1st choice please (and I seem to be seeing students from 2024/2025? if that's no issue, doesn't bother me, just noting) OOPS- TMFritz= Tania Fatovich

This isn't too far from where her and her family had been staying, I wonder if they've settled close to a native society?

Harsh language for sure. I don't like the choice of describing native peoples as "barbarous creatures", but if I were in her position I don't know if I would be thinking about the implications of my presence on these people's land. I would probably also curse whomever kidnapped and beat me.

★ Key idea: Spanish = private/family

★ School ↔ Home

Eliot says that “winter kept us warm,” “covering the earth in forgetful snow,” showing that people felt at peace during winter. Winter is usually associated with depression and slumber, so people may actually feel at peace when they do not have to see reminders of better days. People probably didn’t have very good memories of winter – there was famine, crucial battles, cold, and suffering – but when spring comes, it instantly uncovers the beauty that the world once had. It makes people remember the good things they experienced in the past, so nostalgia hits. It is much harder to be happy when you have good memories that were taken away than when you had nothing in the first place (like winter). This is how the shift from winter to spring feels for many people, especially when spring is no longer the same as it was before.

Going a little bit ahead, in Ezekiel 37 and Ecclesiastes, we can see how important religion is as a source of spiritual fulfillment. The valley of dry bones in Ezekiel 37 symbolizes the Israelites, who have lost their hope and feel as though they are spiritually and physically dead. This connects to Eliot because he also emphasizes the emptiness of people and their loss of spiritual connection. In Ecclesiastes, religion and God are also emphasized, showing the importance of spiritual fulfillment. Worldly things do not ultimately matter because they are temporary, what matters is spiritual meaning. Eliot also explores the possibility of renewal at the very end with “Shantih,” which means “peace”. If people actually begin to change and act, perhaps spiritual renewal is possible (it is not clear to me yet what exactly Eliot means by this, but it could be something like a higher power or a spiritual truth (like the unattainable holy grail) that people need to find within themselves in order to restore the world and save the Waste Land. In this sense, the Waste Land may not be completely hopeless: there is still the possibility of renewal if people can recover some form of spiritual connection)

This line says in German, "I am not a Russian woman at all, I come from Lithuania, genuinely German." This line shows the modernist desire to keep poetry impersonal while still telling a meaningful story. Translated into German, it tells the story of the shifting borders and national identities in Europe around World War I, but only those familiar with that historical context would fully understand it.

The character Marie may connect to Countess Marie Larisch, who grew up in Bavaria, Germany. In her memoir, she describes being the daughter of Duke Ludwig of Bavaria and the niece of the Empress of Austria. These two identities, which come from different parts of Europe, are reflected in the line "genuinely German," as she must explain her complicated background, especially after World War I. She was Bavarian by birth, connected to Austria by family, and part of a European aristocratic world, so she didn't fit into the modern categories of nationality.

I find this reference to Marie Larisch quite interesting as I think it really connects to the ideas of the ideas of impersonality which we discussed in class today. Here, Elliot embodies the real life countess, Marie Larisch, thus separating himself from the poem, in a style quite different from classical poetry. I think that this choice is deliberate, as with many others in the opening of this poem to establish that it is a departure from classical poetry, and a step into modernism. This can also be seen with parallels to other poems such as how Elliot subverts The Canterbury Tales opening by describing april as the cruelest month as opposed to a time of rebirth and new life. Ultimately, I think these deliberate choices lead to an interesting opening to the poem which displays how Elliots writing differs from that of classical poets.

"When the boys said to her: 'Sibly, what do you want?' She answered: 'I want to die.' In relation to My Past, by Countess Marie Louise, the English rendering of The Service for the Burial of the Dead...of the Orthodox Greek Church in London, Sibly's plea for bodily liberation transforms the body into a physical vessel that confines the human soul—the soul being the human's godly half (or fragment of god). Throughout all texts, the soul seems to exist attached to rather than held within the human: lines 25-29 of "The Burial of the Dead" state that, "There is shadow under this red rock/ (Come in under the shadow of this red rock),/ And I will show you something different from either/ Your shadow at morning striding behind you/ Or your shadow at evening rising to meet you..." 'Shadow,' is referred to as a definite object; rather than the product of one's body, 'shadow' is a place for the body to take refuge within.

Further, Tone 2 of the "The Service for the Burial of the Dead..." illustrates a soul departing from its body: "Alas! what strife hath the soul in parting from the body. Alas! what tears, and there is none to pity her; towards angels her helpless eyes are turned in supplication...let us plead with Christ for rest for him who has passed away, and for our souls the great mercy." As the soul is described separate from the body, the female pronoun "her" is attached. She, "turned in supplication," asks for the great mercy (assumed to be entrance to heaven, or a similar plane). The female soul, possibly a symbol of virgin rebirth, awaits return to God, and return to sanctity.

Overall, as suggested in the final lines in the Book of Ezekiel, all texts suggest that the human body is a subaltern vessel, and that the soul is the only kin of God (or the child or product of God).

This is a direct quote from the beginning of Wagner's opera, Tristan und Isolde: a sailor's song, singing about the wind blowing fresh towards home and asking an Irish girl where she was lingering. This song is performed at the beginning of the story, before the fateful encounter of Tristan and Isolde, the couple's passionate and consuming love affair. Eliot places the quote right after the memory of the hyacinth garden which follows. It's a scene full of romance and eroticism and, interestingly enough, one which ends not in consummation but in the speaker's utter silence and paralysis ("I knew nothing... the silence"). The Wagner quotation sets up an expectation of grand romantic union that the poem's own scene refuses to deliver. It's the same technique as the "Shakespeherian Rag" moment later; high art quoted directly, then undercut by what actually happens around it. Here the gap isn't ironic so much as devastating: real operatic passion promised, and then only emptiness delivered in its place.

This particular quotation stood out to me because of its implications when read alongside The Service for the Burial of the Dead According to the Use of the Orthodox Greek Church. According to this book, death is a passage from one state of existence into another instead of an absence or destruction of life. To reflect this, the burial service becomes a ritual. However, the Sibyl in Eliot's chosen quote (taken from Petronius’s Satyricon) experiences the complete opposite of this condition of "death." She has lived for an unnaturally long period of time, and she is suffering to the point where she feels trapped by her own existence and wishes to die. Contrary to the assumption that death is what brings suffering, Sibyl's anguish stems from her inability to reach closure or transition into a new state of being. This reflection upon the purpose of death lends insight into Eliot's deliberate choice of this epigraph over a quote from A Heart of Darkness by Joseph Conrad. The passage Eliot wanted to select focused on the antagonist Kurtz's death. It appears that Eliot originally wanted to choose something that encapsulated the final moments of death and the meaning concentrated in them, but eventually pivoted and decided to publish something that was the complete opposite. This sets the tone for the rest of the poem. In The Waste Land, death is not final but more so a suspension between states of existence. This condition of suspension is what distinguishes Eliot's understanding of death from that of an Orthodox burial. The latter encourages movement and resolution, but The Waste Land constantly raises fragments of experiences without allowing them to settle, dragging them along as the poem progresses. By using Sibyl to introduce this idea, Eliot introduces a more disturbing understanding of death. It is a state without ending, or in this case, a world without teh ability to change or begin again.

wth? I guess we're not going to cover this original problem??

I see part 2 where the photographer snaps a photo at 20 feet and makes a 3/4 rotation, but are we not going to solve this?

sqrt(3)

the

This would have been better worked out as an example

I am curious as to what it will discuss with these tree topics as a student.

Self-discipline is the real key to success.

Concluding sentence.

supporting evidence note

I tend to skim also. Annotation helps me engage more.

I cannot tell what temperature I would prefer. I might just have to try both warm and cold.

I find it interesting that this article mentions temperature. I didn't realise that studying comes down to science.

i also have to do this because of my work schedule. So my availability is blocked out for certain days; I have study time.

i can attest to having to plan my social life around my academic success.

adding why they are important is a helpful supporting sentence so we know exactly why we are discussig this subject.

I see they are stating every individual idea discussed in this article right at the beginning. It's very straight to the point.

This article is a perfect fit for me, as I am an extremely anxious student. It's also a perfect example of a clear main idea.

8:07–9:32 When Dustin Ballard breaks down the three things people should think about when using AI in music, I agree. He discusses whether it is deceptive, whether there is artistic intent, and how it affects musicians. I think these are important because artificial intelligence can be a good creative tool but people should be honest about their use of it and think about if it could take opportunities away from real musicians.

4:02–4:39 I found this part interesting because Ballard explained that the song was created entirely by AI from a text prompt. He also mentioned the strangeness of hearing emotion and pain in the voice of someone who never really existed. This made me think of how artificial intelligence can make something sound really real despite there being no real person behind the music or emotions.

2:16-3:14 I was fascinated to hear Dustin Ballard say that the story about the old blues singer and the song was created entirely by AI. At first I thought the story was true because the music sounded real and emotional. When he told us it was AI, I thought to myself, does knowing how music was made change how we feel about it? I think it does, because I felt differently when I found out that the singer never really existed.

m

This part shows how the introduction of sanitation and better cleaning policies helped reducing the spread of infectious diseases. The main health concern then shifted to chronic illnesses.

This section shows how the health issues shift over time. As infectious diseases become less common, things such as cancer may become more popular.

3.) Three ways to make up for missing notes are to ask a classmate for thier notes and ask the teacher for missed infornmation or handouts or check the textbook powerpoint or online class matirials.

2.) Benifits and potentical problums with taking class notes on a laptop are one benifits you can typr quicly keep notes organized and easliy edit them and problums are laptops can be distrating and you may type too much insted of focusing on the importante information

1.) Two advantages of the cornell system is that it helps organize notes and highlight key concepts two it makes noets easier to review an study later

3.) They would one put it on the board or post it somewhere and would say it.

2.) you should seat where ever your assign seat. Because if you have a assign seating then you proceed to sit somewhere else you will get in trouble.

1.) get out needed martirilals, get seated and wait for activites.

Some things professors do to signal important material are repeat it and emphasize it.

I would sit somewhere where you could focus. This may mean sitting away from friends so you don't get distracted.

Two things you can do to prepare for active listening are to turn your cell phone off and review notes from the pervious class.

whoever started this is getting slimed by me personally

table has so much aura

me with my amethyst and jade frfr

ew

bro was trying to "help" on the group project

important

I was hoping this would be included in the anthology, it's always nice to see what people who analyzed the text before you were thinking.

Especially because America itself is such a diverse nation built on ideals, questioning what America even is is more of a valid question than it might appear at first.

That's us!

Fun, fun.

True, but is this not an early American lit course? Why would we focus on change? Unless we're talking about how the definition changes constantly.

Who would not expect any of these texts to be included in an anthology about American literature? Certainly not our class, as we've already blown to shreds any preconceived ideas one might have about what American literature is.

