Countries can get benefits from bolstering dying cities

This is smart, take advantage of the human disposition already at play

Who gets to allocate then? Just a lottery system, and how does one verify that it is for climate reasons (although maybe that isn't important)

requires so much individual generosity on the part of the country

We need a more strict definition of climate migrant b/c climate change so often works in tandem with other push factors, the scope needs to be narrower

Strategic migration is more economically or individually motivated while managed retreat will happen as that place slowly becomes unlivable (as opposed to rapidly in the case of disaster)

Avoiding the most random and devastating of climate change effects

TLDR: It makes everything worse bozos

This migration would have been internal, will it scale to international

What are the good outcomes for receiving communities

But you are actively fighting against the dominant american thought

Good fucking luck bro

Reviewer #1: Evidentiary Rating: Potentially Informative

Written Review: The authors claim that a spin-enhanced fluorescent nanodiamond (FND) lateral flow test for SARS-CoV-2 antigen detection achieves up to 1,100-fold greater sensitivity than conventional gold nanoparticle LFTs using identical antibodies. In a large, blinded clinical evaluation, the assay demonstrated 95.1% sensitivity (Ct ≤ 30) and 100% specificity, enabling SARS-CoV-2 detection on average two days earlier than conventional LFTs and within 0.6 days of RT-qPCR. They assert that this quantum-enhanced diagnostic platform could be adapted to other infectious and non-infectious diseases. 1. The study represents the first large-scale blinded clinical evaluation of spin-enhanced nanodiamond LFTs, moving beyond proof-of-concept to a performance assessment with real clinical samples. 2. The antibody screening process using biolayer interferometry is well executed, but screening on a single recombinant antigen source introduces potential epitope bias; inclusion of diverse antigen sources could further validate pair robustness. 3. The direct head-to-head comparison with in-house AuNP LFTs using identical reagents is a strong methodological choice, eliminating confounding variables common in cross-platform sensitivity claims. 4. While the assay achieves sub-pg/mL LoDs, residual non-specific binding limits ultimate sensitivity exploitation; more work on membrane chemistry or blocking strategies could push performance closer to the theoretical limit. 5. The sample size is adequate for preliminary evaluation, but a larger, more demographically and geographically diverse cohort is necessary to confirm real-world performance, especially in asymptomatic and early infection cases. 6. The assay’s 95.1% sensitivity at Ct ≤ 30 exceeds WHO “desirable” criteria, but the drop in sensitivity at lower viral loads (<10⁴ copies/mL) should be discussed in terms of balancing infectiousness detection with overdiagnosis risk. 7. The ROC, Bayesian regression, and infection dynamics modelling are sophisticated and well described, but providing raw Ct distribution histograms for positive samples would help readers assess viral load representativeness.

Reviewer #2: Evidentiary Rating: Reliable

Written Review: The authors present the development of a lateral flow assay prototype for the detection of SARS-CoV-2 nucleocapsid antigen, utilizing spin-enhanced nanodiamonds as fluorescent sensors. The manuscript provides sufficient detail regarding the evaluation of fluorescent nanodiamonds, optimization of anti-SARS-CoV-2 nucleocapsid capture and detection antibody pairs, assessment of assay sensitivity, and assay specificity for SARS-CoV-2 in comparison to other coronaviruses. Additionally, the authors include data demonstrating detection of SARS-CoV-2 nucleocapsid antigen in clinically relevant specimens, including nasal and nasopharyngeal swabs. The supplementary material includes a comparative analysis of the assay’s sensitivity relative to published data on various lateral flow tests, highlighting superior sensitivity achieved through the use of fluorescent nanodiamonds. Increased assay sensitivity may lead to earlier detection of SARS-CoV-2 infection thus providing opportunity for early intervention. Overall, the manuscript is well-organized, and the data are scientifically robust. I have a few minor comments for the authors’ consideration: 1. The authors state that the sensitivity of their assay is comparable to that of the Quanterix Simoa assay. Please provide a reference for the Simoa SARS-CoV-2 assay. 2. Could the authors briefly comment on the potential utility of this assay for public health applications such as surveillance or contact tracing?

When I was working in school board, all employees where put into groups to tackle specific issue more precisely. With the huge team broken up into small groups, more focused what put into each committee and its respective workloads.

Angol verzióban "After selecting the break date, by checking "Unique Interest," a manual break interest rate can be specified; otherwise, the system will use the accrued interest associated with the break date." helyett: After selecting the date of early withdrawal, by checking the box "Unique Interest," the interest rate of early withdrawal can be inserted manually; otherwise, the system will use the accrued interest associated with the early withdrawal date.

Haraway introduces a slogan she came up with “Make Kin Not Babies!” to establish her vision in terms of Chthulucene. She emphasizes the urgency of reimagining human relations beyond reproduction and genealogical ties. More so the part of “Make Kin” is to deliver the idea of taking responsibility for the life that we affect around us and to form a deeper connection with species other than ourselves. Haraway's purpose for this slogan is to project more awareness of the Anthropocene we currently live in and Chthulucene.

In this singular sentence, Haraway highlights the destruction of refugia as the “Inflection point” marking the Anthropocene. Building on Anna Tsing’s argument, she emphasizes that without places of refuge for recovery, all life on earth including us humans are living in a displacement and uncertainty of survival in the future. This point broadens the scope of the systemic crisis outlined by Haraway earlier, demonstrating that the Anthropocene is not only characterized by the deterioration of our ecosystem but also by the eradication of the very spaces that would prevent the mass extinction of life.

Haraway begins with a strong compelling opening that frames the argument she is trying to make. She proposes the question of the "inflection point” that changes the “game of life on earth”; she immediately emphasizes the gravity of the ecological and systemic crisis. She expands beyond the understanding of climate change to include toxic chemistry, mining, water depletion, ecosystem simplification, and mass extinctions to establish the interconnect and recursive nature of the collapse of our planet.

I don't completely agree with this thesis. Sure, when the goggles are put on you're tricking your brain into thinking what you are seeing is real. This is not necessarily true. What happens in the virtual world does not coincide with what happens in the world outside of the goggles. If we started to adopt this idea that the VR world is the real world then we would begin to neglect aspects of life and to an extent even ourselves and our health.

When I read this it actually makes sense. Like normally you would think VR is not real because you are putting glasses on to trick your mind a sense, but when you put them on, you are living in that moment/reality for however long the goggles are on. You are doing whatever conquest or activity within the VR making it a sort of reality. I do not know if I believe that the objects we interact with are real just because you normal are holding a controller.

I find it interesting to sya that reality exists just independently of us. I mean everyone lives a completely different live and we tend to forget that. This can also be referred to as sonder. I think sonder can also be applied to concept of if virtual reality is reality and where the truths are within reality. If reality is just wihin our minds, how does one go about trying to find out what is true? Just by living? I think we can create certain realties in our brain that may or may not come true.

My question here is why is virtual reality interaction so appealing to us? Sure using it in a pandemic sense could be nice, but think of the downsides. People could start interacting over virtual reality and never go back to in person conversation, just like a lot of people continued to work at home after COVID. We could lose the genuineness behind a conversation if we're only looking at a virtual version of someone.

While technology can help with traditional questions about value and ethics, some people have different opinions to what's right vs. wrong and better vs. worse. It all depends on each person's mindset.

Virtual worlds and real life worlds are completely different. While in virtual worlds you can make it so that your life is perfect and there would be nothing wrong in your world. However, in virtual you can't feel. You can't feel emotion; you can't feel anything. While in reality your worlds aren't perfect, you're able to feel emotion which is a big part in what makes us human.

I feel that teachers should be able to wear what they want to wear as long as they're covered or aren't wearing super-tight attire. If students are allowed to express themselves, then they should have the same right. I believe the only time this could be challenged is if you're working at a private school where they do have a dress code that you need to follow.

Living in a very diverse area, I feel that teachers need to be aware of how school policies affect all students. Legal decisions help guide schools to provide fair education, so that teachers can support every student’s learning and rights.

It is a bummer when you feel like a book would be great for students to read, but then the fear of repercussions takes over.

I feel like every teacher should build an environment where students feel encouraged to learn whether it's on their own or with classmates.

the society identity comes from peoples character, its environment, and political system, working together to form how that civilization grow and behaves.

I believe that this is why the Chinese became so successful and powerful in time. They have maintained their power for so long and by doing so they in the way that they have done this is so smart. People don't so much come into power just because they were born with it, it has to be earned and to earn it is to prove that they belong. They are well educated people who possibly came from nothing at one point but because they are educated and put in the work to their education they moved up in class and in position.

The speaker is Launfal’s mysterious lover and she is addressing King Arthur. She acknowledges her love for Launfal and defends him. She explains that Launfal was unfairly treated in the court. Her goal is to plead his case and persuade the King to understand Launfal’s actions in a more compassionate way.

The mysterious lady intervenes directly in Launfal’s trial, defending him. The effect on the reader is sympathy and admiration. I really liked these lines because it feels like a really big turning point in the story.

The things I noticed was tone and repetition. The tone of these lines is respectful and obviously thought out. She says "Sire" and "vassals" these both have an elevated tone of respect. There is also a repetive theme of misjudgment in these lines. She says "misprized," "turned to blame," and "over hasty." All of those words lead the reader to the idea that something morally wrong is going on.

Overall, these lines were my really good because Marie de France elevates the voice of a woman. She does this not just to defend Launfal, but to critique a system where justice can be corrupted by jealousy!!

The maiden is speaking to Launfal but is referring him as "fair friend." Before the maiden and Launfal are about to go separate ways the maiden is making a statemen, about how Launfal can see her whenever he wants but his companions can never know of it or her. Where it says "but I must never be known of your comrades, nor must they ever learn my speech" she is setting clear boundaries about how they meet, when they meet, and this secret is clearly between them two. Because the two of them are a secret it gives the idea that their "relationship" is intimate. The words of "Well I deem" and "Without reproach or shame of men" gives the language from the medieval time.

It's not an accident that workers get locked out of knowledge. This hits me hard as a teacher—the gatekeeping of knowledge feels designed. Keep workers (or students) ignorant, and you keep them compliant. How am I supposed to help this be a better world for everyone if I never have the vision of its potential?

high-tech isn't required to enforce domination. Sometimes the simplest tools are the most insidious. We underestimate the genius of simplicity in control—pacing is everything in schools too, from bell schedules to testing windows. Behaviorism teaches us how to condition the masses, and setting forth routines and expectations helps administrators assert a culture of control.

Tech is never neutral. This is exactly why I'm skeptical of ed-tech "solutions". The tool might be brilliant, but who owns it, and how do they wield it? That's the real story. AI as a tutor becomes a uniform process of indoctrination as it guides you to think like the programmers want you to think, and refuses to answer questions you have based on an 'ethical' pretense. Who's ethics matter to me? Someone else's or my own?

The machine isn't just replacing labor, it's erasing identities built on skill. I get why strikes weren't just about wages—they were about dignity and purpose being stripped away.

I'm flagging this because Marx nails the paradox of innovation: tech that could free us instead undermines us. To me, this feels like deja vu in education with AI—we're told these tools are "empowering," but in practice they're just ways to deskill and control teachers. I see the same rhetoric today. Capital sells "efficiency", but the real play is control over labor.

An example would be helpful and appreciated.

for better or worse, probably a reflection of social and cultural priorities. An artifact of the present?

I would like some dressing with this word salad. I have no idea what this means.

So crietria for better or worse organization on this model comes from domain specific practices, producers, assumptions, theories. Sound like this is good for ease of use. Sounds potentially troublesome in that it might lend itself towards conservation of discipline specific categories which are sometimes the product of inherited prejudices, practices, not really reflective of best practices?

What criteria establish which forms of classification are better or worse? Is it a domain specific thing--most useful according to expert concensus of bird watchers, or are there more general criteria that can apply across "knowledge domains". In philosophy, we usually are conducting research in one of major subject areas; metaphysics, epistemology, ethics, or the history of all the above. This makes talking about what you do and finding relevant sources easyish, but it has also had a deep consequence on questions/research topics that seem philosophical but don't quite fit the traditional subject areas.

This reminds me of the ways in which one's theoretical commitments, or interpretive school, in the practice of history, determines which pools of evidence and modes of explanation, will help you account for/ reconstruct your research object. You could be studying the same phenomenon, but use different units of information to understand it. Ie. written published texts, vs. statistics about population health, marriage, etc.

Just figuring out the technology here. I am old and a bit of a luddite.

