics. The revision will add decay time constant and additional waveform descriptors to the amplitude-adjustment models described above in 2.2.

Stimulation periods of differing duration. We will report the dependence of D_sym on window length from the finite-sample bias analysis using synthetic data (see 1.2 above).

Lower signal-to-noise recordings. We will measure the dependence of D_sym on SNR with synthetic data.

The auditory confound. We will add a Discussion paragraph citing Sato et al. (2018), Guo et al. (2023) and Kop et al. (2024), stating that the responses analysed here may reflect an indirect auditory mechanism.

eLife Assessment

This valuable study identifies the IL1R1-IRAK4 axis as a mediator of inflammation-associated ovarian cancer implantation. The evidence is convincing, supported by complementary genetic and pharmacological approaches across multiple in vivo models. These findings advance our understanding of how inflammatory signaling shapes metastatic niche formation and highlight IRAK4 as a potential therapeutic target.

Reviewer #1 (Public review):

Summary:

The manuscript describes a novel mechanism underlying the formation of secondary lesions following treatment-induced injury. Comprehensive analyses were conducted, including bioinformatics, cell culture, and xenograft models, to investigate the role of IL1b/IRAK4 in ovarian cancer. A novel IRAK4 antagonist was designed and tested in these models, demonstrating its effectiveness in reducing metastatic seeding and tumor growth at injury sites.

Strengths:

Mechanistic role of IRAK4 and its small molecule antagonist was demonstrated using several murine and human ovarian cancer cell lines.

Properties of the novel investigational compound UR241-2 were comprehensively assessed.

The data show the effectiveness of UR241-2 in reducing metastatic seeding after injury.

Weaknesses:

An expanded description of the histological types of ovarian cancer used to generate survival curves and demonstration of the expression of total IRAK4 would strengthen the manuscript.

If the information is available, it could be useful to indicate the percentages of different ovarian cancer histotypes analyzed in Figure 1.

Providing panels demonstrating the expression of total IRAK4 in Figure 4 would be informative.

Reviewer #2 (Public review):

This manuscript presents a broad and potentially impactful investigation of inflammation-associated ovarian cancer implantation and identifies IRAK4 as a candidate therapeutic node linking inflammatory signaling to tumor seeding. Major strengths include development of an injury-associated metastasis model, complementary genetic and pharmacological interrogation of IRAK4, extensive characterization of the novel inhibitor UR241-2, and incorporation of both xenograft and immunocompetent models. The observation that genetic or pharmacological disruption of the IL1R1/IRAK4 pathway preferentially affects tumor formation at injured sites rather than generalized omental disease is particularly interesting and potentially novel. However, several conclusions currently exceed the mechanistic evidence. Most importantly, the manuscript does not conclusively demonstrate that the antitumor effects of UR241-2 are mediated through IRAK4, particularly given the concentration differences between pathway inhibition and antiproliferative activity and the compound's measurable off-target kinase activity. In addition, host versus tumor-intrinsic IRAK4 functions are not resolved, the functional contribution of the altered macrophage/neutrophil populations remains unproven, and the needle-injury model should be described more cautiously as a model relevant to rather than fully recapitulating port-site metastasis. Addressing these issues would substantially strengthen the mechanistic foundation and translational significance of the work. The manuscript has potential, particularly if the authors sharpen the central claim and add experiments establishing that UR241-2's phenotypes are actually IRAK4-dependent.

Major concerns:

(1) UR241-2 decreases tumors at the injury site but apparently does not significantly decrease omental tumor burden in the syngeneic MiM model. It argues against simple nonspecific antitumor activity and supports a possible role for IRAK4 specifically within an inflammation/injury-dependent metastatic niche. The authors should make much more of this distinction-but also mechanistically prove it.