Shared identity is probably the single most important aspect of literature.

It seems that for the purpose of this class, we are identifying America as North and South America, along with any American territories, to get as broad a scope as possible.

No one person or group can define what American literature is, there are simply too many different perspectives to consider.

How does it give us the tools to think critically on current events? Are there sections that include other students dissections of texts (not including those made by our class on hypothes.is), or are we given the tools for critical thinking simply by reading the texts ourselves?

What sets this apart from other scholarly conversations on literature? Don't most scholars recognize things like film, music, and other art to be literary?

Diverse how? Diverse ethnically? Religiously? Geographically? Sexually?

As we discussed in class, this is important because of the lack of said voices being taught in primary and secondary schools.

Are there not a lot of these? I'm assuming not. Regardless, it's important that we hear from student voices on what literature should be taught, because the things that made an impact on people our age in recent classes are likely to make an impact on us as well and inspire us to keep learning.

It allows us to understand the events that shaped our country and led to the social and political climate that exists today.

In class, I think we touched on this already. History is a study of events as they happened, while the study of historical literature studies the author's perspective more heavily.

We also question ideas like what makes an author American, and I'd say that it runs deeper than asking what "counts" as American literature. It's about how far we can push the boundaries, and if American literature even exists. If Native American literature can't exist because of the many cultures and areas that could be considered Native American, why can American literature exist when we end up running into the same issues?

This is a long paragraph, consider starting a new paragraph here?

would this section be better titled as Healthcare Practitioners, technologists and technicians?

proactively inquire

I found this statement interesting, as the substitution of Huck and Jim as the ferryman instead of Charon shows that even with his brain as misfiring electricity, he still thought of a story he loved, before resorting to religious or taught context. This suggests that comfort and identity aren't based on what we're taught, but what we love and truly appreciate.

would be helpful if we had more standard python in sanbox..

eg a natural way to answer would be to loop :

train_data can be 'Train_small', 'Train_big', 'Train_tiny'

features should be a list of strings. Features to experiment with are listed below.

['Count_Person'],

['Percent_Person_WithHighBloodPressure'],

['Percent_NoHealthInsurance'],

['Median_Income_Person'],

['Commute_Time']

def run(): features_to_try = ['Count_Person', 'Percent_Person_WithHighBloodPressure', 'Percent_NoHealthInsurance', 'Median_Income_Person', 'Commute_Time'] best_feature, best_mse = None, float('inf') for f in features_to_try: mse = test_analytic_regress(train_data='Train_small', features=[f]) print(f, mse) if mse < best_mse: best_feature, best_mse = f, mse print('best:', best_feature, best_mse) return test_analytic_regress(train_data='Train_small', features=[best_feature])

call this "aggregate cost" or so ?

La célula tiene sensores de defensa que detectan ADN y ARN en el citoplasma porque pueden interpretarlos como una señal de peligro, similar a lo que ocurre durante algunas infecciones virales. Al activarse estos sensores, se pone en marcha la inmunidad innata y se producen moléculas inflamatorias.

En forma sencilla:

Envejecimiento → ↓ heterocromatina → ADN/ARN anormal en citoplasma → activación de sensores de peligro → respuesta inmunitaria → inflamación crónica

A esa inflamación leve, persistente y asociada al envejecimiento se le llama inflammaging (inflammation + aging).

ACLY y ACSS2 → producen acetil-CoA → acetilación de histonas → cromatina más abierta → mayor expresión de ciertos genes

Consider moving this above to replace 1.3 and move 1.3 in this area.

This is labeled as a nursing career ladder but it shows OTA, not nursing

organic compounds characterized by multiple hydroxyl groups (-OH) and an aldehyde functional group (-CHO) at the terminal carbon of their structure.

This timeframe is nearly expired. Is there more current data?

Consider adding CDA= Certified Dental Assistant to this list

In most states, dental assistants can be trained on the job with no formal certificate required

consider adding how much training is needed to achieve the medical professional designation/ credential

in this chapter.

It seems that the equation is missing.

mary is huge bruh

light work no reaction

the chokehold of colord theory bro

brings me back to my art days bro

actually on this unit rn in chem lol

green of the water are we fr

thats pretty interesting lowkey

why were they gatekeeping a snail bro

I like to do this so I can read the section and grasp what the author is saying and make my own annotations before reading what everyone else has to say about the section that I just read. This way I can keep my thought more organized and on topic since I'm not going back and forth between sections.

I like getting to read annotations for this reason. I get to see what other people are thinking when they read something and I feel like it helps me to widen my own perspective by understanding someone else's thoughts.

I love having annotations in my Bible because very often when I have a question about something, there's an answer just like this.

I've always done annotations with other people over a reading like this, but I've never really thought of it as a group thing or that there was much social about it, so this is an interesting perspective.

great way to describe position as a vector

So cool.

I like how modern technology was able to make these new findings, but its also sad that these records were maybe so casual that they didn't really need to be excessively tracked and accounted for.

Overall, I really like how they tied in slavery to incarceration, and showed the trends of post slavery/segregation era, to now.

Still shows how important the parks are.

I remmeber hearing about this and thought it was very bad for our environment and conserving our land for future generations.

NNo entiendo por qué se parte de esta pregunta. La pregunta del poder es en realidad qué tamaño muestral me permite detectar un efecto, dado un diseño específico. Parece un documento genérico y no orientado al diseño específico para el cual se requiere el análisis de poder. Creo que debería ser más conciso, explicando de manera simple paso por paso, y finalmente explicar de manera clara lo de las consecuencias para el tamaño muestral de cada decisión.

What happened to the theta^R notation?

This problem doesn't make clear that the satellite is rotating around the central point of the Earth and that RADIUS of the satellite, here, means R_E+h_S. Not to mention, that the Earth is not perfectly spherical, or circular in our 2-D model that doesn't include the picture. This problem is such a leap from the previous question and information that it's a wonder anyone can get it.

The directions for solving these problems puts these measurements in radians/unit time.

test annotation

test 1

test

Making these connections more efficient, the myelin sheath speeds the communication signal of the nerves

Test

"Design is a social activity." I found this interesting because design is not only about making something look good. Designers work with other people and create things for an audience, so design can have an impact on society.

Theory is all about the question why? This stood out to me because designers usually focus on how to make something, but theory makes us think about why we are making it. I think asking why can help us make better design decisions.

This question made me think about how some people can be left out of design. I think designers should consider different people and make sure their designs are accessible and inclusive.

This stood out to me because I think design can be connected to social change. Designers do not always agree, and debate can help people see different perspectives.

Plato. 1952. 7 Plato. 1st ed. eds. Robert M. Hutchins and Mortimer J. Adler. Chicago, IL: Encyclopedia Britannica, Inc.

Thinking "if I do this closer to the test, it will be at the forefront of my mind". Also, fear of failure.

Challenges: 1. Time Management 2. Personal Challenges 3. Over committing 4. Perfectionsim 5. Lack of intrest

• Dilution and Confusion of the FDE Role

  • The Forward Deployed Engineer (FDE) has emerged as one of the most critical roles across frontier AI labs and startups, yet the title is frequently misapplied to sales engineers, solutions architects, or outsourced consultants.
  • The core distinction: consultants solve isolated customer problems that leave no lasting asset, whereas true FDEs solve last-mile problems specifically to feed insights back into the core platform.

• The End of Pure Out-of-the-Box SaaS

  • The repeatable 80% of generic enterprise software has largely been commoditized and solved.
  • Modern software value has migrated to the remaining 20%—the undocumented, messy, institutional workflows and edge cases that cannot be captured via standard discovery calls.

• Bridging the Gap Between Design and Production Reality

  • Theoretical system design consistently fails when confronted with messy real-world data (illustrated by Palantir’s Phoenix store OOM failure caused by blank timestamps defaulting to the Unix epoch).
  • FDEs close this reality gap by embedding directly within customer environments, directly observing data pipelines, exceptions, and institutional workarounds.

• Organizational Alignment and Incentives

  • FDE organizations should report into Product/Engineering rather than Sales.
  • Aligning FDEs with product development ensures custom fixes translate into reusable core capabilities instead of ad-hoc, unmaintainable client forks.

• The Durable Moat in Enterprise AI

  • Foundational models are depreciating commodities and talent is mobile; the actual defensible moat is the accumulated, battle-tested knowledge of customer operational workflows.
  • Each deployment cycle that absorbs domain errors and converts them into platform features systematically lowers the cost and latency of every subsequent deployment.

https://en.wikipedia.org/wiki/Pithus

https://topostext.org/

Think of your phonebook, the server is like your phonebook, it gives you accses to the peoples' numbers and names (Just like the internet). Typing an URL in the search bar is akin to finding the name of the person you want to contact in your phonebook through the index. Person's name acts as the wbsites domain and the IP adress is like the specidifc print number of your phone book(That might be a strecth). A hyperlink on the otherhand, acts as magical link between two locations. In a phonebook a hyperlink could be a the number of the person you're trying to contact, but once you touch the number with your hand (the mouse) it immedeatly contacts them.

This is key for our students!

How do you accomplish this in modernity, when you can't "get rid" of the opposition? You chill their speech, you erase their language, you erase their history. This seems to be the playbook of the far right in American politics under Donald J. Trump.

Redefine and poison words like DEI and Critical Race Theory (CRT), marginalize and criminalize the people who don't fit in your world view.

I think he is the only one of our public men to start out the right way, for it is right to care first that the young

Cross reference: Teach Your Children Well by Crosby, Stills, Nash & Young

Euthyphro<br /> Stephanus numbers 2a–16a<br /> G. M. A. Grube translation

in <br /> Plato. 1997. Plato: Complete Works. eds. John M. Cooper and D. S. Hutchinson. Indianapolis: Hackett Publishing Company, Inc.

cf. with Benjamin Jowett translation:

A young man who is little known, Euthyphro; and I hardly know him : his name is Meletus, and he is of the deme of Pitthis. Perhaps you may remember his appearance; he has a beak, and long straight hair, and a beard which is ill grown.

There is certainly a charge of "youth" in both translations, but the "beak" and "ill-grown beard" seem to be more belittling in Jowett which be ad hominem appearance attacks on Meletus of Pithos before we've even heard his argument.

Which of these descriptions would have had cultural significance in Ancient Greece?

See: Pithos, Pitthos, Pithus: https://topostext.org/place/380239DPit

Of course if this is the aim, then one also ought to define "morally good" as cleanly as piety, something which might be elusive or which may vary based on an individual person, that person's culture, or even across cultures.

Plato. 1997. Plato: Complete Works. eds. John M. Cooper and D. S. Hutchinson. Indianapolis: Hackett Publishing Company, Inc.

Use this as a basis for comparison when writing your own paper. Also use the template above it that is also highlighted.

test

https://topostext.org/place/380239DPit

cf. https://en.wikipedia.org/wiki/Pithus

I've never heard of this type of literature so it's fascinating to learn about.