Might as well take the occasion to say, as someone with a philosophical background, just learning the vocabulary of Information Science, I am finding the questions at the heart of Knowledge Organization evocative of some of the deepest questions in epistemology, metaphysics, philosophy of mind, and philosophy of language. Is there a mind independant world? How do we know it? What is Knowledge anyways? What role does our dependance on language have in how we know and understand the world? To what extent do our categories of knowledge construct the world? Do they reflect the world as it really is? I keep making associations to Ian Hacking's work, "The Social Construction of What?"

Is the relative frequency of different alleles the best way to measure evolution in action?

Hard to accomplish

This I definitely agree with, and it does not seem as reliant on precedented legal definitions

The counter argument here is that it will sooooooo expand the definition of refugee ironically "opening the floodgates"

Climate change is both a multiplying factor for why families migrate as well a a direct reason why migrating itself is so difficult

Vicious cycle

Talked about this in class

Internal migration

This is persecuation along the lines of a group so it makes them count as refugees

Sort of like what the taliban is doing, you have to bend a knee to power but also the hand that feeds you

Climate change means that cartel life is sometimes the only choice for workers

crazy theme

This feels like maybe it could apply also maybe a particular social group could be those affected by a climate crisis

But because the gov. is saying a blanket no its not persecution?

This unfortunately defo does not count

Could this not qualify as a refugee status because of persecution

What steps would the US need to make so that ehtnic studies is no longer required to fill in the gaps of our eurocentric edu?

• Including grater variety of historical figures and notable individuals in terms of race, gender, and background
• Share lesser known figures, events, and countries that have innfluenced our current society and innovations we enjoy today beyond a eurocentric view

Signing up for classes

Signing up for classes

Being officially a student at school

Signing up for classes

Based on this sentence and really the rest of the page/case, the state of New Jersey can't use tax dollars to support schools that teach a certain religion (ex. like Catholic schools). This would go against the fundamental principles of the First Amendment. Also, you can't force a religion onto people; people are allowed to believe what they believe in (that's a fundamental right and freedom).

First Amendment: Freedom of religion, speech, assembly, the press, etc.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

Horizontal gene transfer is the transmission of genetic material between organisms through ways other than reproduction. Frequent in prokaryotes, this mode of genetic exchange is scarcer in eukaryotes, especially in multicellular eukaryotes. Furthermore, the mechanisms involved in eukaryotic HGT are unknown. This article by Banerjee et al. claims that HGT occurs massively between cells of multicellular organisms. According to this study, the cell free chromatin particles (cfChPs) that are massively released by dying cells are incorporated in the nucleus of neighboring cells.

The reviewer is mistaken. We do not claim that the internalized cfChPs are incorporated into the nucleus. We show throughout the paper that the cfChPs perform their novel functions autonomously outside the genome without being incorporated into the nucleus. This is clearly seen in all our chromatin fibre images, metaphase spreads and our video abstract. Occasionally, when the cfChPs fluorescent signal overlie the chromosomes, we have been careful to state that the cfChPs are associated with the chromosomes without implying that they have integrated.

These cfChPs are frequently rearranged and amplified to form concatemers, they are made of open chromatin, expressed, and capable of producing proteins. Furthermore, the study also suggests that cfChPs transmit transposable elements (TEs) between cells on a regular basis, and that these TEs can transpose, multiply, and invade receiving cells. These conclusions are based on a series of experiments consisting in releasing cfChPs isolated from various human sera into the culture medium of mouse cells, and using FISH and immunofluorescence to monitor the state and fate of cfChPs after several passages of the mouse cell line.

Strengths:

The results presented in this study are interesting because they may reveal unsuspected properties of some cell types that may be able to internalize free-circulating chromatin, leading to its chromosomal incorporation, expression, and unleashing of TEs. The authors propose that this phenomenon may have profound impacts in terms of diseases and genome evolution. They even suggest that this could occur in germ cells, leading to within-organism HGT with long-term consequences.

Again the reviewer makes the same mistake. We do not claim that the internalized cfChPs are incorporated into the chromosomes. We have addressed this issue above.

We have a feeling that the reviewer has not understood our work – which is the discovery of “satellite genomes” which function autonomously outside the nuclear genome.

Weaknesses:

The claims of massive HGT between cells through internalization of cfChPs are not well supported because they are only based on evidence from one type of methodological approach: immunofluorescence and fluorescent in situ hybridization (FISH) using protein antibodies and DNA probes. Yet, such strong claims require validation by at least one, but preferably multiple, additional orthogonal approaches. This includes, for example, whole genome sequencing (to validate concatemerization, integration in receiving cells, transposition in receiving cells), RNA-seq (to validate expression), ChiP-seq (to validate chromatin state).

We disagree with the reviewer that our study is incomplete because we did not perform whole genome sequencing. Tens of thousands of genomes have been sequenced, and yet they have failed to detect the presence of the numerous “satellite genomes” that we describe in our paper. To that extent whole genome sequencing has proved to be an inappropriate approach. Rather, the reviewer should have commended us for the numerous control experiments that we have done to ensure that our FISH probes and antibodies are target specific and do not cross-react.

Should HGT through internalization of circulating chromatin occur on a massive scale, as claimed in this study, and as illustrated by the many FISH foci observed on Fig 3 for example, one would expect that the level of somatic mosaicism may be so high that it would prevent assembling a contiguous genome for a given organism. Yet, telomere-to-telomere genomes have been produced for many eukaryote species, calling into question the conclusions of this study.

The reviewer has raised a related issue below and we have responded to both of them together.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

I thank the reviewers for taking my comments and those of the other reviewer into account and for adding new material to this new version of the manuscript. Among other modifications/additions, they now mention that they think that NIH3T3 cells treated with cfChPs die out after 250 passages because of genomic instability which might be caused by horizontal transfer of cfChPs DNA into the genome of treated cells (pp. 45-46, lines 725-731). However, no definitive formal proof of genomic instability and horizontal transfer is provided.

We mention that the NIH3T3 cells treated with cfChPs die out after 250 passages in response to the reviewer’s earlier comment “Should HGT through internalization of circulating chromatin occur on a massive scale, as claimed in this study, and as illustrated by the many FISH foci observed in Fig 3 for example, one would expect that the level of somatic mosaicism may be so high that it would prevent assembling a contiguous genome for a given organism”.

We have agreed with the reviewer and have simply speculated that the cells may die because of extreme genomic instability. We have left it as a speculation without diverting our paper in a different direction to prove genomic instability.

The authors now refer to an earlier study they conducted in which they Illumina-sequenced NIH3T3 cells treated with cfChPs (pp. 48, lines. 781-792). This study revealed the presence of human DNA in the mouse cell culture. However, it is unclear to me how the author can conclude that the human DNA was inside mouse cells (rather than persisting in the culture medium as cfChPs) and it is also unclear how this supports horizontal transfer of human DNA into the genome of mouse cells. Horizontal transfer implies integration of human DNA into mouse DNA, through the formation of phosphodiester bounds between human nucleotides and mouse nucleotides. The previous Illumina-sequencing study and the current study do not show that such integration has occured. I might be wrong but I tend to think that DNA FISH signals showing that human DNA lies next to mouse DNA does not necessarily imply that human DNA has integrated into mouse DNA. Perhaps such signals could result from interactions at the protein level between human cfChPs and mouse chromatin?

With due respect, our earlier genome sequencing study that the reviewer refers to was done on two single cell clones developed following treatment with cfChPs. So, the question of cfChPs lurking in the culture medium does not arise.

The authors should be commended for doing so many FISH experiments. But in my opinion, and as already mentioned in my earlier review of this work, horizontal transfer of human DNA into mouse DNA should first be demonstrated by strong DNA sequencing evidence (multiple long and short reads supporting human/mouse breakpoints; discarding technical DNA chimeras) and only then eventually confirmed by FISH.

As mentioned earlier, we disagree with the reviewer that our study is incomplete because we did not perform whole genome sequencing. Tens of thousands of genomes have been sequenced, and yet they have failed to detect the presence of the numerous “satellite genomes” that we describe in our paper. To that extent whole genome sequencing has proved to be an inappropriate approach. Rather, the reviewer should have commended us for the numerous control experiments that we have done to ensure that our FISH probes and antibodies are target specific and do not cross-react.

Regarding my comment on the quantity of human cfChPs that has been used for the experiments, the authors replied that they chose this quantity because it worked in a previous study. Could they perhaps explain why they chose this quantity in the earlier study? Is there any biological reason to choose 10 ng and not more or less? Is 10 ng realistic biologically? Could it be that 10 ng is orders of magnitude higher than the quantity of cfChPs normally circulating in multicellular organisms and that this could explain, at least in part, the results obtained in this study?

The reviewer again raises the same issue to which we have already addressed in our revised manuscript. To quote “We chose to use 10ng based on our earlier report in which we had obtained robust biological effects such as activation of DDR and activation of apoptotic pathways using this concentration of cfChPs (Mittra I et. al., 2015)”.

It is also mentioned in the response that RNA-seq has been performed on mouse cells treated with cfChPs, and that this confirms human-mouse fusion (genomic integration). Since these results are not included in the manuscript, I cannot judge how robust they are and whether they reflect a biological process rather than technical issues (technical chimeras formed during the RNA-seq protocol is a well-known artifact). In any case, I do not think that genomic integration can be demonstrated through RNA-seq as junction between human and mouse RNA could occur at the RNA level (i.e. after transcription). RNA-seq could however show whether human-mouse chimeras that have been validated by DNA-sequencing are expressed or not.

We did perform transcriptome sequencing as suggested earlier by the reviewer, but realized that the amount of material required to be incorporated into the manuscript to include “material and methods”, “results”, “discussion”, “figures” and “legends to figures” and “supplementary figures and tables” would be so massive that it will detract from the flow of our work and hijack it in a different direction. We have, therefore, decided to publish the transcriptome results as a separate manuscript.

Given these comments, I believe that most of the weaknesses I mentioned in my review of the first version of this work still hold true.

An important modification is that the work has been repeated in other cell lines, hence I removed this criticism from my earlier review.

Additional changes made

(1) We have now rewritten the “Abstract” to 250 words to fit in eLife’s instructions. (It was not possible to reduce the word count further.

(2) We have provided the Video 1 as separate file instead of link.

(3) Some of Figure Supplements (which were stand-alone) are now given as main figures. We have re-arranged Figures and Figure Supplements in accordance with eLife’s instructions.

(4) We have now provided a list of the various cell lines used in this study, their tissue origin and procurement source in Supplementary File 3.

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

Horizontal gene transfer is the transmission of genetic material between organisms through ways other than reproduction. Frequent in prokaryotes, this mode of genetic exchange is scarcer in eukaryotes, especially in multicellular eukaryotes. Furthermore, the mechanisms involved in eukaryotic HGT are unknown. This article by Banerjee et al. claims that HGT occurs massively between cells of multicellular organisms. According to this study, the cell free chromatin particles (cfChPs) that are massively released by dying cells are incorporated in the nucleus of neighboring cells. These cfChPs are frequently rearranged and amplified to form concatemers, they are made of open chromatin, expressed, and capable of producing proteins. Furthermore, the study also suggests that cfChPs transmit transposable elements (TEs) between cells on a regular basis, and that these TEs can transpose, multiply, and invade receiving cells. These conclusions are based on a series of experiments consisting in releasing cfChPs isolated from various human sera into the culture medium of mouse cells, and using FISH and immunofluorescence to monitor the state and fate of cfChPs after several passages of the mouse cell line.

Strengths:

The results presented in this study are interesting because they may reveal unsuspected properties of some cell types that may be able to internalize free-circulating chromatin, leading to its chromosomal incorporation, expression, and unleashing of TEs. The authors propose that this phenomenon may have profound impacts in terms of diseases and genome evolution. They even suggest that this could occur in germ cells, leading to within-organism HGT with long-term consequences.

Weaknesses:

The claims of massive HGT between cells through internalization of cfChPs are not well supported because they are only based on evidence from one type of methodological approach: immunofluorescence and fluorescent in situ hybridization (FISH) using protein antibodies and DNA probes. Yet, such strong claims require validation by at least one, but preferably multiple, additional orthogonal approaches. This includes, for example, whole genome sequencing (to validate concatemerization, integration in receiving cells, transposition in receiving cells), RNA-seq (to validate expression), ChiP-seq (to validate chromatin state).

We have responded to this criticism under “Reviewer #1 (Recommendations for the authors, item no. 1-4)”.

Another weakness of this study is that it is performed only in one receiving cell type (NIH3T3 mouse cells). Thus, rather than a general phenomenon occurring on a massive scale in every multicellular organism, it could merely reflect aberrant properties of a cell line that for some reason became permeable to exogenous cfChPs. This begs the question of the relevance of this study for living organisms.