(2) The manuscript does not yet establish that UR241-2's antitumor effects are mediated primarily through IRAK4. The compound has measurable activity against additional kinases, including MAP4K2 and LRRK2, and activity against other kinases is reported at higher concentrations. More importantly, there appears to be a substantial concentration disconnect across assays. IRAK4 phosphorylation is inhibited at nanomolar concentrations in some experiments, whereas colony formation/viability phenotypes occur largely in the micromolar range. For example, colony effects are reported at 5-20 µM and viability experiments at 20-60 µM. Thus, are the antiproliferative effects observed at 10-60 µM actually caused by IRAK4 inhibition? The manuscript needs a stronger pharmacological/genetic causality experiment. Ideally, the authors should test UR241-2 in IRAK4-knockdown/knockout cells. If UR241-2 retains essentially identical cytotoxic activity after IRAK4 loss, the mechanistic interpretation would need substantial revision. A rescue experiment with WT versus inhibitor-resistant IRAK4 would be even stronger.

(3) The distinction between host IRAK4 and tumor-cell IRAK4 is insufficiently resolved. The Il1r1 experiments manipulate the host, whereas IRAK4 knockdown manipulates the tumor cell. UR241-2, meanwhile, presumably inhibits IRAK4 in both compartments. Consequently, the current experiments combine at least two mechanistically distinct possibilities: tumor-intrinsic IRAK4 versus host IRAK4. The manuscript would be considerably stronger if these compartments were experimentally separated. For example, IRAK4-deficient tumor cells implanted into WT versus pathway-deficient hosts, or pharmacological treatment of mice bearing IRAK4-deficient tumor cells, could determine how much of UR241-2 efficacy is tumor-intrinsic versus microenvironment-mediated.

(4) The immune conclusions are presently associative. The increase in MHC-II-positive macrophages and neutrophils is interesting, but describing these populations as demonstrating an "antitumor immune response" is stronger than the evidence warrants. MHC-II expression does not itself demonstrate antitumor function. Likewise, neutrophils in ovarian cancer can be either tumor-promoting or tumor-suppressive depending on context. The authors show that UR241-2 changes immune composition/phenotype. They do not yet demonstrate that these cells mediate the therapeutic effect. This could be addressed by macrophage or neutrophil depletion, functional assays, cytokine profiling, T-cell activation measurements, or potentially single-cell profiling.

(5) The drug-development claims are somewhat premature<br /> The ADMET package is useful, but several features deserve more cautious interpretation. The compound shows: very high plasma protein binding, rapid mouse microsomal turnover, evidence of efflux, relatively rapid IV clearance, and measurable off-target kinase activity. The authors report mouse microsomal half-life of only ~8.7 min compared with ~209 min in human microsomes and an efflux ratio of ~4.14. These are not fatal problems for a proof-of-concept molecule, but they make language implying a near-clinical candidate premature. UR241-2 currently looks more convincing as a lead/tool compound demonstrating therapeutic tractability of IRAK4 than as an advanced drug candidate.

(6) Exposure-response relationships need considerably more attention. Analysis of IRAK4 and/or NF-κB pathway in the treated tumors should be examined

(7) Some mechanistic observations need deeper validation. The connections among IRAK4, adhesion, E-cadherin, WNT4 and ECM remodeling are intriguing but currently somewhat descriptive. At present, several pieces of this pathway appear adjacent rather than causally connected.

Author response:

We particularly appreciate the reviewers’ detailed consideration of both the strengths and limitations of the study. The comments have helped us identify areas where the mechanistic interpretation can be further strengthened and where the conclusions should be more precisely defined.

We have carefully considered all of the points raised and provide below our provisional responses and planned revisions. We intend to address these comments comprehensively in the revised manuscript.

Reviewer #1 (Public review):

An expanded description of the histological types of ovarian cancer used to generate survival curves and demonstration of the expression of total IRAK4 would strengthen the manuscript.

If the information is available, it could be useful to indicate the percentages of different ovarian cancer histotypes analyzed in Figure 1.

Providing panels demonstrating the expression of total IRAK4 in Figure 4 would be informative.

We will expand the analysis/description of the ovarian cancer histo-types represented in the survival analyses and, where available, provide the percentages of the different histo-types analyzed in Figure 1. We will also provide additional panels demonstrating total IRAK4 expression in Figure 4. Indeed, these additions will provide greater clarity regarding the histological and molecular characteristics of the models used.