It's ironic to me how Henry VIII tried so hard to ensure that a male heir would succeed him, just for it to be his daughter that would reign for longer.

Is this where that nickname comes from??

King at 9 years old??

This is crazy. He was literally going around and punishing his wives because they didn't produce an heir...as if it was their choice.

Of course it's love and marriage that could end a war between royal dynasties.

This is the only reason I know of Henry VIII...

I wonder if these symbols are still used to represent these counties today?

I don't think this is intuitive when you say "each button" are you talking about the flow chart or the sections below the flow chart? when I click "what is open science" in the flowchart it doesn't take me anywhere

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

Manuscript number: RC-2024-02737

Corresponding author(s): King, Megan

1. General Statements

We thank all reviewers for their comments and recommendations.

In this revised submission we outline compelling evidence that the critical window for polymerase theta (Pol theta) activity is just prior to the G2/M transition, downstream of PLK1 activation but upstream of full CDK1 activation; we arrive at this new focus through our mechanistic dissection of how loss of the DNA repair and replication factor DNA2 compromises Pol theta-mediated end joining (TMEJ). Although a direct role for PLK1 in licensing TMEJ was recently reported, the interpretation of prior studies was that TMEJ repair is restricted to mitosis. Instead, our results highlight that the window in which TMEJ drives most repair is in late G2 during the period of mitotic commitment when PLK1 activity begins to build. In this revision, we provide further evidence to support this key observation, as we now demonstrate that the Aurora A kinase is likewise required for efficient TMEJ. We now propose that during the period of mitotic commitment (but crucially prior to mitosis) Aurora A (not CDK1) primes PLK1 to become active, driving TMEJ competence. This finding is important because although it reinforces that TMEJ is tied to mitosis, it highlights that most TMEJ repair likely occurs prior to mitotic entry itself. We provide further evidence that a minor subset of TMEJ repair events require CDK1 activation and occur in mitosis, but this is largely restricted to complex repair outcomes called templated insertions. Mechanistically, we suspect that these templated insertions events require Pol theta’s ATPase activity (in addition to polymerase activity) and may constitute as second, delayed stage of TMEJ function that drives more complex DSB repair products during mitosis.

In preparing this revision we have made major changes to the manuscript necessary to provide clarity; this includes the removal of data using genetic or pharmacological perturbations to the BLM helicase. Although we observed similar consequences of BLM knock-down or inhibitors on the cell cycle (leading to primarily a G1 arrest) on TMEJ proficiency, we were unable to rescue the genetic effect with a complementation construct while the pharmacological inhibitors may not be sufficiently specific. We therefore felt it best to remove this aspect of the manuscript, which now instead focuses on the cell cycle regulation of TMEJ.

2. Point-by-point description of the revisions

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

*Summary *

*DNA double-strand breaks can be repaired by NHEJ, TMEJ, or HR. While there have been many studies of the factors that regulate double-strand break repair pathway choice, the exact mechanisms by which TMEJ is regulated remain under active study. In this study, McBrine and colleagues conducted experiments in two human cell lines to investigate the contribution of DNA resection to TMEJ. After induction of Cas9 breaks at a single locus, they used NGS amplicon sequencing and a pre-existing computational pipeline to quantify repair by inaccurate NHEJ or TMEJ. Their initial data suggested that loss/inhibition of the BLM DNA helicase or DNA2 nuclease inhibit resection and thereby decrease TMEJ repair. Probing further, they conducted carefully controlled cell cycle analysis and determined that loss of either BLM or DNA2 slows progression to the G2/M transition, when TMEJ is activated through PLK1 phosphorylation of polymerase theta. Based on these findings, they conclude that both BLM and DNA2 promote TMEJ repair, likely by promoting efficient replication and timely progression into G2/M. *

*Major comments *

Overall, the authors' conclusions are well-supported by their data. They used multiple approaches to validate their findings, including the use of siRNAs and small molecule inhibitors that largely produce similar results. However, three major additions/experiments should be included.

1. • First, most of the data from the NGS sequencing results are displayed as TMEJ/NHEJ ratios. While this makes it easy to compare between different experimental conditions, it could mask differences in overall repair efficiency. I recommend that the authors include the initial percentages of TMEJ-like and NHEJ-like repair junctions in supplemental information, so that readers can determine the efficiencies of the different repair pathways in each condition. * We agree with the Reviewer that there is additional information that can be gleaned from the NGS sequencing approach. In the revised manuscript, we now include the full percent of outcomes interpreted as TMEJ and NHEJ (as percent of total sequences) as side-by-side bar graphs in the Supplemental Material for all data (Supplemental Figure S4). As appreciated by the Reviewer, we have retained expressing the TMEJ:NHEJ ratio in the main text for clarity.

2. Second, the DSB repair spectrum (as displayed in Figure 1B) should be provided for each TMEJ:NHEJ plot in supplemental information, at the very least the for knockdown/inhibitor experiments where a significant difference in TMEJ:NHEJ ratio is observed. *

We agree with the Reviewer and now include a representative DSB repair spectrum for each experiment in the Supplemental Material of the revision (Supplemental Figures S1, S2), selecting an experiment that falls in the middle of the distribution from all biological replicates.

• Finally, the conclusion that transition to the G2 stage (where polymerase theta becomes activated through phosphorylation by PLK1) is necessary for TMEJ is logical, based on the data and previously published studies. However, to provide additional support for this model, the authors should consider repeating the experiments from Figure 5B/5C using cells with the PLK1 phosphorylation sites on polymerase theta mutated (e.g., using the 10A construct from Gelot et al., doi.org/10.1038/s41586-023-06506-6). The expectation is that the TMEJ reduction observed with BLM or DNA2 inhibition should not decrease further, if indeed the decrease is largely due to an inability to progress to the point in the cell cycle where polymerase theta is activated by phosphorylation. This experiment should be able to be completed within 1-2 months, if the appropriate cell line can be obtained. *

While we appreciate the intent of the Reviewer’s question, given the consensus discussion between all three reviewers that this was a challenging yet unessential experiment, we have declined to take on this line of questioning as part of this study. We do, however, now provide further evidence (and depth) for this model by demonstrating that Aurora A inhibitors also compromise TMEJ (Figure 5).

*Minor comments *

• The cell cycle plots in Figures 4 and 5 are difficult to interpret given the current color scheme (especially the difference between S and G2). I suggest changing the color scheme and arranging the legend so that the key is in the same order as the plots (G1 on the bottom, S in the middle, G2 on the top).*

We have re-ordered the cell cycle legend and adjusted the colors as suggested.

• In Figure 3B, the resection time course is interesting, but I wondered why they are no time points between 12 and 24 hours? Also, if the time course was performed multiple times, it would be nice to see these data represented in the plot, along with standard error bars. *

The resection time course was performed as a pilot experiment to determine the earliest reasonable time to harvest gDNA and observe sufficiently high resected, ssDNA to make conclusions about relative resection efficiency across the various siRNA-treatment conditions. It was also performed to verify that after ~ 24 hours (the time at which we harvest genomic DNA for endpoint indel scoring) DSBs have been largely repaired. As a pilot experiment, however, we did not collect any timepoints between 12 -24 hours, or multiple replicates for all timepoints. We agree with the Reviewer’s concern about reproducibility and given that these other timepoints are not necessary to support the conclusions of the Figure we have now moved this panel to Supplemental Material (as it may be of interest to readers) in Supplemental Figure S5.

***Referees cross-commenting** *

*I agree with Reviewer #2 that "The primary issue with this manuscript is that it does not present the experimental conclusions in a straightforward manner. Instead of focusing on what was learned, it reads more like a journey." Based on the abstract, I had expected to learn more about the role of BLM and DNA2 in regulating TMEJ via resection, but the crux of the story is about cell cycle effects impacting TMEJ when the levels of these proteins are reduced. Rewriting would help address this. *

Reviewer 2 also asked that that representative spectra of all relevant experiments are presented as supplementary information instead of flattening the entire spectrum in one number. This seems critical in a revised manuscript.

We have endeavored to rewrite the manuscript in a more straightforward manner that focuses on the cell cycle regulation of TMEJ. We now also include representative spectra in the Supplemental Material.

*Reviewer #1 (Significance (Required)): *

*I am a basic scientist studying DNA repair mechanisms and have a long-standing interest in the mechanism of TMEJ. *

*In my opinion, the results from this study will be interesting to scientists studying double-strand break repair pathway choice and those thinking about how inhibition of different DSB repair proteins can be used in cancer treatments. The strongest aspect of the study is the way in which the authors investigated the effect of BLM, DNA2, and EXO1 knockdown/inhibition on cell cycle progression, and used the data to make conclusions about the most important factors influencing TMEJ repair. The experiments are carefully controlled, and the most important conclusions are supported by multiple lines of experimental reasoning. As stated above in the summary, the study can be improved by the inclusion of more of the primary data in the supplemental information, and by the addition of a key experiment that will further support the authors' model. *

*The study supports the recent findings from the Sfier and Ceccaldi labs that the main driver of TMEJ, polymerase theta, operates primarily during mitosis, and its activation depends on PLK1. Further, it presents initial data on the intriguing finding that BRCA1 deficiency is synthetic lethal with BLM deficiency, and this can be enhanced with Olaparib treatment, which will be of interest to translational and clinical scientists. *

*Overall, the study is notable because of its careful attention to cell cycle progression on DSB repair outcomes, and why caution needs to be taken when interpreting how genetic perturbation of DNA repair proteins impacts repair mechanisms. *

We appreciate the Reviewer’s appreciation for our rigorous approach and statement of the context and impact of our work.

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

*In this manuscript, the authors use two cell lines, U2OS and RPE1, to investigate the effects of impeding long-range end resection on alternative end-joining (TMEJ). They monitor repair of a CRISPR-induced DSB, presenting their findings as the ratio of TMEJ to cNHEJ outcomes. Initially, the authors observe that inhibiting BLM and DNA2 produces a TMEJ-deficient phenotype similar to that resulting from pharmacological inhibition of PolQ, suggesting a potential role for long-range end resection. However, this inference is challenged by evidence showing that genetically or pharmacologically perturbed cells fail to progress through the cell cycle, not reaching the previously reported cell cycle phase in which TMEJ typically occurs. Although it remains an open question whether TMEJ acts exclusively in M phase (given MMEJ manifestations in G0/G1 cells), the authors attempt to pinpoint the cell cycle stage of TMEJ-mediated DSB repair by using cell cycle inhibitors. However, in my opinion, these assays and experiments fall short of providing conclusive evidence. I therefore feel that the data is best presented focusing on the cellular consequences of impeding BLM/DNA2, and not on TMEJ, which is basically used as a marker/proxy for mitosis/mitotic cells. In the discussion, the authors themselves suggest several additional experimental approaches that would strengthen their conclusions or deepen their investigations. I believe that implementing such strategies would broaden the impact and interest; in their absence (I do not suggest the authors to start performing these time and resource consuming experimentation to be included in the current manuscript) the study has a more specific target audience. *

We appreciate that the author felt that our initial submission was unsuccessful at straddling two aspects: one related to BLM/DNA2 function and the other to the cell cycle. In this revision, we have chosen to focus entirely on the latter context including through the addition of new experiments and analysis. The revised manuscript highlights a role for Aurora A-PLK1 in activating “simple” TMEJ repair in late G2 with complex TMEJ-driven repair occurring in mitosis. We would argue that these findings indeed provide novel insight into TMEJ regulation.