We have responded to this criticism under “Reviewer #1 (Recommendations for the authors, item no. 6)”.

Should HGT through internalization of circulating chromatin occur on a massive scale, as claimed in this study, and as illustrated by the many FISH foci observed in Fig 3 for example, one would expect that the level of somatic mosaicism may be so high that it would prevent assembling a contiguous genome for a given organism. Yet, telomere-to-telomere genomes have been produced for many eukaryote species, calling into question the conclusions of this study.

The reviewer is right in expecting that the level of somatic mosaicism may be so high that it would prevent assembling a contiguous genome. This is indeed the case, and we find that beyond ~ 250 passages the cfChPs treated NIH3T3 cells begin to die out apparently become their genomes have become too unstable for survival. This point will be highlighted in the revised version (pp. 45-46, lines 725-731).

Reviewer #2 (Public review):

I must note that my comments pertain to the evolutionary interpretations rather than the study's technical results. The techniques appear to be appropriately applied and interpreted, but I do not feel sufficiently qualified to assess this aspect of the work in detail.

I was repeatedly puzzled by the use of the term "function." Part of the issue may stem from slightly different interpretations of this word in different fields. In my understanding, "function" should denote not just what a structure does, but what it has been selected for. In this context, where it is unclear if cfChPs have been selected for in any way, the use of this term seems questionable.

We agree. We have removed the term “function” wherever we felt we had used it inappropriately.

Similarly, the term "predatory genome," used in the title and throughout the paper, appears ambiguous and unjustified. At this stage, I am unconvinced that cfChPs provide any evolutionary advantage to the genome. It is entirely possible that these structures have no function whatsoever and could simply be byproducts of other processes. The findings presented in this study do not rule out this neutral hypothesis. Alternatively, some particular components of the genome could be driving the process and may have been selected to do so. This brings us to the hypothesis that cfChPs could serve as vehicles for transposable elements. While speculative, this idea seems to be compatible with the study's findings and merits further exploration.

We agree with the reviewer’s viewpoint. We have replaced the term “predatory genome” with a more realistic term “satellite genome” in the title and throughout the manuscript. We have also thoroughly revised the discussion section and elaborated on the potential role of LINE-1 and Alu elements carried by the concatemers in mammalian evolution. (pp. 46-47, lines 743-756).

I also found some elements of the discussion unclear and speculative, particularly the final section on the evolution of mammals. If the intention is simply to highlight the evolutionary impact of horizontal transfer of transposable elements (e.g., as a source of new mutations), this should be explicitly stated. In any case, this part of the discussion requires further clarification and justification.

As mentioned above, we have revised the “discussion” section taking into account the issues raised by the reviewer and highlighted the potential role of cfChPs in evolution by acting as vehicles of transposable elements.

In summary, this study presents important new findings on the behavior of cfChPs when introduced into a foreign cellular context. However, it overextends its evolutionary interpretations, often in an unclear and speculative manner. The concept of the "predatory genome" should be better defined and justified or removed altogether. Conversely, the suggestion that cfChPs may function at the level of transposable elements (rather than the entire genome or organism) could be given more emphasis.

As mentioned above, we have replaced the term “predatory genome” with “satellite genome” and revised the “discussion” section taking into account the issues raised by the reviewer.

Reviewer #1 (Recommendations for the authors):

(1) I strongly recommend validating the findings of this study using other approaches. Whole genome sequencing using both short and long reads should be used to validate the presence of human DNA in the mouse cell line, as well as its integration into the mouse genome and concatemerization. Breakpoints between mouse and human DNA can be searched in individual reads. Finding these breakpoints in multiple reads from two or more sequencing technologies would strengthen their biological origin. Illumina and ONT sequencing are now routinely performed by many labs, such that this validation should be straightforward. In addition to validating the findings of the current study, it would allow performance of an in-depth characterization of the rearrangements undergone by both human cfChPs and the mouse genome after internalization of cfChPs, including identification of human TE copies integrated through bona fide transposition events into the mouse genome. New copies of LINE and Alu TEs should be flanked by target site duplications. LINE copies should be frequently 5' truncated, as observed in many studies of somatic transposition in human cells.

(2) Furthermore, should the high level of cell-to-cell HGT detected in this study occur on a regular basis within multicellular organisms, validating it through a reanalysis of whole genome sequencing data available in public databases should be relatively easy. One would expect to find a high number of structural variants that for some reason have so far gone under the radar.

(3) Short and long-read RNA-seq should be performed to validate the expression of human cfChPs in mouse cells. I would also recommend performing ChIP-seq on routinely targeted histone marks to validate the chromatin state of human cfChPs in mouse cells.

(4) The claim that fused human proteins are produced in mouse cells after exposing them to human cfChPs should be validated using mass spectrometry.

The reviewer has suggested a plethora of techniques to validate our findings. Clearly, it is neither possible to undertake all of them nor to incorporate them into the manuscript. However, as suggested by the reviewer, we did conduct transcriptome sequencing of cfChPs treated NIH3T3 cells and were able to detect the presence of human-human fusion sequences (representing concatemerisation) as well as human-mouse fusion sequences (representing genomic integration). However, we realized that the amount of material required to be incorporated into the manuscript to include “material and methods”, “results”, “discussion”, “figures” and “legends to figures” and “supplementary figures and tables” would be so massive that it will detract from the flow of our work and hijack it in a different direction. We have, therefore, decided to publish the transcriptome results as a separate manuscript. However, to address the reviewer’s concerns we have now referred to results of our earlier whole genome sequencing study of NIH3T3 cells similarly treated with cfChPs wherein we had conclusively detected the presence of human DNA and human Alu sequences in the treated mouse cells. These findings have now been added as an independent paragraph (pp. 48, lines. 781-792).

(5) It is unclear from what is shown in the paper (increase in FISH signal intensity using Alu and L1 probes) if the increase in TE copy number is due to bona fide transposition or to amplification of cfChPs as a whole, through mechanisms other than transposition. It is also unclear whether human TEs end up being integrated into the neighboring mouse genome. This should be validated by whole genome sequencing.

Our results suggest that TEs amplify and increase their copy number due to their association with DNA polymerase and their ability to synthesize DNA (Figure 14a and b). Our study design cannot demonstrate transposition which will require real time imaging.

The possibility of incorporation of TEs into the mouse genome is supported by our earlier genome sequencing work, referred to above, wherein we detected multiple human Alu sequences in the mouse genome (pp. 48, lines. 781-792).

(6) In order to be able to generalize the findings of this study, I strongly encourage the authors to repeat their experiments using other cell types.

We thank the reviewer for this suggestion. We have now used four different cell lines derived from four different species and demonstrated that horizontal transfer of cfChPs occur in all of them suggesting that it is a universal phenomenon. (pp. 37, lines 560-572) and (Supplementary Fig. S14a-d).

We have also mentioned this in the abstract (pp. 3, lines 52-54).

(7) Since the results obtained when using cfChPs isolated from healthy individuals are identical to those shown when using cfChPs from cancer sera, I wonder why the authors chose to focus mainly on results from cancer-derived cfChPs and not on those from healthy sera.

Most of the experiments were conducted using cfChPs isolated from cancer patients because of our especial interest in cancer, and our earlier results (Mittra et al., 2015) which had shown that cfChPs isolated from cancer patients had significantly greater activity in terms of DNA damage and activation of apoptotic pathways than those isolated from healthy individuals. We have now incorporated the above justification on (pp. 6, lines. 124-128).

(8) Line 125: how was the 10-ng quantity (of human cfChPs added to the mouse cell culture) chosen and how does it compare to the quantity of cfChPs normally circulating in multicellular organisms?

We chose to use 10ng based on our earlier report in which we had obtained robust biological effects such as activation of DDR and apoptotic pathways using this concentration of cfChPs (Mittra I et. al. 2015). We have now incorporated the justification of using this dose in our manuscript (pp. 51-52, lines. 867-870).

(9) Could the authors explain why they repeated several of their experiments in metaphase spreads, in addition to interphase?

We conducted experiments on metaphase spreads in addition to those on chromatin fibres because of the current heightened interest in extra-chromosomal DNA in cancer, which have largely been based on metaphase spreads. We were interested to see how the cfChP concatemers might relate to the characteristics of cancer extrachromosomal DNA and whether the latter in fact represent cfChPs concatemers acquired from surrounding dying cancer cells. We have now mentioned this on pp. 7, lines 150-155.

(10) Regarding negative controls consisting in checking whether human probes cross-react with mouse DNA or proteins, I suggest that the stringency of washes (temperature, reagents) should be clearly stated in the manuscript, such that the reader can easily see that it was identical for controls and positive experiments.

We were fully aware of these issues and were careful to ensure that washing steps were conducted meticulously. The careful washing steps have been repeatedly emphasized under the section on “Immunofluorescence and FISH” (pp. 54-55, lines. 922-944).

(11) I am not an expert in Immuno-FISH and FISH with ribosomal probes but it can be expected that ribosomal RNA and RNA polymerase are quite conserved (and thus highly similar) between humans and mice. A more detailed explanation of how these probes were designed to avoid cross-reactivity would be welcome.

We were aware of this issue and conducted negative control experiment to ensure that the human ribosomal RNA probe and RNA polymerase antibody did not cross-react with mouse. Please see Supplementary Fig. S4c.

(12) Finally, I could not understand why the cfChPs internalized by neighboring cells are called predatory genomes. I could not find any justification for this term in the manuscript.

We agree and this criticism has also been made by #Reviewer 2. We have now replaced the term “predatory” genomes with “satellite” genomes.

Reviewer #2 (Recommendations for the authors):

(1) P2 L34: The term "role" seems to imply "what something is supposed to do" (similar to "function"). Perhaps "impact" would be more neutral. Additionally, "poorly defined" is vague-do you mean "unknown"?

We thank the reviewer for this suggestion. We have now rephrased the sentence to read “Horizontal gene transfer (HGT) plays an important evolutionary role in prokaryotes, but it is thought to be less frequent in mammals.” (pp. 2, lines. 26-27).

(2) P2 L35: It seems that the dash should come after "human blood."

Thank you, we have changed the position of the dash (pp. 2, line. 29).

(3) P2 L37: Must we assume these structures have a function? Could they not simply be side effects of other processes?

We think this is a matter of semantics, especially since we show that cfChPs once inside the cell perform many functions such as replication, DNA synthesis, RNA synthesis, protein synthesis etc. We, therefore, think the word “function” is not inappropriate.

(4) Abstract: After reading the abstract, I am unclear on the concept of a "predatory genome." Based on the summarized results, it seems one cannot conclude that these elements provide any adaptive value to the genome.

We agree. We have now replaced the term “predatory” genomes with a more realistic term viz. “satellite” genomes.

(5) Video abstract: The video abstract does not currently stand on its own and needs more context to be self-explanatory.

Thank you for pointing this out. We have now created a new and much more professional video with more context which we hope will meet with the reviewer’s approval.

(6) P4 L67: Again, I am uncertain that HGT should be said to have "a role" in mammals, although it clearly has implications and consequences. Perhaps "role" here is intended to mean "consequence"?

We have now changed the sentence to read as follows “However, defining the occurrence of HGT in mammals has been a challenge” (pp. 4, line. 73).

(7) P6 L111: The phrase "to obtain a new perspective about the process of evolution" is unclear. What exactly is meant by this statement?

We have replaced this sentence altogether which now reads “The results of these experiments are presented in this article which may help to throw new light on mammalian evolution, ageing and cancer” (pp. 5-6, lines 116-118).

(8) P38 L588: The term "predatory genome" has not been defined, making it difficult to assess its relevance.

This issue has been addressed above.

(9) P39 L604: The statement "transposable elements are not inherent to the cell" suggests that some TEs could originate externally, but this does not rule out that others are intrinsic. In other words, TEs are still inherent to the cell.

This part of the discussion section has been rewritten and the above sentence has been deleted.

(10) P39 L609: The phrase "may have evolutionary functions by acting as transposable elements" is unclear. Perhaps it is meant that these structures may serve as vehicles for TEs?

This sentence has disappeared altogether in the revised discussion section.

(11) P41 L643: "Thus, we hypothesize ... extensively modified to act as foreign genetic elements." This sentence is unclear. Are the authors referring to evolutionary changes in mammals in general (which overlooks the role of standard mutational processes)? Or is it being proposed that structural mutations (including TE integrations) could be mediated by cfChPs in addition to other mutational mechanisms?