Reviewer #2 (Public review):

(1) UR241-2 decreases tumors at the injury site but apparently does not significantly decrease omental tumor burden in the syngeneic MiM model. It argues against simple nonspecific antitumor activity and supports a possible role for IRAK4 specifically within an inflammation/injury-dependent metastatic niche. The authors should make much more of this distinction-but also mechanistically prove it.

We agree that the differential response to UR241-2 at the injury site versus the omentum is an important finding and will make this distinction substantially more prominent in the revised manuscript. We appreciate that reviewer considers this pattern arguing against interpreting UR241-2 simply as a nonspecific inhibitor of ovarian tumor growth. Rather, the preferential reduction of tumor formation at the injury site is consistent with a role for IRAK4 in the establishment of tumors within an injury-associated inflammatory niche.

This interpretation is also consistent with our data that genetic IRAK4 depletion significantly prolonged survival than null control. This newly generated data will be added to the revised manuscript.

We will also clarify an important limitation our study pertaining to the omental comparison. Because the omentum is thin and multilayered, direct needle implantation does not provide the same spatial control as implantation at an injury site in the peritoneum; needle penetration may result in tumor-cell deposition beyond the intended omental compartment and complicate quantitative assessment of omental tumor burden. Thus, while we agree that the differential response may be informative, we will avoid interpreting the absence of a significant omental response as definitive evidence that IRAK4 is not involved in omental disease. This is stated so because, importantly, clinical observations have been reported indicating that surgically injured or partially resected omental tissue can support tumor-cell adhesion (Dong X et al. https://doi.org/10.1186/s13048-024-01401-8). This provides an additional rationale for considering injury/inflammation, rather than anatomical site alone, as the relevant biological feature needed for inflammation-mediated seeding. We will incorporate this evidence and provide the published literature citations to show that residual injured omentum, when present, could constitute an inflammatory niche where seeding can occur. Nevertheless, omentum is generally resected in advanced EOC (omentectomy), so it is not available for seeding during the recurrence and hence is largely irrelevant to our study theme.

(2) The manuscript does not yet establish that UR241-2's antitumor effects are mediated primarily through IRAK4. The compound has measurable activity against additional kinases, including MAP4K2 and LRRK2, and activity against other kinases is reported at higher concentrations. More importantly, there appears to be a substantial concentration disconnect across assays. IRAK4 phosphorylation is inhibited at nanomolar concentrations in some experiments, whereas colony formation/viability phenotypes occur largely in the micromolar range. For example, colony effects are reported at 5-20 µM and viability experiments at 20-60 µM. Thus, are the antiproliferative effects observed at 10-60 µM actually caused by IRAK4 inhibition? The manuscript needs a stronger pharmacological/genetic causality experiment. Ideally, the authors should test UR241-2 in IRAK4-knockdown/knockout cells. If UR241-2 retains essentially identical cytotoxic activity after IRAK4 loss, the mechanistic interpretation would need substantial revision. A rescue experiment with WT versus inhibitor-resistant IRAK4 would be even stronger.

We agree that establishing pharmacological causality is central to interpreting UR241-2’ s mechanism of action and designating it as a drug-like candidate. In particular, the reviewer appropriately points out that biochemical inhibition of IRAK4 occurs at lower concentrations than some of the antiproliferative and colony-formation phenotypes and that UR241-2 has measurable activity against additional kinases. However, nanoBret assay (Figure-2M) identifies UR241-1 as a very selective IRAK4 kinase inhibitor. The homologue IRAK1 is inhibited at ~1092-fold higher doses than IRAK4. Similarly, FLT3, MAP4K1, MAP4K2, MAP4K3, MAP4K5 and LRRK2 are inhibited at over 18-800 folds. We agree that doses of UR241-2’s at which kinase inhibition and anti-proliferative actions occur do not match. We will attempt to carefully distinguish the concentrations required for direct IRAK4 pathway inhibition from those producing broader cellular phenotypes and will strengthen the pharmacological-genetic evidence linking UR241-2 activity to IRAK4. We will specifically address whether the cellular effects of UR241-2 are diminished when IRAK4 is genetically depleted or otherwise functionally absent. These analyses will help determine the extent to which the higher-concentration antiproliferative effects can be attributed to IRAK4 versus potential off-target activities.