*Major comments *

1. *i) The primary issue with this manuscript is that it does not present the experimental conclusions in a straightforward manner. Instead of focusing on what was learned, it reads more like a "journey." This issue is apparent from the abstract: 13 out of 16 lines are devoted to describing the authors' initial intent to investigate the role of long-range end resection enzymes in TMEJ, only to reveal that the observed effect is actually an indirect consequence of cell cycle perturbations. The manuscript uses TMEJ as a proxy for cells not reaching mitosis, thus supporting the conclusions reached from the more direct cell cycle analyses. While the majority of the experiments address the effects of inhibiting BLM and DNA2, the abstract suggests that these findings reveal new insights about TMEJ, which is doubtful or at most limited. The only potential novelty regarding TMEJ, aside from confirming its regulation by the cell cycle, is the narrowing down of the specific substage in which it operates. However, this conclusion is inferred from indirect effects rather than direct measurement, and also somewhat debatable by being dependent on a very explicit interpretation of the cell cycle inhibitors that are being used (whose action in my experiences are not as strict/narrow as here considered). * In response to this point and the critiques of the other reviewers, we have now extensively edited the manuscript. Given concerns raised about the interpretation of our data on knock-down of the BLM helicase given our inability to rescue TMEJ by complementation as described above, we have rewritten the manuscript to have a clear focus on: 1) what can be learned from the NGS approach to interrogating repair outcomes in response to the perturbations we explore; and 2) the effect of cell cycle progression on TMEJ proficiency, particularly in response to inhibitors of PLK1 and Aurora A compared to CDK1 and nocodazole blocks. We agree with the Reviewer that our initial description of the cell cycle inhibitors was overly binary. However, given that inhibitors of these kinases are actively being pursued in the clinic, and effects on TMEJ could contribute to their efficacy, we would argue that our insights will be important to the field.

2. *ii) I do not have an immediate solution to this concern but feel the data itself is valuable. I also appreciate the authors' thorough approach in conclusively establishing that the observed disruption of TMEJ upon BLM/DNA2 depletion is a downstream consequence of cellular phenotypes, rather than an anticipated resection issue at CRISPR-induced DNA breaks. I would thus recommend rewriting the manuscript to reflect this perspective. * We appreciate that the reviewer feels that the data is valuable and highlights the need to take cell cycle effects into account when assessing the genetic or pharmacological inhibition of DNA repair factors.

*iii) Some conclusions are overinterpreted, especially regarding the timing of DNA repair based on mutant spectra analysis. For instance, in lines 257-258, the authors conclude, "by the same timepoints, we find over 65% of DSBs have been repaired with mutagenic indels, indicating high transfection efficiency and rapid DSB induction." This statement is inaccurate for several reasons: (1) Given the iterative cycles of DSB induction, error-free repair, and recutting, it's possible that, for example, only 15% of DSBs have actually been repaired with mutagenic indels; (2) The assay detects only intact molecules, so unrepaired DSBs/broken molecules are not accounted for, especially those that are not resected and thus escape detection in Figure 3; and (3) The assay captures only deletions that retain the primer binding site. A more accurate statement would thus be something like, "65% of intact alleles at this timepoint contain mutations indicative of error-prone repair, indicating high transfection efficiency and robust (rather than rapid) DSB induction." The use of "rapid" is questionable here, as I consider 24 hours a long time, given that NHEJ typically occurs within an hour. *

We agree with the Reviewer. This sentence was meant to indicate that the ‘final’ repair product (i.e. a mutagenic indel) was robustly observed and indicates that the transfection/induction was successful, rather than establishing a value reflecting the outcome of a single DNA cutting and repair event. As perfect repair is indistinguishable from uncut chromosomes in our assay, we can infer that the total percentage of DSBs induced per genome is likely higher than 65%. Moreover, persistent DSBs or repaired DSBs that deleted enough flanking sequence to lose the primer binding region would also be invisible in our assay. Given this, we have now edited this statement to more accurately state: “65% of intact alleles at this timepoint contain mutations indicative of error-prone repair, indicating high transfection efficiency and robust DSB induction” as suggested by the Reviewer.

1. *iv) While I'm hesitant to request additional experiments, I believe the HR measurements of siDNA2 through the mClover assay, alongside the siNT condition, require additional replicates. Currently, these results are marked as non-significant in Figure 3D but labeled with a "+" instead of "+++" in Figure 3E. Additionally, the combination siBLM+siEXO1 is marked with a "-" in Figure 3E, though a statistical comparison of single siDNA2 to the double combination would likely result in a non-significant outcome. This makes it difficult to draw firm conclusions, and further biological replicates would help clarify the data; two of the current data points are wildtype, while one aligns with siBLM levels. * We appreciate the concern of the Reviewer and agree. We now include additional biological replicates of the LMNA-mClover assay with siDNA2 treatment to reinforce this result.

2. *v) Another major concern is the potential off-target effects of siRNA, as the reported phenotypes might partly stem from this issue. None of the experiments have been validated using complementing constructs resistant to targeting. Although the inhibitor data is useful, not all experiments have been validated with this method. Adding these controls would be beneficial but is ultimately "optional". In their absence, I recommend the authors temper their conclusions regarding siRNA-only data. * We took this concern of this and the other reviewers to heart and attempted to carry out key experiments of siBLM conditions with rescue constructs that are insensitive to the siRNA. As described above, we were unable to achieve a rescue of TMEJ. While this could be a consequence of effects of BLM under- or over-expression, as it is challenging to match to the endogenous levels, or a potential complication from different BLM splice variants, we are not sufficiently confident at this point to include this data and have therefore removed it from the manuscript. This revision instead focuses on the cell cycle regulation of TMEJ. We remain confident in the results of the siDNA2 and DNA2i given our ability to recapitulate the expected delays in S-phase upon this perturbation and the alignment of these changes with the DNA repair outcome.

*Minor comments *

• *

*1) The title is unclear: "its" refers to a repair pathway, which is here not demonstrated to be "sensitive" but instead not employed in cells where BLM-DNA2 is inhibited and/or downregulated. It is to me also somewhat off point: it turns out that TMEJ is a proxy for reading out a cell cycle stage, and I feel that the title should reflect the more direct consequences induced by loss of BLM-DNA. Otherwise one could substitute TMEJ for any other mitotic process creating a "true" statement. *

Given the shift in focus of the manuscript, the manuscript has been retitled: “Activation of Pol theta-mediated end joining begins during mitotic commitment”.

*2) To me, the readability of the manuscript would be improved by shortening the text: Especially the intro is very lengthy and reads like a review instead of focusing on providing the information necessary to understand the experiments/logic of the result section. Additional information that is relevant for discussing the outcomes of the experiments can be introduced at that stage. I realize this is also a matter of style but I felt that I had to go through a lot of context before getting to the actual data, which did not necessarily brought new insight into all what was discussed before. *

As described above, we have endeavored to rewrite the manuscript in more direct and concise fashion.

*3) Typo's: Line 242, missing "on" (Based on...); line 590, missing "to" (important to test) *

Thank you, we have corrected this error.

*4) I suggest to have the y-axis of the histograms in the figures to be identical. For example Figure 1 C en D have three histograms with Y-axis going to 4, but one going to 3. For consistency and comparison it is more useful to have all of them going to 4. Idem for Figure 2 where D deviates from A-C (I understand that e.g. has no datapoint exceeding the arbitrary mark 3 but it allows for a better comparison of D to A-C if the Y-axes are identical. *

We agree and have made all y-axes consistent across the Figures for each relevant comparison.

*5) Please specify, upon first usage, e.g. in the legend of a Figure how the ratio TMEJ:NHEJ is defined (instead of only in the method section). Also mention in the text that this is an arbitrary and subjective unit used for clarity which is indicative but not an absolute measure for the underlying biology: all product that have a MH of 2 and higher are binned under TMEJ, but surely non-sequence guided NHEJ will also produce deletions that, just by pure chance, have junctions in which 2 bp overlap. Also, not all TMEJ outcomes have 2bp of MH. NHEJ outcomes are thus in the TMEJ subclass and vice versa. This is not problematic to me but it should be kept in mind, especially in interpreting mutant backgrounds. *

We agree with the Reviewer and now discuss our categorization scheme in far greater detail including the rationale and caveats in the revised manuscript.

*6) Related to the above I also want to propose that representative spectra of all relevant experiments are presented as supplementary information instead of flattening the entire spectrum in one number, in which all nuance but also details may be lost, which can be informative to the TMEJ and NHEJ afficionados. *

We agree that this is likely one aspect of our work that will be useful to experts. We therefore now include a representative DSB mutation spectrum for each experimental condition in the Supplemental Material (Supplemental Figures S1, S2).

***Referees cross-commenting** *

*I agree with all the points brought up by the other 2 reviewers (and glad to see so much overlap). And thus feel that the authors need to address all them. *

*I initially was somewhat reserved on asking for additional experiments addressing potential off-targets of the siRNAs underlying some of the phenotypes (no experience myself using si's as opposed to sg's and clean knock-out models) wanting to see how the other reviewers potentially more experienced with this technology would weigh this concern. Having read their reports I thus als underwrite reviewer #3's remark: 2. I would strongly recommend that the authors confirm their BLM siRNA results (experiments in Fig. 1C, 3C and 4A) by examining whether add-back of exogenous, siRNA-resistant BLM can rescue the phenotypes they see on cell cycle progression and TMEJ. Alternatively, they could also make use of at least one of the many BLM knockout cell models that have been generated previously by many labs (including in both RPE-1 (Tsukada et al., Mol. Cell 2024) and U2OS (Jiang et al., Mol. Cell 2024) backgrounds), where exogenous BLM and helicase-dead BLM have been added back. This would be especially important to do if the authors cannot confirm the potency and selectivity of BLM-IN-1, as suggested above." *

Final comment from my end: I perhaps was somewhat unclear or went too fast: I've read concern #3 of reviewer 1 in the light of "the authors should consider..." thinking it was "optional". I agree with reviewer #3 that it is not essential.

Again, we thank the Reviewer for their expert advice and have addressed these issues in the the point-by-point responses above.