We have replaced this sentence which now reads “Thus, “within-self” HGT may occur in mammals on a massive scale via the medium of cfChP concatemers that have undergone extensive and complex modifications resulting in their behaviour as “foreign” genetic elements” (pp. 47, lines 763-766).

(12) P41 L150: The paragraph beginning with "It has been proposed that extreme environmental..." transitions too abruptly from HGT to adaptation. Is it being proposed that cfChPs are evolutionary processes selected for their adaptive potential? This idea is far too speculative at this stage and requires clarification.

We agree. This paragraph has been removed.

(13) P43 L681: This summary appears overly speculative and unclear, particularly as the concept of a "predatory genome" remains undefined and thus cannot be justified. It suggests that cfChPs represent an alternative lifestyle for the entire genome, although alternative explanations seem far more plausible at this point.

We have now replaced the term “predatory” genome with “satellite” genome. The relevant part of the summary section has also been partially revised (pp. 49-50, lines 817-831).

Changes independent of reviewers’ comments.

We have made the following additions / modifications.

(1) The abstract has been modified and it’s “conclusion” section has been rewritten.

(2) Section 1.14 has been newly added together with accompanying Figures 15 a,b and c.

(3) The “Discussion” section has been greatly modified and parts of it has been rewritten.

Self regulation = using reading & cognitive skills that work for each iniviual, an being aware of these strategies.

In this way, models could be said to be dynamic according to how often they're 'fed' or designed around training data. Does this mean every time a ML model or AI resource is 'updated,' it is referring to the model having been given more data to model and extract patterns from?

As a bioinformatics student, I often find that most Bioinformaticians have assumed the role of experienced artists. It seems the 'composing' in industry tends to fall to software engineers, and 'listening' to subject-matter expert scientists well-versed in theory and application. As a Bioinformatician, you are almost like a middleman between the two other professionals, translating algorithms and leveraging their outputs. It would appear then that a valuable skill to have as a bioinformatics specialist lies in your ability to describe and explain the inner workings of the ML models. This is certainly something I'll pay special attention to during my degree at Guelph.

but there is no difference between positive and negative interaction with a social media post

talk about the social media platform that tried adding a dislike button

you will see these questions 3 times before the test!

This course provides great insight and learning into the world of web design and focuses on user satisfaction, which is important when designing any web application

locke not accepted by christian theologians

Author is trying to tell the audience that this isn't right. Balfour explains what the Egyptians might feel but have we ever heard from the Egyptians themselves?

Does it come from the people who want to override or by a particular person.

Orientalism is what causes the conflict not the place itself.

Wasn't just an idea.

The way westerners view the east, which turns into colonialism.

Challenging to address high error rate for minority groups

minorities under-represented in data used for training leads to performance issues

Would this issue persists if two human translators (one translating English -> Turkish, the other translating Turkish -> English) were involved instead?

ML emphasizes societal stereotypes.

Further discussions can be had about cameras that capture infra-red (thermal/night vision) and ultra-violet or x-ray wavelengths. Often these images are "colorized" based on the preference of some graphic artist (e.g., colours added to black-hole images, Cosmic micro-wave background, etc.). The colour scheme used can also be subjective.

I have similar gripe about the multi-factor authentication systems (MFAs) being used ubiquitously.

This directed graph is similar to ones used for mathematical modelling, except "Individuals" usually replaced by "Computations"

definition: statistical bias

flaws in only relying on examples.

The need for "learning" over "branching"

Abolitionist against expansionism

Southern support for expansion

Key Point.

National Security concerns over Manifest Destiny

Key Point

interesting

eLife Assessment

This study presents valuable findings on the ability of a state-of-the-art method – temporally delayed linear modelling (TDLM) – to detect the replay of sequences in human memory. The investigation provides convincing evidence that TDLM has limitations in its sensitivity to detect replay when being applied to extended (minutes-long) rest periods, though a more thorough treatment of the relationship to prior positive findings would make the demonstration even stronger. The work will be of particular interest to researchers investigating memory reactivation in humans, especially using iEEG, MEG, and EEG.

Reviewer #1 (Public review):

Summary:

Participants learned a graph-based representation, but, contrary to the hypotheses, failed to show neural replay shortly after. This prompted a critical inquiry into temporally delayed linear modeling (TDLM)--the algorithm used to find replay. First, it was found that TDLM detects replay only at implausible numbers of replay events per second. Second, it detects replay-to-cognition correlations only at implausible densities. Third, there are concerning baseline shifts in sequenceness across participants. Fourth, spurious sequences arise in control conditions without a ground truth signal. Fifth, when reframing simulations previously published, similar evidence is apparent.

Strengths:

(1) This work is meticulous and meets a high standard of transparency and open science, with preregistration, code and data sharing, external resources such as a GUI with the task and material for the public.

(2) The writing is clear, balanced, and matter-of-fact.

(3) By injecting visually evoked empirical data into the simulation, many surface-level problems are avoided, such as biological plausibility and questions of signal-to-noise ratio.

(4) The investigation of sequenceness-to-cognition correlations is an especially useful add-on because much of the previous work uses this to make key claims about replay as a mechanism.

Weaknesses:

Many of the weaknesses are not so much flaws in the analyses, but shortcomings when it comes to interpretation and a lack of making these findings as useful as they could be.

(1) I found the bigger picture analysis to be lacking. Let us take stock: in other work, during active cognition, including at least one study from the Authors, TDLM shows significance sequenceness. But the evidence provided here suggests that even very strong localizer patterns injected into the data cannot be detected as replay except at implausible speeds. How can both of these things be true? Assuming these analyses are cogent, do these findings not imply something more destructive about all studies that found positive results with TDLM?

(2) All things considered, TDLM seems like a fairly 'vanilla' and low-assumption algorithm for finding event sequences. It is hard to see intuitively what the breaking factor might be; why do the authors think ground truth patterns cannot be detected by this GLM-based framework at reasonable densities?

(3) Can the authors sketch any directions for alternative methods? It seems we need an algorithm that outperforms TDLM, but not many clues or speculations are given as to what that might look like. Relatedly, no technical or "internal" critique is provided. What is it about TDLM that causes it to be so weak?

Addressing these points would make this manuscript more useful, workable, and constructive, even if they would not necessarily increase its scientific breadth or strength of evidence.

Reviewer #2 (Public review):

Summary:

Kern et al. investigated whether temporally delayed linear modeling (TDLM) can uncover sequential memory replay from a graph-learning task in human MEG during an 8-minute post-learning rest period. After failing to detect replay events, they conduct a simulation study in which they insert synthetic replay events, derived from each participant's localizer data, into a control rest period prior to learning. The simulations suggest that TDLM only reveals sequences when replay occurs at very high densities (> 80 per minute) and that individual differences in baseline sequenceness may lead to spurious and/or lackluster correlations between replay strength and behavior.

Strengths:

The approach is extremely well documented and rigorous. The authors have done an excellent job re-creating the TDLM methodology that is most commonly used, reporting the different approaches and parameters that they used, and reporting their preregistrations. The hybrid simulation study is creative and provides a new way to assess the efficacy of replay decoding methods. The authors remain measured in the scope/applicability of their conclusions, constructive in their discussion, and end with a useful set of recommendations for how to best apply TDLM in future studies. I also want to commend this work for not only presenting a null result but thoroughly exploring the conditions under which such a null result is expected. I think this paper is interesting and will be generally quite useful for the field, but I believe it also has a number of weaknesses that, if addressed, could improve it further.

Weaknesses:

The sample size is small (n=21, after exclusions), even for TDLM studies (which typically have somewhere between 25-40 participants). The authors address this somewhat through a power analysis of the relationship between replay and behavioral performance in their simulations, but this is very dependent on the assumptions of the simulation. Further, according to their own power analysis, the replay-behavior correlations are seriously underpowered (~10% power according to Figure 7C), and so if this is to be taken at face value, their own null findings on this point (Figure 3C) could therefore just reflect undersampling as opposed to methodological failure. I think this point needs to be made more clearly earlier in the manuscript. Relatedly, it would be very useful if one of the recommendations that come out of the simulations in this paper was a power analysis for detecting sequenceness in general, as I suspect that the small sample size impacts this as well, given that sequenceness effects reported in other work are often small with larger sample sizes. Further, I believe that the authors' simulations of basic sequenceness effects would themselves still suffer from having a small number of subjects, thereby impacting statistical power. Perhaps the authors can perform a similar sort of bootstrapping analysis as they perform for the correlation between replay and performance, but over sequenceness itself?

The task paradigm may introduce issues in detecting replay that are separate from TDLM. First, the localizer task involves a match/mismatch judgment and a button press during the stimulus presentation, which could add noise to classifier training separate from the semantic/visual processing of the stimulus. This localizer is similar to others that have been used in TDLM studies, but notably in other studies (e.g., Liu, Mattar et al., 2021), the stimulus is presented prior to the match/mismatch judgment. A discussion of variations in different localizers and what seems to work best for decoding would be useful to include in the recommendations section of the discussion. Second, and more seriously, I believe that the task design for training participants about the expected sequences may complicate sequence decoding. Specifically, this is because two images (a "tuple") are shown together and used for prediction, which may encourage participants to develop a single bound representation of the tuple that then predicts a third image (AB -> C rather than A -> B, B -> C). This would obviously make it difficult to i) use a classifier trained on individual images to detect sequences and ii) find evidence for the intended transition matrix using TDLM. Can the authors rule out this possibility?

Participants only modestly improved (from 76-82% accuracy) following the rest period (which the authors refer to as a consolidation period). If the authors assume that replay leads to improved performance, then this suggests there is little reason to see much task-related replay during rest in the first place. This limitation is touched on (lines 228-229), but I think it makes the lack of replay finding here less surprising. However, note that in the supplement, it is shown that the amount of forward sequenceness is marginally related to the performance difference between the last block of training and retrieval, and this is the effect I would probably predict would be most likely to appear. Obviously, my sample size concerns still hold, and this is not a significant effect based on the null hypothesis testing framework the authors employ, but I think this set of results should at least be reported in the main text. I was also wondering whether the authors could clarify how the criterion over six blocks was 80% but then the performance baseline they use from the last block is 76%? Is it just that participants must reach 80% within the six blocks *at some point* during training, but that they could dip below that again later?

Because most of the conclusions come from the simulation study, there are a few decisions about the simulations that I would like the authors to expand upon before I can fully support their interpretations. First, the authors use a state-to-state lag of 80ms and do not appear to vary this throughout the simulations - can the authors provide context for this choice? Does varying this lag matter at all for the results (i.e., does the noise structure of the data interact with this lag in any way?) Second, it seems that the approach to scaling simulated replays with performance is rather coarse. I think a more sensitive measure would be to scale sequence replays based on the participants' responses to *that* specific sequence rather than altering the frequency of all replays by overall memory performance. I think this would help to deliver on the authors' goal of simulating an "increase of replay for less stable memories" (line 246). On the other hand, I was also wondering whether it is actually necessary to use the real memory performance for each participant in these simulations - couldn't similar goals (with a better/more full sampling of the space of performance) be achieved with simulated memory performance as well, taking only the MEG data from the participant? Finally, Figure 7D shows that 70ms was used on the y-axis. Why was this the case, or is this a typo?

Because this is a re-analysis of a previous dataset combined with a new simulation study on that data aimed at making recommendations about how to best employ TDLM, I think the usefulness of the paper to the field could be improved in a few places. Specifically, in the discussion/recommendation section, the authors state that "yet unknown confounders" (line 295) lead to non-random fluctuations in the simulated correlations between replay detection and performance at different time lags. Because it is a particularly strong claim that there is the potential to detect sequenceness in the baseline condition where there are no ground-truth sequences, the manuscript could benefit from a more thorough exploration of the cause(s) of this bias in addition to the speculation provided in the current version. In addition, to really provide that a realistic simulation is necessary (one of the primary conclusions of the paper), it would be useful to provide a comparison to a fully synthetic simulation performed on this exact task and transition structure (in addition to the recreation of the original simulation code from the TDLM methods paper). Finally, I think the authors could do further work to determine whether some of their recommendations for improving the sensitivity of TDLM pan out in the current data - for example, they could report focusing not just on the peak decoding timepoint but incorporating other moments into classifier training.

Lastly, I would like the authors to address a point that was raised in a separate public forum by an author of the TDLM method, which is that when replays "happen during rest, they are not uniform or close". Because the simulations in this work assume regularly occurring replay events, I agree that this is an important limitation that should be incorporated into alternative simulations to ensure the lack of findings is not because of this assumption.