We will also revise the manuscript so that conclusions regarding UR241-2 mechanism are proportional to the available evidence and do not imply that all cellular effects at higher concentrations necessarily reflect IRAK4 inhibition.

(3) The distinction between host IRAK4 and tumor-cell IRAK4 is insufficiently resolved. The Il1r1 experiments manipulate the host, whereas IRAK4 knockdown manipulates the tumor cell. UR241-2, meanwhile, presumably inhibits IRAK4 in both compartments. Consequently, the current experiments combine at least two mechanistically distinct possibilities: tumor-intrinsic IRAK4 versus host IRAK4. The manuscript would be considerably stronger if these compartments were experimentally separated. For example, IRAK4-deficient tumor cells implanted into WT versus pathway-deficient hosts, or pharmacological treatment of mice bearing IRAK4-deficient tumor cells, could determine how much of UR241-2 efficacy is tumor-intrinsic versus microenvironment-mediated.

We agree that this is an important mechanistic distinction. The current experiments interrogate different compartments: IL1R1 manipulation primarily addresses the host inflammatory environment, whereas IRAK4 depletion in tumor cells addresses tumor-intrinsic signaling. UR241-2, in contrast, can potentially affect IRAK4 in both compartments.

We will therefore more explicitly separate these mechanisms in the revised manuscript. In particular, we will examine the available genetic and pharmacological data in the context of a model in which host IL1R1/inflammatory signaling establishes a permissive injury-associated environment, while tumor-cell IRAK4 contributes to tumor-cell responses within that environment. Where feasible, we will incorporate additional experiments to better distinguish tumor-intrinsic from host-mediated contributions to UR241-2 activity.

(4) The immune conclusions are presently associative. The increase in MHC-II-positive macrophages and neutrophils is interesting, but describing these populations as demonstrating an "antitumor immune response" is stronger than the evidence warrants. MHC-II expression does not itself demonstrate antitumor function. Likewise, neutrophils in ovarian cancer can be either tumor-promoting or tumor-suppressive depending on context. The authors show that UR241-2 changes immune composition/phenotype. They do not yet demonstrate that these cells mediate the therapeutic effect. This could be addressed by macrophage or neutrophil depletion, functional assays, cytokine profiling, T-cell activation measurements, or potentially single-cell profiling

We agree that the changes in macrophage and neutrophil populations are currently associative, and do not by themselves establish that these cells mediate the therapeutic response of UR241-2. MHC-II expression, for example, indicates a change in macrophage phenotype but is not sufficient evidence of antitumor activity, and neutrophil function can be context-dependent.

We will therefore revise the terminology to distinguish changes in immune-cell composition or phenotype from demonstrated immune-mediated tumor suppression. We will integrate these findings more appropriately into the inflammatory-niche model and will limit causal conclusions regarding macrophages and neutrophils unless supported by additional functional evidence.

(5) The drug-development claims are somewhat premature

The ADMET package is useful, but several features deserve more cautious interpretation. The compound shows: very high plasma protein binding, rapid mouse microsomal turnover, evidence of efflux, relatively rapid IV clearance, and measurable off-target kinase activity. The authors report mouse microsomal half-life of only ~8.7 min compared with ~209 min in human microsomes and an efflux ratio of ~4.14. These are not fatal problems for a proof-of-concept molecule, but they make language implying a near-clinical candidate premature. UR241-2 currently looks more convincing as a lead/tool compound demonstrating therapeutic tractability of IRAK4 than as an advanced drug candidate.

We agree that, although the ADMET characterization of UR241-2 is comprehensive and addresses key parameters relevant to drug development, its potential as a drug candidate should be interpreted cautiously.