*Reviewer #2 (Significance (Required)): *

*This study is of interest to those studying the long range end resection enzymes including DNA2, BLM and EXO1, and to researchers with in depth knowledge and interest into the regulation of TMEJ. The study is limited in providing very concrete new information - it is somewhat descriptive (e.g. the study doesn't provide insight into why cells arrest at a given stage in the absence of BLM or DNA2 functionality). Hence, I feel that the outcomes are of interest to specialist as I feel that the individual observations are of relevance but there is not a impactful overarching gain of knowledge. The data can be used by others to further design experiments aimed to elucidate the roles of specific DNA repair enzymes. *

*Of note and as requested, my field of interest is repair of DNA breaks: EJ, cNHEJ, altEJ. *

We would suggest that we have enhanced the impact of our work in this revision through the addition of new data highlighting our evidence for a privileged late G2 window for TMEJ prior to mitotic entry while complex, TMEJ-driven templated insertions occur in mitosis itself.

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

*Summary and significance: *

• *

*In this manuscript, the authors describe mutational outcomes at a single site-specific DNA double-strand break induced by Cas9 in RPE-1 and U2OS cells. They find that depletion of BLM or DNA2 using siRNA or treatment of cells with small molecule inhibitors causes a shift from DNA polymerase theta (POLQ)-dependent repair outcomes to classical non-homologous end-joining (NHEJ)-mediated ones. In contrast, EXO1 depletion has no effect. They go on to show that this is not due to any potential role for BLM and DNA2 in DNA-end resection, but rather because under their experimental conditions, loss of BLM or DNA2 leads to cell cycle disruption such that fewer cells make it into mitosis, where POLQ is most active. *

*The key conclusions the authors draw are in large part convincing, as most experiments in the manuscript are replicating previously published findings. There is no reason to believe that long-range DNA-end resection would be required for POLQ-mediated end-joining (TMEJ), because TMEJ does not need longer overhangs than those MRE11 and CtIP can already provide via initial end-processing (50-100 nt). The fact that BLM and EXO1 do not promote microhomology-mediated end-joining was already shown in 2013 by Truong et al., PNAS. *

*The manuscript reads a bit like a warning that cell cycle progression defects must be taken into account when measuring POLQ-mediated end-joining (TMEJ) repair events; but this is not really a novel insight, given the recent reports that POLQ is most active in mitosis (work from Löbrich, Sfeir and Ceccaldi labs). *

We appreciate that the Reviewer found our work largely convincing. We have substantially edited the manuscript to make it more straightforward. In addition, taken together our data make an important addition to prior reports by highlighting the concept that TMEJ becomes fully activated in late G2 prior to mitosis (given that CDK1 inhibition, which blocks mitosis but not the Aurora A-PLK1 circuit, leads to TMEJ that is as efficient as in unperturbed cells).

*The manuscript would be suitable for publication if the authors could address the points below. *

*Major comments: *

1. • Major conclusions are drawn about effects of BLM loss on cell cycle progression based on the use of only a single siRNA, and the use of reported chemical inhibitors of BLM DNA-binding activity. Unfortunately, the ML216 inhibitor the authors use was found not to be a selective or potent inhibitor of BLM (Oliver lab, eLife 2021), and BLM-IN-1 has not been sufficiently well-characterised in cells yet as far as I am aware. The authors need to remove the ML216 data, as any effects they see are likely just due to the toxic effects of this compound rather than any effect on BLM specifically. Have the authors checked SCEs in cells treated with BLM-IN-1? If they do not see the characteristic 5-10x increase in SCEs that is observed in all BLM-deficient cells, then they can also not trust any results they get with that compound either. * As highlighted above, we agree with the Reviewer and endeavored to further validate our siBLM knock-down results through a variety of means. Ultimately, as described above, we were unable to achieve a rescue of TMEJ. We suspect that this is a consequence of effects of BLM under- or over-expression, as it is challenging to match to the endogenous levels, or a potential complication from different BLM splice variants. We also agree about the toxicity of the BLM inhibitors. Given these concerns, we have now removed this data from the manuscript, which has been refocused to directly address the cell cycle regulation of TMEJ.

2. I would strongly recommend that the authors confirm their BLM siRNA results (experiments in Fig. 1C, 3C and 4A) by examining whether add-back of exogenous, siRNA-resistant BLM can rescue the phenotypes they see on cell cycle progression and TMEJ. Alternatively, they could also make use of at least one of the many BLM knockout cell models that have been generated previously by many labs (including in both RPE-1 (Tsukada et al., Mol. Cell 2024) and U2OS (Jiang et al., Mol. Cell 2024) backgrounds), where exogenous BLM and helicase-dead BLM have been added back. This would be especially important to do if the authors cannot confirm the potency and selectivity of BLM-IN-1, as suggested above. *

Please see the response to Point 1.

• How do the authors know whether their EXO1 depletion is working in Figure S4C? EXO1 loss is synthetic lethal with BRCA1 deficiency (van de Kooij et al., Mol. Cell 2024 and Tsukada et al., Mol. Cell 2024), so I would have expected to see a drop in cell viability when EXO1 is depleted in BRCA1-deficient cells. Also, western blots for RPE-1 in Fig. 1C are missing; as are ones for Figures S4A-C. In Figure S4D, blots are not of sufficient quality, alternative ones should be provided. What does "stain-free" mean here? *

Given the removal of data involving siBLM treatments, this Western blot has been removed. Instead, we validate target transcript knockdown by qRT-PCR. In the case of DNA2, the effects of an siRNA knockdown are corroborated with a small molecule inhibitor.

*Minor comments: *

1. • I find the title of the manuscript somewhat lacking clarity/precision. It is not clear what the word "its" is referring to. * Given the shift in focus of the manuscript, the manuscript has been retitled: “Activation of Pol theta-mediated end joining begins during mitotic commitment”.

2. Lines 23-24: TMEJ is not just a back-up for HR, but also for NHEJ. *

We have edited this text in the revised manuscript and now focus on TMEJ’s ability to act on resected, unresolved DSBs that persist to the G2/M transition .

Lines 27-28: who has proposed this? Consider re-phrasing this (see second paragraph of summary above).

We have rephased this section in the revised manuscript (paragraph beginning at line 71). Our rationale was not based on long-range resection being a strict, biochemical requirement for TMEJ, but rather was based on the observation that many of the proposed substrates for TMEJ (overly resected DSBs that cannot be ligated by NHEJ, or aborted HR intermediates) are downstream of long-range resection. For example, in Vergara, et al. 2023, Nat Comm loss of BLM (or RMI2) suppresses MMEJ repair in an MMEJ vs NHEJ reporter assay. Indeed, Truong et al. 2013 PNAS indicates that BLM loss actually promotes MMEJ, but notably this assay only measures competition between HR and MMEJ outcomes, rather than MMEJ vs NHEJ.

• Line 32: DNA should be DNA2. *

Thank you, this error has been corrected.

• Line 71: Sgs1 should be in italics. *

Thank you, this error has been corrected.

• Line 147: NHEJ is not active throughout the cell cycle; it is inhibited during mitosis. *

Thank you, this has been corrected to “throughout interphase” (Line 92).

• Line 242: the word "on" is missing after the word "Based". *

Thank you, this error has been corrected.

• Line 282: replace resolution with dissolution. *

Thank you, this text was removed in revision.

• Line 482: it is already known that BRCA-deficient tumours accumulate mutational signatures attributed to TMEJ. *

Thank you, this text was removed in revision.

• Line 484: any role of BRCA1 in resection is highly controversial; but anyway no-one has claimed that BRCA1 is required for short-range resection by MRE11, which is all that would be required for TMEJ. *

While not strictly required for either short or long-range resection, we are quite open to the model of BRCA1 stimulating resection via its interaction with CtIP and general antagonism of 53BP1 recruitment (Cruz-Garcia, et al. 2014 Cell Rep, Densham et al. 2016 Nat Struct Mol Biol). Regardless, this section has been removed to better focus on the cell cycle control of TMEJ.

• Line 500: deficient is mis-spelled. *

Thank you, this error has been corrected.

*Lines 500-520: work by van de Kooij et al. and Tsukada et al. is not properly referenced here. Both already showed that BLM loss is synthetic lethal with BRCA1 deficiency, but interestingly, van de Kooij et al. showed that 53BP1 loss rescues the BRCA1-BLM synthetic lethality, whereas Tsukada et al. found that it does not, despite the fact that it rescues olaparib sensitivity. The authors' results seem to support the latter. Also, hypersensitivity to olaparib in BLM-depleted cells was already shown previously by Gottipati et al. Cancer Res. 2010. *

We thank the reviewer for their comments; however, this data and discussion of BRCA1 and BLM has been removed.

• Lines 519-520: it is not clear to me why the results in Figure 6 provide an alternative mechanism contributing to synthetic lethality between BLM and BRCA1 from that shown previously. *

See Point 12.

• Methods: siRNA sequences should be provided. *

We apologize for the oversight. While some siRNA sequences are proprietary, we have included the source for each commercially available reagent in the Materials and Methods.

***Referees cross-commenting** *

*I agree with most of the comments from the other reviews. As stated by Reviewer 2, it is nice to see significant overlap between the three reviews. *

*It seems all three of us think the manuscript needs a significant re-write for clarity purposes, to make it read less like a "journey" and more like the synthesis of existing observations that it actually is. It is important for the field to know that EXO1, BLM and DNA2 do not contribute directly to TMEJ, and that any effects that can be observed indicating that they do, can likely be put down to cell-cycle progression defects. *

*Where I differ from reviewer 1 and 2, is in the need to carry out experiments with the POLQ-10A mutant from the Ceccaldi paper. There are multiple reasons why that might not work, so I think that is unnecesary. But I otherwise agree with reviewer 2 that the authors need to address all the other points from all three of us. *

We have endeavored to address the general critiques of the reviewers by rewriting the manuscript and focusing directly on the cell cycle regulation of TMEJ.

*Reviewer #3 (Significance (Required)): *

*The key conclusions the authors draw are in large part convincing, as most experiments in the manuscript are replicating previously published findings. There is no reason to believe that long-range DNA-end resection would be required for POLQ-mediated end-joining (TMEJ), because TMEJ does not need longer overhangs than those MRE11 and CtIP can already provide via initial end-processing (50-100 nt). The fact that BLM and EXO1 do not promote microhomology-mediated end-joining was already shown in 2013 by Truong et al., PNAS. *

The manuscript reads a bit like a warning that cell cycle progression defects must be taken into account when measuring POLQ-mediated end-joining (TMEJ) repair events; but this is not really a novel insight, given the recent reports that POLQ is most active in mitosis (work from Löbrich, Sfeir and Ceccaldi labs).