Reviewer #3 (Public review):

Summary:

Kern et al. critically assess the sensitivity of temporally delayed linear modelling (TDLM), a relatively new method used to detect memory replay in humans via MEG. While TDLM has recently gained traction and been used to report many exciting links between replay and behavior in humans, Kern et al. were unable to detect replay during a post-learning rest period. To determine whether this null result reflected an actual absence of replay or sensitivity of the method, the authors ran a simulation: synthetic replay events were inserted into a control dataset, and TDLM was used to decode them, varying both replay density and its correlation with behavior. The results revealed that TDLM could only reliably detect replay at unrealistically (not-physiological) high replay densities, and the authors were unable to induce strong behavior correlations. These findings highlight important limitations of TDLM, particularly for detecting replay over extended, minutes-long time periods.

Strengths:

Overall, I think this is an extremely important paper, given the growing use of TDLM to report exciting relationships between replay and behavior in humans. I found the text clear, the results compelling, and the critique of TDLM quite fair: it is not that this method can never be applied, but just that it has limits in its sensitivity to detect replay during minutes-long periods. Further, I greatly appreciated the authors' efforts to describe ways to improve TDLM: developing better decoders and applying them to smaller time windows.

The power of this paper comes from the simulation, whereby the authors inserted replay events and attempted to detect them using TDLM. Regarding their first study, there are many alternative explanations or possible analysis strategies that the authors do not discuss; however, none of these are relevant if, under conditions where it is synthetically inserted, replay cannot be detected.

Additionally, the authors are relatively clear about which parameters they chose, why they chose them, and how well they match previous literature (they seem well matched).

Finally, I found the application of TDLM to a baseline period particularly important, as it demonstrated that there are fluctuations in sequenceness in control conditions (where no replay would be expected); it is important to contrast/calculate the difference between control (pre-resting state) and target (post-resting state) sequenceness values.

Weaknesses:

While I found this paper compelling, I was left with a series of questions.

(1) I am still left wondering why other studies were able to detect replay using this method. My takeaway from this paper is that large time windows lead to high significance thresholds/required replay density, making it extremely challenging to detect replay at physiological levels during resting periods. While it is true that some previous studies applying TDLM used smaller time windows (e.g., Kern's previous paper detected replay in 1500ms windows), others, including Liu et al. (2019), successfully detected replay during a 5-minute resting period. Why do the authors believe others have nevertheless been able to detect replay during multi-minute time windows?

For example, some studies using TDLM report evidence of sequenceness as a contrast between evidence of forwards (f) versus backwards (b) sequenceness; sequenceness was defined as ZfΔt - ZbΔt (where Z refers to the sequence alignment coefficient for a transition matrix at a specific time lag). This use case is not discussed in the present paper, despite its prevalence in the literature. If the same logic were applied to the data in this study, would significant sequenceness have been uncovered? Whether it would or not, I believe this point is important for understanding methodological differences between this paper and others.

(2) Relatedly, while the authors note that smaller time windows are necessary for TDLM to succeed, a more precise description of the appropriate window size would greatly improve the utility of this paper. As it stands, the discussion feels incomplete without this information, as providing explicit guidance on optimal window sizes would help future researchers apply TDLM effectively. Under what window size range can physiological levels of replay actually be detected using TDLM? Or, is there some scaling factor that should be considered, in terms of window size and significance threshold/replay density? If the authors are unable to provide a concrete recommendation, they could add information about time windows used in previous studies (perhaps, is 1500ms as used in their previous paper a good recommendation?).

(3) In their simulation, the authors define a replay event as a single transition from one item to another (example: A to B). However, in rodents, replay often traverses more than a single transition (example: A to B to C, even to D and E). Observing multistep sequences increases confidence that true replay is present. How does sequence length impact the authors' conclusions? Similarly, can the authors comment on how the length of the inserted events impacts TDLM sensitivity, if at all?

For example, regarding sequence length, is it possible that TDLM would detect multiple parts of a longer sequence independently, meaning that the high density needed to detect replay is actually not quite so dense? (example: if 20 four-step sequences (A to B to C to D to E) were sampled by TDLM such that it recorded each transition separately, that would lead to a density of 80 events/min).

The red wheelbarrow seems like an unimportant item, but it is a foundational piece that this farm relies on. It may not seem like much to us, but to a farmer and his farm, it is essential.

clear instructions of accessibility features for cognitive accessibility features

Text summary for audio accessibility

Closed captions on all IMDb orignals

The Budweiser advert for a CD with songs sung by Louie and the ferret

For the Thailand Snapshot Please replace the Technology from Thermal battery to the already exisiting Thermal storage

Can the main image of the Thailand snapshot be the first or second image? Because right now we are showing the 3rd image and it is the least relevant one.

can all of this be hidden and only appear on the mouse hover over "sources" in "Snapshot Thailand from the following sources"

There is structure in the content we perceive. For example the content layout is the same which makes it cohesive. Not only people with information processing differences benefit from it but also someone like myself that can get distracted from "loud" or "unorganized" information.

The core idea of the website is very clear and easy to read. Doesn't overstimulate the viewer. On the bottom right side of the screen there is a small person icon in a blue circle. When you click it it gives the user several options to change the accessibility features of the webpage like screen reader mode for blind users, enhances visuals for visually impaired individuals, even keyboard navigation with those with motor disabilities, and so much more. I think the home page itself markets the service they provide by incorporating accessibility features someone might need.

The images here for example have descriptive alt tags making it Perceivable when used by Screen Reading features. * alt="Participants at a nonprofit’s adaptive skiing event, sponsored by accessiBe. (right image) * alt="accessiBe employees volunteering at the Special Olympics World Games in Berlin, June 2023." (middle image) * alt="Chandra Smith, Ms. Wheelchair America 2024, and Josh Basile, accessiBe's Community Relations Manager, sitting in their power wheelchairs and wearing accessiBe swag at an adaptive skiing event."> (left image)

I have rendered the screen of the website as well as viewed the same website website from my mobile device. The screen renders similarly and seamlessly. The same content is on both platforms and displayed in the same accessible way demonstrating robustness of the website.

The photo is missing alt text. This makes it inaccessible to users that rely on screen readers.

The website is keyboard accessible. When pressing the Tab key users are able to navigate the site, and when pressing Enter users can open the links.

The heading ‘The Latest’ is in all caps, a different font, and a different colour, making it easy for users to quickly identify and navigate the latest news.

The heading is large, bold and highly visible, which helps with readability.

For the Uruguay snapshot, please replace the Thematic focus flexibility with the already exisiting Renewable energy integration/flexibility

where she could have flaws

goal of the book

By recording that more than 90% of participants regularly experienced discrimination, prejudice, or stereotyping related to aging, the study demonstrates that these are not uncommon, but rather a universal issue. The prevalence shows that ageism is a widespread social issue rather than a result of individual bias. The study also found that because of how frequently the participants encounter ageism, it causes poorer physical and mental health outcomes. As a result, the number is a key to the article's claim that everyday ageism is both common and damaging to health.

1_Flooded with information. This page's design is overloaded with text and images, which can create confusion for users trying to understand it.

2_Poor contrast. The multiple colours contrast with each other; an example of this is the white word on a yellow highlight.

3_No headlines. Texts are scattered around the website.

4_Inconsistent image descriptions, some images have descriptions while others don't.

Can the figures be seperated by Module? Please group all demand figures together under the GO ones.

Can the filter options for Technology, Sector and Thematic focus update depending on the Snapshot. Meaning to show which tech/sector/theme each snapshot belongs to.

Can the main page of Snapshot be the map only? Right now when you click on "Snapshot" on the top bar you go to the UK snapshot (which was the first snapshot we uploaded).

Each event has a headline and a subheading that contains all the important information about the event so that a person using a screen reader can obtain information about the type of event, location, date, and time. Each event must meet these criteria so that all events are displayed uniformly.

symbols are used to represent the different categories not only with text but also visually, making them easier to recognize and distinguish.

EID 100: The website also provides direct links to customer service and licensed vets on the home page, making it easy for users to find help with either the website or their pet's needs.

EID 100: The "popular categories" menu provides shortcuts to the most frequently accessed pages on the site, which eliminates the need for users to type in the search bar. Moreover, the icons used to represent the shortcuts in this section are simple, high contrast drawings that are easy to distinguish.

EID 100: The menu used to navigate through different animal categories provides both text and images, allowing users to distinguish between options in multiple ways. This improves readability, provides an option for non-readers, and even accommodates those who may be using a text-to-speech feature.

eLife Assessment

This important study fills a gap in our knowledge of the evolution of GPCRs in holozoans, as well as the phylogeny of associated signaling pathway components such as G proteins, GRKs, and RIC8 proteins. The evidence supporting the conclusions is compelling, with the analysis of extensive new genomic data from choanoflagellates and other non-animal holozoans. Overall, the study is thorough and well-executed. It will be a resource for researchers interested in both the comparative genomics of multicellularity and GPCR biology more broadly, especially given the importance of GPCRs as highly druggable targets

Reviewer #1 (Public review):

Summary:

The authors strived for an inventory of GPCRs and GPCR pathway component genes within the genomes of 23 choanoflagellates and other close relatives of metazoans.

Strengths:

The authors generated a solid phylogenetic overview of the GPCR superfamily in these species. Intriguingly, they discover novel GPCR families, novel assortments of domain combinations, novel insights into the evolution of those groups within the Opisthokonta clade. A particular focus is laid on adhesion GPCRs, for which the authors discover many hitherto unknown subfamilies based on Hidden Markov Models of the 7TM domain sequences, which were also reflected by combinations of extracellular domains of the homologs. In addition, the authors provide bioinformatic evidence that aGPCRs of choanoflagellates also contained a GAIN domain, which are self-cleavable thereby reflecting the most remarkable biochemical feat of aGPCRs.

Weaknesses:

The chosen classification scheme for aGPCRs may require reassessment and amendment by the authors in order to prevent confusion with previously issued classification attempts of this family.

Reviewer #2 (Public review):

Summary:

The authors set out to characterise the GPCR family in choanoflagellates (and other unicellular holozoans). GPCRs are the most abundant gene family in many animal genomes, playing crucial roles in a wide range of physiological processes. Although they are known to evolve rapidly, GPCRs are an ancient feature of eukaryotic biology. Identifying conserved elements across the animal-protist boundary is therefore a valuable goal, and the increasing availability of genomes from non-animal holozoans provides new opportunities to explore evolutionary patterns that were previously obscured by limited taxon sampling. This study presents a comprehensive re-examination of GPCRs in choanoflagellates, uncovering examples of differential gene retention and revealing the dynamic nature of the GPCR repertoire in this group. As GPCRs are typically involved in environmental sensing, understanding how these systems evolved may shed light on how our unicellular ancestors adapted their signalling networks in the transition to complex multicellularity.

Strengths:

The paper combines a broad taxonomic scope with the use of both established and recently developed tools (e.g. Foldseek, AlphaFold), enabling a deep and systematic exploration of GPCR diversity. Each family is carefully described, and the manuscript also functions as an up-to-date review of GPCR classification and evolution. Although similar attempts of understanding GPCR evolution were done over the last decade, the authors build on this foundation by identifying new families and applying improved computational methods to better predict structure and function. Notably, the presence of Rhodopsin-like GPCRs in some choanoflagellates and ichthyosporeans is intriguing, even though they do not fall within known animal subfamilies. The computational framework presented here is broadly applicable, offering a blueprint for surveying GPCR diversity in other non-model eukaryotes (and even in animal lineages), potentially revealing novel families relevant to drug discovery or helping revise our understanding of GPCR evolution beyond model systems.

Weaknesses:

While the study contributes several interesting observations, it does not radically revise the evolutionary history of the GPCR family. However, in an era increasingly concerned with the reproducibility of scientific findings, this is arguably a strength rather than a weakness. It is encouraging to see that previously established patterns largely hold, and that with expanded sampling and improved methods, new insights can be gained-especially at the level of specific GPCR subfamilies. Then, no functional follow ups are provided in the model system Salpingoeca rosetta, but I am sure functional work on GPCRs in choanoflagellates is set to reveal very interesting molecular adaptations in the future.