Importantly, the available data supports appropriate cellular selectivity for IRAK4. Although the initial HotSpot kinase panel identified additional kinase interactions, most of these activities were substantially weaker than IRAK4 inhibition and were effectively dialed out in the cell-based NanoBRET assay, with target engagement generally requiring approximately 50–1000-fold higher concentrations than those required for IRAK4. Thus, the NanoBRET data provides a more physiologically relevant assessment of cellular target selectivity and supports preferential engagement of IRAK4 by UR241-2 at pharmacologically relevant concentrations.

We also interpret the high plasma protein binding of UR241-2 in the context of its intended pharmacological profile. Rather than necessarily representing a liability, extensive plasma binding may be compatible with development of a formulation or dosing strategy designed to provide sustained, controlled exposure. This could be advantageous for IRAK4, an important component of innate immune signaling, because prolonged and complete suppression may increase susceptibility to infectious complications, particularly in patients with advanced or treatment-exposed EOC who may already have compromised host defenses. Accordingly, a sustained but incomplete degree of IRAK4 inhibition may be preferable to continuous, complete target blockade. This hypothesis will require direct validation during further drug-development studies.

The relatively short microsomal half-life observed in mouse microsomes is recognized as a limitation for preclinical development and may complicate exposure and toxicity studies in rodents. However, UR241-2 demonstrated a substantially longer microsomal half-life in human microsomes (209 min) (the intended species), which supports further consideration of the scaffold for human drug development. Collectively, these findings support UR241-2 as a pharmacological proof-of-concept and a tractable lead scaffold, while acknowledging that additional medicinal chemistry and pharmacokinetic optimization will be necessary to establish a suitable development candidate

However, we will revise the manuscript to position UR241-2 as an investigational lead/tool compound demonstrating the therapeutic tractability of IRAK4 while clearly acknowledging its current development limitations. We will also distinguish proof-of-concept efficacy from the properties required for progression toward a clinical candidate.

(6) Exposure-response relationships need considerably more attention. Analysis of IRAK4 and/or NF-κB pathway in the treated tumors should be examined

We agree that linking systemic exposure to tumor pharmacodynamics would strengthen interpretation of the in vivo findings. We will further examine the available tumor samples and pharmacodynamic data for evidence of IRAK4 and/or NF-κβ pathway modulation following UR241-2 treatment and relate these findings, where possible, to compound exposure and tumor response.

This analysis should help distinguish target engagement and pathway modulation from nonspecific effects occurring at higher systemic or tissue concentrations.

(7) Some mechanistic observations need deeper validation. The connections among IRAK4, adhesion, E-cadherin, WNT4 and ECM remodeling are intriguing but currently somewhat descriptive. At present, several pieces of this pathway appear adjacent rather than causally connected.

We agree that these observations currently provide a mechanistic framework but that some relationships are not yet established as directly causal. We will therefore more clearly distinguish findings that demonstrate pathway activity from those that represent downstream associations.

In particular, we will strengthen the discussion of how IRAK4 signaling may influence tumor-cell adhesion and establishment following tissue injury and will avoid presenting changes in E-cadherin, WNT4, or ECM-associated factors as independently proven causal steps unless directly supported by the data. Where appropriate, additional analyses will be used to clarify the relationship among these processes.

We appreciate the reviewers' careful evaluation of the manuscript. The comments have helped us sharpen the central model: that tissue injury creates an inflammatory environment that promotes tumor establishment and that IRAK4 represents an important signaling node within this process. The revised manuscript will strengthen the mechanistic evidence for this model, clarify the distinction between injury-associated metastatic establishment and generalized tumor growth, and more appropriately define the translational potential and limitations of UR241-2.

https://journals.plos.org/ploscompbiol/article/authors?id=10.1371/journal.pcbi.1014707

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a rare and progressive nervous system disease that destroys nerve cells in the brain and spinal cord, leading to a loss of muscle contro

annotation!

this is a test

these are prtty cool, but technically it would be all three? xD might need to rethink what we say in the regualr banner.