We thank the Reviewer for their comments and have endeavored to refocus, clarify and emphasize the novel aspects of our study, namely evidence for a period of late G2 during which TMEJ is licensed through Aurora A-PLK1 with additional complex, TMEJ-mediated repair occurring in mitosis.

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

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Referee #3

Evidence, reproducibility and clarity

Summary and significance:

In this manuscript, the authors describe mutational outcomes at a single site-specific DNA double-strand break induced by Cas9 in RPE-1 and U2OS cells. They find that depletion of BLM or DNA2 using siRNA or treatment of cells with small molecule inhibitors causes a shift from DNA polymerase theta (POLQ)-dependent repair outcomes to classical non-homologous end-joining (NHEJ)-mediated ones. In contrast, EXO1 depletion has no effect. They go on to show that this is not due to any potential role for BLM and DNA2 in DNA-end resection, but rather because under their experimental conditions, loss of BLM or DNA2 leads to cell cycle disruption such that fewer cells make it into mitosis, where POLQ is most active.

The key conclusions the authors draw are in large part convincing, as most experiments in the manuscript are replicating previously published findings. There is no reason to believe that long-range DNA-end resection would be required for POLQ-mediated end-joining (TMEJ), because TMEJ does not need longer overhangs than those MRE11 and CtIP can already provide via initial end-processing (50-100 nt). The fact that BLM and EXO1 do not promote microhomology-mediated end-joining was already shown in 2013 by Truong et al., PNAS.

The manuscript reads a bit like a warning that cell cycle progression defects must be taken into account when measuring POLQ-mediated end-joining (TMEJ) repair events; but this is not really a novel insight, given the recent reports that POLQ is most active in mitosis (work from Löbrich, Sfeir and Ceccaldi labs).

The manuscript would be suitable for publication if the authors could address the points below.

Major comments:

1. Major conclusions are drawn about effects of BLM loss on cell cycle progression based on the use of only a single siRNA, and the use of reported chemical inhibitors of BLM DNA-binding activity. Unfortunately, the ML216 inhibitor the authors use was found not to be a selective or potent inhibitor of BLM (Oliver lab, eLife 2021), and BLM-IN-1 has not been sufficiently well-characterised in cells yet as far as I am aware. The authors need to remove the ML216 data, as any effects they see are likely just due to the toxic effects of this compound rather than any effect on BLM specifically. Have the authors checked SCEs in cells treated with BLM-IN-1? If they do not see the characteristic 5-10x increase in SCEs that is observed in all BLM-deficient cells, then they can also not trust any results they get with that compound either.
2. I would strongly recommend that the authors confirm their BLM siRNA results (experiments in Fig. 1C, 3C and 4A) by examining whether add-back of exogenous, siRNA-resistant BLM can rescue the phenotypes they see on cell cycle progression and TMEJ. Alternatively, they could also make use of at least one of the many BLM knockout cell models that have been generated previously by many labs (including in both RPE-1 (Tsukada et al., Mol. Cell 2024) and U2OS (Jiang et al., Mol. Cell 2024) backgrounds), where exogenous BLM and helicase-dead BLM have been added back. This would be especially important to do if the authors cannot confirm the potency and selectivity of BLM-IN-1, as suggested above.
3. How do the authors know whether their EXO1 depletion is working in Figure S4C? EXO1 loss is synthetic lethal with BRCA1 deficiency (van de Kooij et al., Mol. Cell 2024 and Tsukada et al., Mol. Cell 2024), so I would have expected to see a drop in cell viability when EXO1 is depleted in BRCA1-deficient cells. Also, western blots for RPE-1 in Fig. 1C are missing; as are ones for Figures S4A-C. In Figure S4D, blots are not of sufficient quality, alternative ones should be provided. What does "stain-free" mean here?

Minor comments:

1. I find the title of the manuscript somewhat lacking clarity/precision. It is not clear what the word "its" is referring to.
2. Lines 23-24: TMEJ is not just a back-up for HR, but also for NHEJ.
3. Lines 27-28: who has proposed this? Consider re-phrasing this (see second paragraph of summary above).
4. Line 32: DNA should be DNA2.
5. Line 71: Sgs1 should be in italics.
6. Line 147: NHEJ is not active throughout the cell cycle; it is inhibited during mitosis.
7. Line 242: the word "on" is missing after the word "Based".
8. Line 282: replace resolution with dissolution.
9. Line 482: it is already known that BRCA-deficient tumours accumulate mutational signatures attributed to TMEJ.
10. Line 484: any role of BRCA1 in resection is highly controversial; but anyway no-one has claimed that BRCA1 is required for short-range resection by MRE11, which is all that would be required for TMEJ.
11. Line 500: deficient is mis-spelled.
12. Lines 500-520: work by van de Kooij et al. and Tsukada et al. is not properly referenced here. Both already showed that BLM loss is synthetic lethal with BRCA1 deficiency, but interestingly, van de Kooij et al. showed that 53BP1 loss rescues the BRCA1-BLM synthetic lethality, whereas Tsukada et al. found that it does not, despite the fact that it rescues olaparib sensitivity. The authors' results seem to support the latter. Also, hypersensitivity to olaparib in BLM-depleted cells was already shown previously by Gottipati et al. Cancer Res. 2010.
13. Lines 519-520: it is not clear to me why the results in Figure 6 provide an alternative mechanism contributing to synthetic lethality between BLM and BRCA1 from that shown previously.
14. Methods: siRNA sequences should be provided.

Referees cross-commenting

I agree with most of the comments from the other reviews. As stated by Reviewer 2, it is nice to see significant overlap between the three reviews.

It seems all three of us think the manuscript needs a significant re-write for clarity purposes, to make it read less like a "journey" and more like the synthesis of existing observations that it actually is. It is important for the field to know that EXO1, BLM and DNA2 do not contribute directly to TMEJ, and that any effects that can be observed indicating that they do, can likely be put down to cell-cycle progression defects.

Where I differ from reviewer 1 and 2, is in the need to carry out experiments with the POLQ-10A mutant from the Ceccaldi paper. There are multiple reasons why that might not work, so I think that is unnecesary. But I otherwise agree with reviewer 2 that the authors need to address all the other points from all three of us.

Significance

The key conclusions the authors draw are in large part convincing, as most experiments in the manuscript are replicating previously published findings. There is no reason to believe that long-range DNA-end resection would be required for POLQ-mediated end-joining (TMEJ), because TMEJ does not need longer overhangs than those MRE11 and CtIP can already provide via initial end-processing (50-100 nt). The fact that BLM and EXO1 do not promote microhomology-mediated end-joining was already shown in 2013 by Truong et al., PNAS.

The manuscript reads a bit like a warning that cell cycle progression defects must be taken into account when measuring POLQ-mediated end-joining (TMEJ) repair events; but this is not really a novel insight, given the recent reports that POLQ is most active in mitosis (work from Löbrich, Sfeir and Ceccaldi labs).

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Referee #2

Evidence, reproducibility and clarity

In this manuscript, the authors use two cell lines, U2OS and RPE1, to investigate the effects of impeding long-range end resection on alternative end-joining (TMEJ). They monitor repair of a CRISPR-induced DSB, presenting their findings as the ratio of TMEJ to cNHEJ outcomes. Initially, the authors observe that inhibiting BLM and DNA2 produces a TMEJ-deficient phenotype similar to that resulting from pharmacological inhibition of PolQ, suggesting a potential role for long-range end resection. However, this inference is challenged by evidence showing that genetically or pharmacologically perturbed cells fail to progress through the cell cycle, not reaching the previously reported cell cycle phase in which TMEJ typically occurs. Although it remains an open question whether TMEJ acts exclusively in M phase (given MMEJ manifestations in G0/G1 cells), the authors attempt to pinpoint the cell cycle stage of TMEJ-mediated DSB repair by using cell cycle inhibitors. However, in my opinion, these assays and experiments fall short of providing conclusive evidence. I therefore feel that the data is best presented focusing on the cellular consequences of impeding BLM/DNA2, and not on TMEJ, which is basically used as a marker/proxy for mitosis/mitotic cells

In the discussion, the authors themselves suggest several additional experimental approaches that would strengthen their conclusions or deepen their investigations. I believe that implementing such strategies would broaden the impact and interest; in their absence (I do not suggest the authors to start performing these time and resource consuming experimentation to be included in the current manuscript) the study has a more specific target audience.

Major comments

i) The primary issue with this manuscript is that it does not present the experimental conclusions in a straightforward manner. Instead of focusing on what was learned, it reads more like a "journey." This issue is apparent from the abstract: 13 out of 16 lines are devoted to describing the authors' initial intent to investigate the role of long-range end resection enzymes in TMEJ, only to reveal that the observed effect is actually an indirect consequence of cell cycle perturbations. The manuscript uses TMEJ as a proxy for cells not reaching mitosis, thus supporting the conclusions reached from the more direct cell cycle analyses. While the majority of the experiments address the effects of inhibiting BLM and DNA2, the abstract suggests that these findings reveal new insights about TMEJ, which is doubtful or at most limited. The only potential novelty regarding TMEJ, aside from confirming its regulation by the cell cycle, is the narrowing down of the specific substage in which it operates. However, this conclusion is inferred from indirect effects rather than direct measurement, and also somewhat debatable by being dependent on a very explicit interpretation of the cell cycle inhibitors that are being used (whose action in my experiences are not as strict/narrow as here considered).

ii) I do not have an immediate solution to this concern but feel the data itself is valuable. I also appreciate the authors' thorough approach in conclusively establishing that the observed disruption of TMEJ upon BLM/DNA2 depletion is a downstream consequence of cellular phenotypes, rather than an anticipated resection issue at CRISPR-induced DNA breaks. I would thus recommend rewriting the manuscript to reflect this perspective.

iii) Some conclusions are overinterpreted, especially regarding the timing of DNA repair based on mutant spectra analysis. For instance, in lines 257-258, the authors conclude, "by the same timepoints, we find over 65% of DSBs have been repaired with mutagenic indels, indicating high transfection efficiency and rapid DSB induction." This statement is inaccurate for several reasons: (1) Given the iterative cycles of DSB induction, error-free repair, and recutting, it's possible that, for example, only 15% of DSBs have actually been repaired with mutagenic indels; (2) The assay detects only intact molecules, so unrepaired DSBs/broken molecules are not accounted for, especially those that are not resected and thus escape detection in Figure 3; and (3) The assay captures only deletions that retain the primer binding site. A more accurate statement would thus be something like, "65% of intact alleles at this timepoint contain mutations indicative of error-prone repair, indicating high transfection efficiency and robust (rather than rapid) DSB induction." The use of "rapid" is questionable here, as I consider 24 hours a long time, given that NHEJ typically occurs within an hour.

iv) While I'm hesitant to request additional experiments, I believe the HR measurements of siDNA2 through the mClover assay, alongside the siNT condition, require additional replicates. Currently, these results are marked as non-significant in Figure 3D but labeled with a "+" instead of "+++" in Figure 3E. Additionally, the combination siBLM+siEXO1 is marked with a "-" in Figure 3E, though a statistical comparison of single siDNA2 to the double combination would likely result in a non-significant outcome. This makes it difficult to draw firm conclusions, and further biological replicates would help clarify the data; two of the current data points are wildtype, while one aligns with siBLM levels.

v) Another major concern is the potential off-target effects of siRNA, as the reported phenotypes might partly stem from this issue. None of the experiments have been validated using complementing constructs resistant to targeting. Although the inhibitor data is useful, not all experiments have been validated with this method. Adding these controls would be beneficial but is ultimately "optional". In their absence, I recommend the authors temper their conclusions regarding siRNA-only data.