Comments on the latest version:

The authors have done a good job answering my questions and suggestions.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1: 

“I am sorry to dwell on the point of naming the newly identified families of adhesion GPCRs in choanoflagellates. I commented: "Can the authors suggest another scheme (mind to avoid the subfamily I-IX or the alternative ADGRA-G,L,V subfamily schemes of metazoan aGPCRs) and adapt their numbering throughout the text and all figures/supplementary figures/supplementary files." Now the authors have changed the Roman numeral numbering (previously used by the adhesion GPCR field to denominate metazoan receptor families) to the other option that I explicitly said should be obsolete, the numbering by capital letters (which is in use since its introduction in 2015 in Hamann et al., Pharmacol Rev, 2015). The authors write: "Phylogenetic analysis of the 7TM domains of choanoflagellates uncovered at least 19 subfamilies of aGPCRs (subfamilies A-S ...". I am thus afraid this has not addressed my point at all. For example, in the revised numbering scheme for Choanoflagellates aGPCR subfamilies of the authors the now used "A" descriptor, which are predicted to contain a HYR domain, can be mistaken for ADGRA homologs (abbreviated as "A" receptors, previously termed subfamily III aGPCRs) of metazoan aGPCRs, which contain HRM and LRR domains. Likewise, choanoflagellate "E" receptors are predicted to harbour LRR repeats, but metazoan ADGRE (abbreviated as "E" too) are characterised by their EGF domains. This clearly underlines the need to devise a numbering scheme for the newly described choanoflagellate aGPCR homologs so they cannot be confused with the receptors from other kingdoms, for which identical naming conventions exist. Please change this, e.g. by numbering/denominating the choanoflagellate subfamilies by greek letters (or your pick of any other ordering system that does not lend itself to be mistaken with the previous and existing aGPCR classifications) and change the manuscript and figures accordingly.”

We have now re-labeled the choanoflagellate aGPCR subfamilies, previously numbered from A to S, using Greek alphabetical enumeration (from α to τ). Changes have been made throughout the main text, in Figure 5, and in Supplementary Figures  S6 and S7.

This website provides great content in various languages, as well as ensuring that it can be easily translated judging by the amount of options it provides, therefore making it (Robust). It provides proper accessibility for language identification that allows screen readers and translation tools to switch seamlessly between the various languages that it provides. It empowers users in multilingual regions where English or other dorminant languages may not be the main language that is widely spoken.

The ‘Create Account’ button stands out as one of the most important elements on the page, designed with strong visibility and bold contrast that makes it easy to spot among other links

The chatbot asks contextual, user-specific questions e.g., “Are you interested in cloud training, pricing, or documentation?”. This is a good practice as some people may have a hard time navigating the page

Having the ability to change language is good practice used by AWS. With around 16 different language people from different background have no barrier to access

A built-in feedback tool that lets users share their experience with the site’s accessibility, helping highlight areas for improvement

The text here is very hard to see. While this is only the date of the announcement, its small size and faint grey text make it hard to see and understand

The use of colour contrast work well for accessibility. It makes the white text stand out to understand the different categories on this page. The webpage is still complex though.

These links are cluttered and have no relation to each other besides being for students. Better labeling or categorizing of the links would go a long way for accessibility. This text is not structured logically

Not very operable as there is no major indication pointing towards this link being the primary one to access courses. It is buried within 2 headings.

Category Icons (Good Practice)

These icons are accessible because they include both an image and a text label, so users who rely on screen readers or can’t identify the image alone still understand the category.

Good Practice

These icons (like the cart and account icons) are well-designed because they include visible labels/tooltips. This makes them understandable for all users, including those using screen readers, and avoids confusion from relying only on visuals.

Product Image Without Alt Text (Bad Practice)

This image doesn’t have an alt text description, so screen reader users can’t understand what’s being shown.

Headings Are Organized (Good Practice)

The headings here follow a clear order, which helps screen reader users navigate the page.

The video is closed captioned, making it accessible to people with hearing disabilities.

A good contrast ratio between the page content.

Includes an option to view website in both English and French so it can be understood by both people who speak English and French. It is placed at the top of the website so it can be easily seen and selected if needed.

Offers online service to renew documents online or in person, which makes it available for those who may not be able to go renew documents in-person. Also has accompanied pictures of the types of documents that can be renewed online for understanding.

Has a section that lists the most visited pages for resources available in a clear and easily accessible way, with clear text and simple language. Also includes a design that lets you easily differentiate between topics and resource pages.

Has clear branding and instruction on what the page is for.

eLife Assessment

This important study convincingly shows that Vibrio bacteria act as predators of ecologically significant algae that contribute to harmful blooms in the lab, as well as in their natural habitat. While the data strongly suggest that starvation may induce predation, further work is needed to fully establish this link. Similarly, the evidence for a social component in the predation process remains incomplete. This study will be very impactful to those interested in the diversity of microbial predator-prey interactions and controlling toxic algal bloom, but the paper could be strengthened by more clearly showing the degree of replication, by better defining the terms used to describe the observed behaviour, and by providing better support for starvation and collective behaviour.

Reviewer #1 (Public review):

Summary:

Rolland and colleagues investigated the interaction between Vibrio bacteria and Alexandrium algae. The authors found a correlation between the abundance of the two in the Thau Lagoon and observed in the laboratory that Vibrio grows to higher numbers in the presence of the algae than in monoculture. Time-lapse imaging of Alexandrium in coculture with Vibrio enabled the authors to observe Vibrio bacteria in proximity to the algae and subsequent algae death. The authors further determine the mechanism of the interaction between the two and point out similarities between the observed phenotypes and predator-prey behaviours across organisms.

Strengths:

The study combines field work with mechanistic studies in the laboratory and uses a wide array of techniques ranging from co-cultivation experiments to genetic engineering, microscopy and proteomics. Further, the authors test multiple Vibrio and Alexandria species and claim a wide spread of the observed phenotypes.

Weaknesses:

In my view, the presentation of the data is in some cases not ideal. The phrasing of some conclusions (e.g., group-attacks and wolf-pack-hunting by the bacteria) is in my opinion too strong based on the herein provided data.

Reviewer #2 (Public review):

Goal summary:

The authors sought to (i) demonstrate correlations between the dynamics of the dinoflagellate Alexandrium pacificum and the bacterim Vibrio atlanticus in natural populations, ii) demonstrate the occurrence of predation in laboratory experiments, iii) claim coordinated action by the predators in the predation process, iv) demonstrate that predation is induced by predator starvation, and v) test for effects of quorum sensing and iron-uptake genes on the predation process.

Strengths include:

(1) Data indicating correlated dynamics in a natural environment that increase the motivation for the study of in vitro interactions.

(2) Experimental design allowing clear inference of predation based on population counts of both prey and predators in addition to microscopy-based evidence.

(3) Supplementation of population-level data with molecular approaches to test hypotheses regarding possible involvement of quorum sensing and iron uptake in predation.

Weaknesses include:

(1) A lack of early, clear definitions for several important terms used in the paper, including 'predation', 'coordination' and 'coordinated action', 'group attack', and 'wolf-pack hunting', along with a corresponding lack of criteria for what evidence would warrant use of some of these labels. (For example, does mere simultaneity of attacks of an A. pacificum cell by many V. atlanticus cells constitute "coordination"? Or, as it seems to us, does coordination require some form of signalling between predator cells?)

(2) Absence of controls for cell density in the test for starvation effects on predatory behavior; unclear how the length of incubation affects the density of V. atlanticus cells.

(3) Lack of clarity in some of the methodological descriptions

Appraisal:

The authors convincingly achieve their aim of demonstrating that V. atlanticus can prey on A. pacificum, provide strongly suggestive evidence that such predation is induced by starvation, and clearly demonstrate that both iron availability and, correspondingly, the presence of genes involved in iron uptake, strongly influence the efficacy of predation. However, the evidence for starvation-induction of predation can be strengthened with cell-density controls; evidence for a social component to predation - positive interactions between attacking predators - is lacking.

Discussion of impact:

This paper will interest those interested in how microbial behaviour responds to environmental fluctuations, in particular predatory behaviour, but will do so more strongly if the evidence of starvation-induction of predation is strengthened. It will also interest those investigating bacteria-algae interactions and potential ecological controls of algal blooms. It has the potential to interest researchers of microbial cooperation, should the authors be able to provide any evidence of coordination between predator cells.

the feature where the button darkens when you hover over it is good as it can help people visually see where they are on the page. This helps those who are not that efficient in using the mouse.

Users are able to change the language. This helps if they do not speak english then they have the options to switch the language and the icon next to the Change Language text is good in case you can't even understand "Change Language."

This dropdown feature is this. While you can hover over it to look at the menu options, you can also click on it which is good for someone who can use the mouse or scroll much.

Page includes many colours. May be inaccessible to individuals with visual impairments, however is cohesive with the brand and it's products.

not text heavy, very image focused.

small body text, no function visible to increase size.

from the POUR acronym, this is perceivable because the arrows also point up and down and does not rely on colour.

Closed captioning on videos

The video settings allow for closed captioning options

Appropriate Text Alternatives

The Ontario website allows for a French option. This is important as it is the second language of Canada, where many speak french as a first language. This allows those who only speak french, or are french profficient to read/understand

Structure Text Logically

The Ontario website uses bold text and left page centring to allow for easy readability (from left to right). It also has a bold header, under it showing what the page is about as well as links to each of the topics

Distinguishable Content

The Ontario website uses bold black text on a white background. This allows those with colourblindness to view the appropriate messaging.

wow the fact that he's doing this with the light off for a bunch of the time is wild - I find it hard enough to follow a drop with our little video setup

can it obtain an ion basically immediately when the field is turned on due to the ions being pushed out of the way and potentially into the drops?

how does he know this? Also, my understanding is that because the size of the drop is obtained from the fall, we're assuming that the ions have no mass, or at least negligible mass (because the fall under gravity does not change...)

!!!! в примере промпта они пишут что все-таки ему можно их параллельно вызывать

Launch multiple agents concurrently whenever possible, to maximize performance; to do that, use a single message with multiple tool uses

WTF для приветсвенной шутки?

<example_agent_descriptions> "code-reviewer": use this agent after you are done writing a signficant piece of code

"greeting-responder": use this agent when to respond to user greetings with a friendly joke </example_agent_description>

это как раз про то, что копию самого себя с более простой задачей создает

Мб нам такое же для вопросов по банку сделать?

не до конца понял, но звучит как будто они дают ему возможность читать его же документацию (а там достаточно дофига инфы)

например https://docs.anthropic.com/en/docs/claude-code/memory

в тревеле планер(аналог TODO) генерит MODE и по MODE в систем-промпте расписаны сценарии действий

мб стоит это попробовать как-то красивее раскидать по xml тэгам и как-то красивее напишется

https://openreview.net/pdf?id=y1SnRPDWx4 -- в этой статье показывали, что любое форматирование, не обязательно markdown работает

но мб с markdown легче какую-то согласованность среди команд заиметь

отсылка к эссе Р. Саттона The Bitter Lesson: в долгую выигрывают простые, масштабируемые методы, которые лучше всего используют вычисления и данные, а не ручные правила и хитрую инженерную логику.

This sticks out to me, I enjoy a lot of French music, film, and literature (Simone de Beauvoir is the best). Being in art history much of the critical history is originally in French with France massively contributing to the development of "fine art", so I very much agree with point on culture.

darkness here showing not skin colour or mood, but isolation, further from others.

"smart" is a racialised trait for east asians it becomes a colour line, also talking abt east asians being the model minority in america, and how fitting into that is also separating u from these ppl, but it is also the best way to get to proximity to whiteness

double consciousness, going on, she feels like she is white andar se, cause u cant rlly be american if ur not white, so she feels from the inside she is compatible with whiteness, but isnt so, so being poc but raised american is a complete attack on your personhood and identity and thats what she felt growing up

purple is opposite to yellow on colour wheel. but next to the mountains are east asian ppl so she wants to be near them,but also away from them

talks about yellow ppl. and east asian context during world war 2, and internment era druring world war 2, andtalks about opium crisis ineast asian people, at the time, and this issue in bad neighbourhoods, and w the shooting up of their arms and talks abt dirtiness and feeling dirty because she isnt white

style is like similar to how a child would write, and cyclical poem, it ends the way it starts, very disjointed word vomit type of poem, reads like a confession also, we dont know where things start or end, plus lack of capitalisation,

At the bottom of the page the website offers the same page but instead in French and additional languages/regions. This feature highlights the site's equal accessibility for all the different languages spoken in Canada. Although the feature may seem minor it is extremely beneficial to Canada's diverse demographic and its non-English speaking citizens.