Minor comments

1. The title is unclear: "its" refers to a repair pathway, which is here not demonstrated to be "sensitive" but instead not employed in cells where BLM-DNA2 is inhibited and/or downregulated. It is to me also somewhat off point: it turns out that TMEJ is a proxy for reading out a cell cycle stage, and I feel that the title should reflect the more direct consequences induced by loss of BLM-DNA. Otherwise one could substitute TMEJ for any other mitotic process creating a "true" statement.
2. To me, the readability of the manuscript would be improved by shortening the text: Especially the intro is very lengthy and reads like a review instead of focusing on providing the information necessary to understand the experiments/logic of the result section. Additional information that is relevant for discussing the outcomes of the experiments can be introduced at that stage. I realize this is also a matter of style but I felt that I had to go through a lot of context before getting to the actual data, which did not necessarily brought new insight into all what was discussed before.
3. Typo's: Line 242, missing "on" (Based on...); line 590, missing "to" (important to test)
4. I suggest to have the y-axis of the histograms in the figures to be identical. For example Figure 1 C en D have three histograms with Y-axis going to 4, but one going to 3. For consistency and comparison it is more useful to have all of them going to 4. Idem for Figure 2 where D deviates from A-C (I understand that e.g. has no datapoint exceeding the arbitrary mark 3 but it allows for a better comparison of D to A-C if the Y-axes are identical.
5. Please specify, upon first usage, e.g. in the legend of a Figure how the ratio TMEJ:NHEJ is defined (instead of only in the method section). Also mention in the text that this is an arbitrary and subjective unit used for clarity which is indicative but not an absolute measure for the underlying biology: all product that have a MH of 2 and higher are binned under TMEJ, but surely non-sequence guided NHEJ will also produce deletions that, just by pure chance, have junctions in which 2 bp overlap. Also, not all TMEJ outcomes have 2bp of MH. NHEJ outcomes are thus in the TMEJ subclass and vice versa. This is not problematic to me but it should be kept in mind, especially in interpreting mutant backgrounds.
6. Related to the above I also want to propose that representative spectra of all relevant experiments are presented as supplementary information instead of flattening the entire spectrum in one number, in which all nuance but also details may be lost, which can be informative to the TMEJ and NHEJ afficionados.

Referees cross-commenting

I agree with all the points brought up by the other 2 reviewers (and glad to see so much overlap). And thus feel that the authors need to address all them.

I initially was somewhat reserved on asking for additional experiments addressing potential off-targets of the siRNAs underlying some of the phenotypes (no experience myself using si's as opposed to sg's and clean knock-out models) wanting to see how the other reviewers potentially more experienced with this technology would weigh this concern. Having read their reports I thus als underwrite reviewer #3's remark: 2. I would strongly recommend that the authors confirm their BLM siRNA results (experiments in Fig. 1C, 3C and 4A) by examining whether add-back of exogenous, siRNA-resistant BLM can rescue the phenotypes they see on cell cycle progression and TMEJ. Alternatively, they could also make use of at least one of the many BLM knockout cell models that have been generated previously by many labs (including in both RPE-1 (Tsukada et al., Mol. Cell 2024) and U2OS (Jiang et al., Mol. Cell 2024) backgrounds), where exogenous BLM and helicase-dead BLM have been added back. This would be especially important to do if the authors cannot confirm the potency and selectivity of BLM-IN-1, as suggested above."

Final comment from my end: I perhaps was somewhat unclear or went too fast: I've read concern #3 of reviewer 1 in the light of "the authors should consider..." thinking it was "optional". I agree with reviewer #3 that it is not essential.

Significance

This study is of interest to those studying the long range end resection enzymes including DNA2, BLM and EXO1, and to researchers with in depth knowledge and interest into the regulation of TMEJ. The study is limited in providing very concrete new information - it is somewhat descriptive (e.g. the study doesn't provide insight into why cells arrest at a given stage in the absence of BLM or DNA2 functionality). Hence, I feel that the outcomes are of interest to specialist as I feel that the individual observations are of relevance but there is not a impactful overarching gain of knowledge. The data can be used by others to further design experiments aimed to elucidate the roles of specific DNA repair enzymes.

Of note and as requested, my field of interest is repair of DNA breaks: EJ, cNHEJ, altEJ.

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Referee #1

Evidence, reproducibility and clarity

Summary

DNA double-strand breaks can be repaired by NHEJ, TMEJ, or HR. While there have been many studies of the factors that regulate double-strand break repair pathway choice, the exact mechanisms by which TMEJ is regulated remain under active study. In this study, McBrine and colleagues conducted experiments in two human cell lines to investigate the contribution of DNA resection to TMEJ. After induction of Cas9 breaks at a single locus, they used NGS amplicon sequencing and a pre-existing computational pipeline to quantify repair by inaccurate NHEJ or TMEJ. Their initial data suggested that loss/inhibition of the BLM DNA helicase or DNA2 nuclease inhibit resection and thereby decrease TMEJ repair. Probing further, they conducted carefully controlled cell cycle analysis and determined that loss of either BLM or DNA2 slows progression to the G2/M transition, when TMEJ is activated through PLK1 phosphorylation of polymerase theta. Based on these findings, they conclude that both BLM and DNA2 promote TMEJ repair, likely by promoting efficient replication and timely progression into G2/M.

Major comments

Overall, the authors' conclusions are well-supported by their data. They used multiple approaches to validate their findings, including the use of siRNAs and small molecule inhibitors that largely produce similar results. However, three major additions/experiments should be included.

1. First, most of the data from the NGS sequencing results are displayed as TMEJ/NHEJ ratios. While this makes it easy to compare between different experimental conditions, it could mask differences in overall repair efficiency. I recommend that the authors include the initial percentages of TMEJ-like and NHEJ-like repair junctions in supplemental information, so that readers can determine the efficiencies of the different repair pathways in each condition.
2. Second, the DSB repair spectrum (as displayed in Figure 1B) should be provided for each TMEJ:NHEJ plot in supplemental information, at the very least the for knockdown/inhibitor experiments where a significant difference in TMEJ:NHEJ ratio is observed.
3. Finally, the conclusion that transition to the G2 stage (where polymerase theta becomes activated through phosphorylation by PLK1) is necessary for TMEJ is logical, based on the data and previously published studies. However, to provide additional support for this model, the authors should consider repeating the experiments from Figure 5B/5C using cells with the PLK1 phosphorylation sites on polymerase theta mutated (e.g., using the 10A construct from Gelot et al., doi.org/10.1038/s41586-023-06506-6). The expectation is that the TMEJ reduction observed with BLM or DNA2 inhibition should not decrease further, if indeed the decrease is largely due to an inability to progress to the point in the cell cycle where polymerase theta is activated by phosphorylation. This experiment should be able to be completed within 1-2 months, if the appropriate cell line can be obtained.

Minor comments

1. The cell cycle plots in Figures 4 and 5 are difficult to interpret given the current color scheme (especially the difference between S and G2). I suggest changing the color scheme and arranging the legend so that the key is in the same order as the plots (G1 on the bottom, S in the middle, G2 on the top).
2. In Figure 3B, the resection time course is interesting, but I wondered why they are no time points between 12 and 24 hours? Also, if the time course was performed multiple times, it would be nice to see these data represented in the plot, along with standard error bars.

Referees cross-commenting

I agree with Reviewer #2 that "The primary issue with this manuscript is that it does not present the experimental conclusions in a straightforward manner. Instead of focusing on what was learned, it reads more like a journey." Based on the abstract, I had expected to learn more about the role of BLM and DNA2 in regulating TMEJ via resection, but the crux of the story is about cell cycle effects impacting TMEJ when the levels of these proteins are reduced. Rewriting would help address this.

Reviewer 2 also asked that that representative spectra of all relevant experiments are presented as supplementary information instead of flattening the entire spectrum in one number. This seems critical in a revised manuscript.

Significance

I am a basic scientist studying DNA repair mechanisms and have a long-standing interest in the mechanism of TMEJ.

In my opinion, the results from this study will be interesting to scientists studying double-strand break repair pathway choice and those thinking about how inhibition of different DSB repair proteins can be used in cancer treatments. The strongest aspect of the study is the way in which the authors investigated the effect of BLM, DNA2, and EXO1 knockdown/inhibition on cell cycle progression, and used the data to make conclusions about the most important factors influencing TMEJ repair. The experiments are carefully controlled, and the most important conclusions are supported by multiple lines of experimental reasoning. As stated above in the summary, the study can be improved by the inclusion of more of the primary data in the supplemental information, and by the addition of a key experiment that will further support the authors' model.

The study supports the recent findings from the Sfier and Ceccaldi labs that the main driver of TMEJ, polymerase theta, operates primarily during mitosis, and its activation depends on PLK1. Further, it presents initial data on the intriguing finding that BRCA1 deficiency is synthetic lethal with BLM deficiency, and this can be enhanced with Olaparib treatment, which will be of interest to translational and clinical scientists.

Overall, the study is notable because of its careful attention to cell cycle progression on DSB repair outcomes, and why caution needs to be taken when interpreting how genetic perturbation of DNA repair proteins impacts repair mechanisms.

This comes across as strictly positive, but if you look at the evidence, I believe there was also some less positive news about finance.

The developments reported below make it look like things have only gone in a positive direction, but I believe that on the finance side, things may have been scaled back? This might be adjusted a bit to not present the wrong impression.

This last clause is a bit vague - what do we mean, "the kind of food people would buy"?

Quantify and link, potentially in tooltips to avoid too much clutter

"wide" is a vague term. Can't you be more quantitative and reference something specific here, or at least link it?

In a tooltip link direct quotes/sections - everything stated here needs some sort of attribution.

Accurately exacly

Bijvoorbeeld art. 2 Gw (‘de wet bepaalt.....’) De wet staat voor formele wetgever. Deze bevoegdheid is dus geattribueerd aan de formele wetgever (Staten Generaal en Regering)

Originally annotated everything in a private group and couldn't figure out how to unprivate it, so I just moved them all into the public one.

The underlying message being that 'white' is superior, and if you can't afford high-end beauty products, pure straight-from-the-garden foods, and all the trendy outfits, then don't bother showing yourself.

Let's not exclude another factor, everyone's favorite topic: patriarchy. in the manosphere it has been emphasized that men should only want girls who don't wear a lot of makeup, they want 'natural' looks, obviously not too natural though. In that area of the internet though the demand for women who look effortlessly flawless is high and this trend could definitely be the response to that demand

I know this one in particular was popular with the '13-year-olds taking over Sephora'. different issue with kids and the lack of third spaces.

The clean girl aesthetic likely is what caused the rise of people taking interest in maximalism, stories of rot, and a break in the same formula of content. There was another microtrend surrounding forgotten supermarkets and the gross things inside. In a way, romanticized bugs, rot, and decay.

A lot of social things are geared to the slow erasure of things outside societal norms or things that don't resonate with the perfect format. TikTok, Instagram, Facebook, they all prefer particular curated short form videos that hit every mark and are easily marketable. That means therapy talk, routines, the perfect set, and a white person telling you whats fashionable and whats not.

Its all part of a system, conform or be excluded and alienated. This behavior dates back to hunter/gatherer times where stepping outside of line means slow exile until you cooperate. of course, that was life and death and this is completely social however our brains are still hardwired to crave acceptance from the group

This makes sense since Cambridge is known to be a wealthier school. It would make sense for somewhere like Dalton State to not have this problem because most of the students aren't that wealthy or white. It's easier for Cambridge to fall subject to trends like this because a lot of the trend's demographic goes there.

Very true. Things like Femininity and Masculinity are always changing definitions while trying to maintain core attributes. Lately, society has been trying to subtly shift those definitions back to the traditional ones but using new words that sound freeing. Think the 'male gaze' vs the 'female gaze' or 'feminine energy' vs 'masculine energy'

There is always something that women are doing that is critiqued by the rest of the world and judged on whether or not women are allowed to like. is this the case here?

How?

arguably most are

Sad beige aesthetic may be connected. Spend more to have less but look like you have more. popularized by celebrity houses. a vast number of aesthetics are likely connected

Does this show for everyone or just admin users?

Remove all caps

Remove all caps

Is the body text configurable in the dashboard?

General question: Does this show for everyone or just those who are signed in?

Move buttons to extreme right.

Remove subtitle. Replace title text with: "Explore the history of the university, Knoxville, East Tennessee, and beyond."

Remove all caps (should be site-wide to not have all caps)

.

"This is a humorous, practical stage direction that reflects Bottom’s blunt, literal-minded nature. He is so focused on the physical details of the play that he overlooks the fact that the lion character is meant to be a beast, not a human. The instruction about the lion’s nails is absurd in a human context — a human’s nails won’t “hang out” like claws — but it’s meant to be funny because it’s taken so literally. It also shows Bottom’s lack of theatrical subtlety; he’s more concerned with the literal appearance of the lion than with the character’s role or the audience’s expectations."

This ai search is extremely bland and feels to take the emotions out of the plays words.

"This exchange happens after Bottom has been “transported” (possessed by the fairy Puck) and is now in a different state of mind. The other craftsmen are trying to convince him to join their play. Flute is complimenting Bottom’s intelligence, good character, and appreciation for beauty (in this case, a beautiful voice) to encourage him to participate."

This part of the play seems to have a change of heart on bottom since he I think he is more portrayed as kind of a cocky mean person now he is being shown as smart good man.

Adding "social well-being" to the definition sounds like a broad, humanistic goal, but it actually started as bureaucratic turf-war posturing against the ILO. The term is so vague that it's nearly impossible to measure clinically, showing how political posturing over organizational scope shaped the language we still use today.

We usually take the WHO's definition for granted as an innocent, progressive standard, but its wording was actually an aggressive political push for universal healthcare. Framing health as a human right that requires government action directly challenged the private medical model, which is why it had to be smuggled past Cold War opposition.

"The key part is “Mine own and not mine own.” This reflects Helena’s confusion and uncertainty. She has just realized that Demetrius’s love for her is not entirely real — it has been altered by the magical spell. On one level, she feels he is “mine own” in that he is her beloved and she has found him; on another, he is “not mine own” because the spell has changed his affections, making them part of a dream or illusion"

This scene seems like Helena is really having an internal conflict with herself about the love Demetrius has for her and the love she may or may not have for him

Demetrius is explaining the plan Helena has told him while also showing his loss of self-control due to the love spell puck put him under. This is also the start of his speech talking about the woman he once loved... Hermia is now someone and something he hates.

"Oberon is ordering Puck to take the “transformed scalp” (the donkey’s head) from Bottom’s head so that Bottom can return to his normal human form. Once Bottom is restored, he will wake up alongside the other Athenian lovers (Hermia, Lysander, Demetrius, and Helena) and be able to “all to Athens back again repair” — return to their city and lives "

This ai assumption of the play gives a very literal explanation where it also has another meaning as if the fairy are going to return everyone to their normal lives and leave them alone.

In Eilot's original draft, his epigraph was "Did he live his life again in every detail of desire, temptation, and surrender during that supreme moment of complete knowledge? He cried in a whisper at some image, at some vision,—he cried out twice, a cry that was no more than a breath—'The horror! the horror!'" The original epigraphs frames the poem through Kurtz’s final vision of absolute horror and the collapse of European ideals which I think was intentional, considering the timeframe of this writing. In essence, it was dramatic, personal, and nonetheless modern. The published epigraph replaces that with the image of the Cumaean Sibyl suspended in a bottle, reduced to a voice that only wants to die. This is reinforced by the Ovid passage, which supplies the full back-story: the Sibyl asked Apollo for as many years as the grains of dust but forgot to ask for perpetual youth; she is condemned to shrink into pure voice. With this switch from Conrad to Petronius/Ovid, the poem inhabits a more classical, timeless image of exhuasted longevity and the desire for extinction rather than just a psychological horror. I believe that both epigraphs share a sense of terminal knowledge and desolation, but the final choice is cooler, more allusive, and more tightly integrated with the classical underworld and fertility-myth material that structures the rest of the poem

The original title of this poem was derived from Dicken's Our Mutual Friend ( the Betty Higden/ Sloppy scene. Higden praises sloppy's newspaper-reading: "He do the Police in different voices." The phrase signals to a single speaker who can adopt multiple registers, accents, and personae—which is exactly the polyvocal technique that is implemented in TWL. With Eliot discarding the Dickensian title, the final title shifts the emphasis from a sort of dramatic ventriloquism to a more broad cultural and sprirtual naming of modern sterility.

• the eighth design: a suburban cottage in the Italian style reminds me of new England homes with this grand central hallway
• grand porch is appealing and is giving Beecher

• homes make people invested which means they are not fucking crazy
• homes have a moral influence that keep men tethered
• rural space affords architects to approach home building as an art and not a science, more personal expression
• Houses are artistic and investing in the culture of a people by living in one
• we take the art of architecture for granted, especially given that it is art owned by many many people
• the level of thought and intentionality that goes into building a home equates to the level of civilization
• it is a privilege to be able to treat your home as art or self expression beyond a messaging of your material means
• the art must bow to natural laws, materials constraints, and livability
• the useful is the largest satisfaction to be gained by architecture
• humans do desire to beautify their dwellings
• absolute beauty in architecture: proportion, symmetry, variety, harmony, and unity
• the beauty pf architecture is taken for granted because it is often unfelt if done well
• houses in STL are often slaves to the symmetry
• symmetry = balance, evoke justice , related to harmony, a feeling that all elements make up some cohesive whole
• also getting this sense that if you have a small house or an apartment out of necessity, some of these elements of beauty are closed off to you
• unity is one single feeling that unites all the other abstract elements relative beauty expresses moral, social, or intellectual idea and it is self expression, that is, different for each person. There is some central goal or feeling to evoke
• regularity and uniformity -> satisfaction. relative beauty -> respect
• do I see the character of my friends in their faces or in their actions
• absolute beauty is only the base for true self expression
• different styles of architecture are national characters which have become permanent, an implementable shorthand for personality
• picturesque is a commodification of the beautiful
• the general truth is the usefulness of any house, the local truth situates it within a community and the specific truth to the individual
• the relationship of the house, and maybe expression of the owner, to larger society must adhere also to the abstract ideas of beauty
• know your means and stay within the personality you actually have, limits to the soft launch
• humbleness, or at least matching means to expression is more desirable and beautiful than baseless grandeur
• there is an implication that the dreams of self expression must be anchored by the reality of building materials and such

Studies require one to back up their data with scientific data and real-life examples; otherwise, it won't be reliable. What happens when a theory is not accepted by others or lacks evidence?

I had no idea it had been around that long! But I suppose in the long run that's not actually that old, but I was under the impression it was much younger than that for some reason.

Hello

Really enjoyed this — the range of demos, from phantoms to whole Xenopus embryos, was pretty compelling to see!

One thing I was wondering about wrt the phantom characterization is that it only tests resolution at a single scattering strength. Since the biological samples afterward span a much wider scattering mean free path, it seems like it'd add a lot to turn that into an actual sweep: same phantom, more tape layers --> FWHM plotted against ℓₛ.

Also noticed the skin/VML numbers (Fig 3n,o) land pretty close to the 1 ℓₛ phantom result, which hints performance might hold up flat across a decent scattering range — but that's more a hunch from comparing separate experiments than something directly demonstrated. Curious if this is something you're planning to add, or if you already have data that speaks to it.

Hello

Hello

Labeling obesity as an individual disease completely ignores things like food deserts and how corporations market junk food. It turns a systemic food access problem into a personal medical issue, pushing high-cost surgeries instead of fixing neighborhood food equity.

This assumes that the increase in contested illness diagnoses comes mostly from patients pushing for answers, but it feels more like systemic burnout and cost-cutting. Giving someone a vague label and basic palliative care seems like a convenient way for systems to stop running expensive tests on "difficult" cases.

hello ?

Using pre-pandemic death rates as the baseline benchmark assumes that "normal conditions" were equitable to begin with. In maternal-child health, baseline mortality for Black and Indigenous communities was already elevated by systemic racism, meaning this metric risks masking how existing structural inequities compounded during the crisis.

Framing vaccine hesitancy around individual beliefs assumes the core issue is a knowledge deficit rather than a rational response to structural abandonment. In rural areas, chronic clinic closures and spatial isolation destroy provider trust long before a vaccine ever reaches the market.

lac and thyA fail at 1 MOI but J23119 and thyA succeeds at 1 MOI. Why does thyA help mitigate the toxicity effects with only J23119? I theorize it has something to do with the higher baseline expression levels of J23119 compared to lac. Because lac is inducible, any gene driven by it will take some time after induction to ramp up while any gene driven by J23119 is always highly expressed.

Segregation and racism began and continued due to the color of skin, because of a legal system determining if their skin was non-white. But why did it matter so much?