Something the website could benefit from, instead of these 'learn more' tabs, is an actual live chat or call feature on the main page, to assist customers with general inquiries and navigation. Many older users often struggle with navigating online webpages and would prefer to speak to a live agent. Therefore, by adding this feature Apple would be more accessible to users who require more assistance on the website. Moreover, this form of accessibility would also benefit all consumers, as customers with special issues or inquiries that would typically require emailing or going to a store in person, can now resolve their issue in a more convenient manner

Apple's use of large graphics, text, and its 'learn more' function allows for greater accessibility for those with learning disabilities or limited literacy. In more detail, these features allow for easy comprehension and navigation as it clearly depicts what the website is selling/advertising, as well as provides users with step-by-step paths to additional information.

this things move along the curve because they are already on the X/Y axis, other factors are not

company overproducing, the price exceeds the equilibrium price (the price they should be asking for) ex. they produce 35 million and $8, but buyers consume 15 million at that price surplus = 20 million lbs

The following are unchecked exceptions: IndexOutOfBoundsException NullPointerException NumberFormatException

The remaining two are checked exceptions.

They are no longer with the world so they don't have any more ties or duties.

the reason Eurydice accepted hades offer was because she wanted to be safe when Orpheus was blind to what was happening

She is finally free from him and is able to do what she wants

She is saying goodbye to all of the things on earth because she is leaving them and going with Hades.

Uhh, det giver mening

Her laver vi jo type declaration

This particular point really intrigued me because he is expressing the fact that people aren’t always necessarily paying attention to one individual specifically even though we think they might be, which is what makes people less focused on others around them, this being the issue addressed throughout the speech.

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

Reviewer #1

Summary: The authors have previously published Mass-spectrometry data that demonstrates a physical interaction between Sall4 and the BAF chromatin complex in iPSC derived neurectodermal cells that are a precursor cell state to neural crest cells. The authors sought to understand the basis of this interaction and investigate the role of Sall4 and the BAF chromatin remodelling complex during neural crest cell specification. The authors first validate this interaction with a co-IP between ARID1B subunit and Sall4 confirming the mass spec data. The authors then utilise in silico modelling to identify the specific interaction between the BAF complex and Sall4, suggesting that this contact is mediated through the BAF complex member DPF2. To functionally validate the role of Sall4 during neural crest specification, the authors utilsie CRISPR-Cas9 to introduce a premature stop codon on one allele of Sall4 to generate iPSCs that are haploinsufficient for Sall4. Due to the reports of Sall4's role in pluripotency, the authors confirm that this model doesn't disrupt pluripotent stem cells and is viable to model the role of Sall4 during neural crest induction. The authors expand this assessment of Sall4 function further during their differentiation model to cranial neural crest cells, assessing Sall4 binding with Cut+Run sequencing, revealing that Sall4 binds to motifs that correspond to key genes in neural crest differentiation. Moreover, reduction in Sall4 expression also reduces the binding of the BAF complex, through Cut and Run for BRG1. Overall, the authors then propose a model by which Sall4 and BRG1 bind to and open enhancer regions in neurectodermal cells that enable complete differentiation to cranial neural crest cells.

Overall, the data is clear and reproducible and offers a unique insight into the role of chromatin remodellers during cell fate specification.

We thank the Reviewer for the nice words of appreciation of our manuscript.

However, I have some minor comments.

1- Using AlphaFold in silico modelling, he authors propose the interaction between the BAF complex with Sall4 is mediated by DPF2, but don't test it. Does a knockout, or knockdown of DPF2 prevent the interaction?

We agree with the Reviewer that we are not functionally validating our computational prediction that DPF2 is the specific BAF subunit directly linking SALL4 with BAF. We chose not to perform the validation experiment for two main reasons:

1) This would be outside of the scope of the paper. In fact, from a mechanistic point of view, we have confirmed via both Mass-spectrometry and co-IP with ARID1B that SALL4 and BAF interact in our system. Moreover, mechanistically we also extensively demonstrate that the interaction with SALL4 is required to recruit BAF at the neural crest induction enhancers and we further demonstrate that depletion of SALL4 impairs this. In our view, this was the focus of the manuscript. On the other hand, detecting with certainty which BAF subunit mediates the interaction with SALL4 would be outside the scope of the paper.

2) Moreover, after careful consideration, we don’t think that even a knock-out of DPF2 would provide a definite answer to which exact BAF subunit mediates the interaction with SALL4. In fact, knock out of DPF2 could potentially disrupt BAF assembly or stability, and this could result in a disruption of the interaction with SALL4 even if DPF2 is not the very subunit mediating it (in other words the experiment could provide a false positive result). In our opinion, the only effective experiment would be mutating the DPF2 residues that we computationally predicted as responsible for the interaction with SALL4, but again this would be very laborious and out of the scope.

That being said, we agree with the Reviewer that while the SALL4-BAF interaction was experimentally validated with robust approaches, the role of DPF2 in the interaction was only computationally predicted, which comes as a limitation of the study. We have now added a dedicated paragraph in the discussion to acknowledge such limitation.

2- OPTIONAL: Does knockout of DPF2 phenocopy the Sall4 ko? This would be very interesting to include in the manuscript, but it would perhaps be a larger body of work.

See point-1.

3- Figure 1, the day of IP is not clearly described until later in the test. please outline during in the figure.

We thank the Reviewer for pointing this out. This has been fixed.

3- What is the expression of Sall1 (and other Sall paralogs) during differentiation. The same with the protein levels of Sall4, does this remain at the below 50%, or is this just during pluripotency?

As Recommend by the Reviewer, we have performed time-course WB of SALL1 and SALL4. These experiments revealed that SALL1 remains very lowly expressed in wild-type conditions across time points and all the way through differentiation until CNCC (See updated supplementary Fig. S9). This is consistent with previous studies that demonstrated that SALL4, but not SALL1, is required for early mammalian development (see for example Miller et al. 2016, Development, and Koulle et al. 2025, Biorxiv). We performed the same time-course WB for SALL4 which revealed that SALL4 expression progressively decreases after day-5 (as expected) and it’s very low at CNCC stage (day-14), therefore we would expect the KO to remain at even lower level at this stage.

4- The authors hypothesise that Sall4 binds to enhancers- with the criteria for an enhancer being that these peaks > 1KB from the TSS are enhancers. Can this be reinforced by overlaying with other ChIP tracks that would give more confidence in this? There are several datasets from Joanna Wysocka's lab that also utilise this protocol which can give you more evidence to reinforce the claim and provide further detail as to the role of Sall4.

We thank the Reviewer for this great suggestion. As recommended, we have used publicly available ChIP-seq data generated by the Wysocka lab (H3K4me1, H3K4m3) and also generated new H3K27ac CHIP-seq data as well. These experiments and analyses confirmed that these regions are putative CNCC enhancers (and a minority of them putative promoters), decorated with H3K4me1 and with progressive increase in H3K27ac after CNCC induction (day-5). See new Supplementary Figure S6.

5- The authors state that cells fail to become cranial neural crest cells, however they do not propose what the cells do instead. do they become neural? Or they stay at pluriopotent, which is one option given the higher expression of Nanog, OCT4 and OTX2 that are all expressed in pluripotent stem cells.

We think that it is likely a mix of both. There is a mixed bag of expression of pluripotency markers, but also high expression of neuroectodermal markers. This suggests that most cells safely reach the neuroectodermal stage but fail to go beyond that, while some of the cells simply do not differentiate or regress back to pluripotency. We would rather refrain on overinterpreting what the KO-cells become, as it is likely an aberrant cell type, but following the Reviewer’s indication we have added a paragraph in the discussion to speculate on this.

6- In general, I would like to see the gating strategy and controls for the flow cytometry in a supplemental figure.

As Recommended by the Reviewer, we have added the gating strategy in the Supplementary Fig. S4.

7- For supplementary figure 1- please include the gene names in the main image panels rather than just the germ layer.

Done. The figure is now Supplementary Figure S3 since two supplementary figures were added before.

Reviewer #2

Summary In this manuscript, the authors build on their previous work (Pagliaroli et al., 2021) where they identified an interaction between the transcription factor SALL4 and the BAF chromatin remodeling complex at Day-5 of an iPSC to CNCC differentiation protocol. In their current work, the authors begin by exploring this interaction further, leveraging AlphaFold to predict interaction surfaces between SALL4 and BAF complex members, considering both SALL4 splice isoforms: a longer SALL4A (associated with developmental processes) and a shorter SALL4B (associated with pluripotency). They propose that SALL4A may interact with DPF2, a BAF complex member, in an isoform-dependent manner. The authors next explore the role of SALL4 in craniofacial development, motivated by patient heterozygous loss of function mutations, leveraging iPSC cells with an engineered SALL4 frameshift mutation (SALL4-het-KO). Using this model, the authors first demonstrate that a reduced expression of SALL4 does not impact the iPSC identity, perhaps due to compensation via upregulation of SALL1. Upon differentiation to neuroectoderm, SALL4 haploinsufficiency causes a reduction in newly accessible sites which are associated with a reduction in SALL4 binding and therefore a loss of BAF complex recruitment. Interestingly, however, there were few transcriptional changes at this stage. Later in the CNCC differentiation at Day-14 when the wildtype cells have switched expression of CNCC markers, the SALL4-het-KO cells fail to switch cadherin expression associated with a transition from epithelial to mesenchymal state, and fail to induce CNCC specification and post-migratory markers. Together the authors propose that SALL4 recruits BAF to CNCC enhancers as early as the neuroectodermal stage, and failure of BAF recruitment in SALL4-het-KO lines results in a loss of open chromatin at regulatory regions required later for induction of the CNCC programme. The failure of the later differentiation is compelling in the light of the early stages of the differentiation progressing normally, and the authors outline an interesting proposed mechanism whereby SALL4 recruits BAF to remodel chromatin ahead of CNCC enhancer activation, a model that can be tested further in future work. The link between SALL4 DNA binding and BAF recruitment is nicely argued, and very interesting as altered chromatin accessibility at Day 5 in the neuroectodermal stage is associated with only few changes in gene expression, while gene expression is greatly impacted later in the CNCC stage at Day 14. The in silico predictions of SALL4-BAF interaction interfaces are perhaps less convincing, requiring experimental follow-up outside the scope of this paper. Some of the associated figures could perhaps be moved to the supplement to enhance the focus on the later functional genomics experiments.

We thank the Reviewer for the nice words of appreciation of our manuscript.

Major comments

1. A lot of emphasis is placed on the AlphaFold predictions in Figure 1, however the predictions in Figure 1B appear to be mostly low or very low confidence scores (coloured yellow and orange). It is unclear how much weight can be placed on these predictions without functional follow-up, e.g. mutating certain residues and showing impact on the interaction by co-IP. The latter parts of the manuscript are much better supported experimentally, and therefore perhaps some of the Figure 1 could move to a Supplemental Figure (e.g. the right-hand part of 1B, and the lower part of Figure 1C showing SALL4B predicted interactions). The limitations of AlphaFold predictions should be acknowledged and the authors should discuss how these predicted interactions could be experimentally explored further in the future.

As recommended by the Reviewer, we have moved part of the AlphaFold predictions to Supplementary Figure S1, and we added a paragraph in the discussion to acknowledge the limitations of AlphaFold.

The authors only show data for one heterozygous knockout clone for SALL4. It is usual to have more than one clone to mitigate potential clonal effects. The authors should comment why they only have one clone and include any data for a second clone for key experiments if they already have this. Alternatively, the authors could provide any quality control information generated during production of this line, for example if any additional genotyping was performed.

We apologize for the confusion and for our lack of clarify on this. We have used two clones (one generated with a 11 bp deletion, one with a 19 bp deletion, both in exon-1, see also the point 6 of your minor points). The two clones were used as biological replicates, so for example the two ATAC-seq replicates performed in each time point were performed with the two different clones, and the three RNA-seq replicates were performed with two technical replicates of the clone with the 11bp deletion and one replicate with the clone with 19 bp deletion. We have clarified this in the methods section of the manuscript and added a Supplementary Figure (S2) showing the editing strategy for the two clones. Thank you for catching it.

The authors show all genomics data (ATAC-seq, CUT&RUN and ChIP-seq) as heatmaps and average profiles. It would be valuable to see some representative loci for the ATAC seq (perhaps along with SALL4 and BRG1 recruitment) at some representative and interesting loci.

As recommended by the Reviewer, we have added Genome Browser screenshots of representative loci in Fig. 6.

Figure 4A. The schematic could be improved by including brightfield or immunofluorescent images at the three stages of the differentiation. Are the iPS cells seeded as single cells, or passaged as colonies before starting the differentiation. Further details are required in the methods to clarify how the differentiation is performed, for example at what Day are the differentiating cells passaged, this is not shown on the schematic in Figure 4A.

As recommended, we added IF images in the Fig. 4A schematic, and added more details in the methods.

There is likely some heterogeneity of cell types in the differentiation at Day 5 and Day 14. Can the authors comment on this from previous publications or perhaps conduct some IF for markers to demonstrate what proportions of cells are neuroectoderm at Day 5 and CNCCs at Day 14.

The differentiation starts with single cells that aggregate to form neuroectodermal clusters, as per original protocol. The CNCCs that we obtain with this protocol homogeneously express CNCC markers, as shown by IF of SOX9 in Fig. 4A. For the day-5, as recommended we have added IF for PAX6 also showing homogeneous expression (Fig. 4A).

For the motif analysis for Day 5-specific SALL4 binding sites (Figure 4E), was de novo motif calling performed? Were any binding sites reminiscent of a SALL4 binding site observed (e.g. an AT-rich motif)? Could the authors comment on this in the text - if there is no SALL4 binding motif, does this suggest SALL4 is recruited indirectly to these sites via interaction with another transcription factor for example?

Similar to SALL4, SALL1 also recognizes AT-rich motifs. However, while we found AT-rich motifs as enriched in our day-5 motif analysis (in the regions that gain SALL4 binding upon differentiation), the enrichment is not particularly strong, and several other motifs are significantly more enriched, suggesting that, like the Reviewer mentioned, SALL4 might be recruited indirectly at these sites by other factors. We have added a paragraph on this in the discussion.

Does SALL1 remain upregulated at Day-5 and Day-14 of the differentiation for the SALL4-het-KO line? Are binding sites known for this TF and were they detected in the motif analysis performed? Further discussion of the impact of the overexpression of SALL1 on the phenotypes observed is warranted - e.g. for Figure 5F, could the sites associated with a gain of BRG1 peaks upon loss of SALL4 be associated with SALL1 being upregulated and 'hijacking' BAF recruitment to distinct sites associated with nervous system development? Is SALL1 still upregulated at Day 5?

As mentioned above, SALL1 also recognizes AT-rich motifs but similar to SALL4 also binds unspecifically, likely in cooperation with other TFs. Like the Reviewer suggested, it is certainly possible that some of the sites associated with a gain of BRG1 peaks upon loss of SALL4 could be associated with SALL1 being upregulated and 'hijacking' BAF recruitment to distinct sites. While this is speculative, we have added a paragraph on this in the discussion.

Related to the point above, SALL4A is proposed to have an isoform-specific interaction with the BAF complex. It would be valuable to plot SALL4A and SALL4B expression from the available RNA-seq data at Day 0, 5 and 14 to explore whether stage-specific isoform expression matches with the proposed role of SALL4A to interact with BAF at Day 5. It could be valuable to also look at expression of SALL1, 2 and 3 across the time course to see whether additional compensation mechanisms are at play during the differentiation.

Thanks for suggesting this. We performed a time course analysis of isoform specific gene expression, which showed that SALL4B expression remains low throughout differentiation, while SALLA4A expression increases upon differentiation cues and it remains at high levels until the end. We have added this to supplementary Fig. S9. Moreover, we have performed an additional experiment, using pomalidomide, which is a thalidomide derivative that selectively degrades SALL4A but not SALL4B. Notably, SALL4A degradation recapitulated the main findings obtained with the CRISPR-KO of SALL4, further supporting that SALL4A is the isoform involved in CNCC induction (see new Fig. 8).

At line 264, The authors state "SALL4 recruits the BAF complex at CNCC developmental enhancers to increase chromatin accessibility". Given that this analysis is performed at Day 5 of the differentiation, which is labelled as neuroectoderm what evidence do the authors have that these are specifically CNCC enhancers? Statements relating to enhancers should generally be re-phrased to putative enhancers (as no functional evidence is provided for enhancer activity), and further evidence could be provided to support that these are CNCC-specific regulatory elements, e.g. showing representative gene loci from CNCC-specific genes. Discussion of the RNA-seq presented in Supplementary Figure 2B may also be appropriate to introduce here given that large numbers of accessible chromatin sites are detected while the expression of very few genes is impacted, suggesting these sites may become active enhancers at a later developmental stage.

As also recommended by the other Reviewer, to further characterize these sites, we have used publicly available histone modification CHIP-seq data (H3K4me1, H3K4me3) generated by the Wysocka lab (H3K4me1, H3K4m3) and also generated new H3K27ac CHIP-seq data as well. These experiments and analyses confirmed that these regions are putative CNCC enhancers (and a minority of them putative promoters), all decorated with H3K4me1, and all showing progressive increase in H3K27ac after CNCC induction (day-5). See new Supplementary Figure S6.

1. Do any of the putative CNCC enhancers detected at Day 5 as being sensitive to SALL4 downregulation and loss of BAF recruitment overlap with previously tested VISTA enhancers (https://enhancer.lbl.gov/vista/)?

Yes, we have found examples of overlap and have included two of them in the updated Figure 6 as Genome Browser screenshots.

Minor comments

1. The authors are missing references in the introduction "a subpopulation of neural crest cells that migrate dorsolaterally to give rise to the cartilage and bones of the face and anterior skull, as well as cranial neurons and glia".

Fixed, thank you.

The discussion of congenital malformations associated with SALL4 haploinsufficiency is brief in the introduction. From OMIM, SALL4 heterozygous mutations are implicated with the condition Duane-radial ray syndrome (DRRS) with "upper limb anomalies, ocular anomalies, and, in some cases, renal anomalies... The ocular anomalies usually include Duane anomaly". That Duane anomaly is one phenotype among a number for patients with SALL4 haploinsufficiency could be clarified in the introduction. Of note, this is stated more clearly in the discussion but needs re-wording in the introduction.

Done, thank you.

The statements "show that the SALL4A isoform directly interacts with the BAF complex subunit DPF2 through its zinc-finger-3 domain" and "this interaction occurs between the zinc-finger-cluster-3 (ZFC3) domain of SALL4A and the plant homeodomains (PHDs) of DPF2" in the introduction appear overstated and should be toned down. To show this the authors would need to mutate or delete the proposed important zinc-finger domains from SALL4A, which is outside the scope of this work. Notably, this is less strongly-stated elsewhere in the manuscript, e.g "predict that this interaction is mediated by the BAF subunit DPF2", Line 162.

Done, thank you.

Could the authors clarify why 3 Alphafold output models are shown for SALL4B in Figure 1C, and only one output model for SALL4A?

AlphaFold3 produces five separate predicted models per protein combination (e.g., Model_1 … Model_4), each derived from slightly different network parameters or initializations. The final output prioritizes the model with the highest confidence score. This multi-model strategy enables the identification of the most robust conformation while providing a measure of structural uncertainty (as per GitHub documentation for AlphaFold3). wE have conducted the same analysis for SALL4A as we did for SALL4B. Specifically, SALL4A interacts with the AT-rich DNA in models 0, 1, and 2, therefore models 3 and 4 were excluded. When analysing models 1 and 2, we found a higher number of residues involved in the interaction (>800 instead of 396). Similarly to model 0, only the interactions between residues belonging to an annotated functional domain (ZFs and PHDs) were considered.

In Model 1: SALL4A and DPF2 interact mainly through ZF6 and 7, and not 5 as Model 0.

In Model 2: SALL4A and DPF2 interact mainly through ZF5 and 6, and not 7 as Models 0. In contrast, this model shows an interaction with ZF1 not shown in the other two models, but with a higher PAE (31 average compared to 25 to 27 average of the other two ZFs.

Therefore, we considered Model 0 as it is the model with higher confidence and representative of all significant models (includes ZF5, 6, and 7).

Line 121. The authors state "DPF2, a broadly expressed BAF subunit,", but don't show expression during their CNCC differentiation. It would be good to include expression of DPF2 in Figure 1E.

Done, thank you.

The text states "a 11 bp deletion within the 3'-terminus of exon 1 of SALL4", while the figure legend states, "Sanger sequencing confirming the 19 bp deletion in one allele of SALL4 is displayed". The authors should clarify this disparity and experimentally confirm the deletion, e.g. by TA-cloning the two alleles and sequencing these separately to show that one allele is wildtype and the other has a frameshift deletion.

We apologize for the confusion. As stated above (point-2 of the major comments), we have used two clones (one generated with a 11 bp deletion, one with a 19 bp deletion, both in exon-1, see also the point 6 of your minor points). The two clones were used as biological replicates (see response above for details). The deletion for both clones was experimentally confirmed by Sanger sequencing by the company that generated the lines for us (Synthego). The strategy for the two clones is now shown also in Supplementary Fig. S2.

The authors generate an 11-bp (or 19-bp?) deletion in exon-1 - it would be valuable to include a discussion whether patients have been identified with deletions and frame-shift mutations in this region of SALL4 exon-1. And also clarify, if not clearly stated in the text, that both SALL4A and SALL4B will be impacted by this mutation. Are there examples of patient mutations which only impact SALL4A?

As requested, we have added a discussion paragraph to discuss this. And, yes, both SALL4A and SALL4B are impacted by both deletions in both clones (11 bp and 19 bp deletion).

Regarding patient variants on exon-1 and patient variants that only impact SALL4A. We could only find one published pathogenic 170bp deletion in exon 1 (VCV000642045.7). The majority of the pathogenic or likely pathogenic variances are located on exon2. In particular, of the 63 reported pathogenic (or likely pathogenic) clinical variants, 42 were located on exon 2. Among these, 28 are located in the portion shared by both SALL4A and SALL4B, while the remaining 14 were SALL4A specific.

For the SALL4 blots in Figure 2B, is the antibody expected to detect both isoforms (SALL4A and SALL4B), and which isoform is shown? If two isoforms are detected, they should both be presented in the figure.

Yes, the antibody detects both isoforms, and we now present both in the figure 2, as recommended.

SALL4 expression should be shown for Figure 2C to see whether the >50% down-regulation of SALL4 at the protein level may be partially driven by transcriptional changes.

Done, thank you. As expected, we observed the SALL4 mRNA expression in the KO line is comparable to wild-type conditions, but still this results in a significant decrease of the SALL4 protein level likely because of autoregulatory mechanisms coupled with non-sense mediated decay of the mutated allele. Also, we note that SALL4 usually makes homodimers, therefore lack of sufficient amount of protein could also lead to degradation of the monomers.

The number of experimental replicates should be indicated in all figure legends where relevant. Raw data points should be plotted visibly over the violin plots (e.g. Figure 2C).

Done, thank you.

For Figure 3A, the images of the DAPI and NANOG/OCT4 staining should be shown separately in addition to the overlay.

Done, thank you.

The metric 'Corrected Total Cell Fluorescence (CTCF)' should be described in the methods. The number of images used for the quantification in Figure 3A should be

Done, thank you.

Figure 3C - what are the 114 differentially expressed genes? Some interesting genes could be labelled on the plot and the data used to generate this plot should be included as a Supplementary Table. Supplementary Tables should similarly be provided for Figure 6C, Day 14 and Supplementary Figure 2B, Day 5.

As recommended, we have highlighted some interesting genes in the volcano plot and also included all the expression data for all genes in Supplementary Table S3.

Figure 4B. The shared peaks are not shown. For completeness, it would be ideal to show these sites also.

Done, thank you.

Figure 4C is difficult to interpret. Why is the plot asymmetric to the left versus right? What does the axis represent - % of binding sites?

The asymmetry is due to the fact that there is a larger number of peaks that are downstream of the TSS than peaks that are upstream of TSS. This is consistent with the fact that many SALL4 peaks are in introns, likely representing intronic enhancers.

Line 224-225. What do n= 3,729 and n= 6,860 refer to? There appear to be many more binding sites indicated in Figure 4B, therefore these numbers cannot represent 86% and 97% of sites?

Thank you for pointing this out, we should have specified in the text. Those numbers refer to the genes whose TSS is closest to each SALL4 peak. Notably, multiple peaks can share the same closest TSS, hence the discrepancy between # of peaks and # of nearest genes.

Raw numbers:

• Day-0 RAW = 6,104 (peaks = 6,114);
• Day-5 RAW = 17,131 (peaks = 17,137). Now raw data reported in Supplementary Table 4.

Figure 4E. Several TFs mentioned in the text (Line 243) are not shown in the figure, it would be good to show all TFs motifs mentioned in the text in this figure. Again, there is no mention of whether a sequence-specific motif is detected for SALL4 (e.g. an AT-rich sequence) from this motif analysis.

Done, thank you. An AT-rich sequence, resembling the SALL4 motif, was detected in a small minority of sites (this is now shown in Supplementary Figure S5), suggesting that SALL4 engages chromatin in a broad manner, going beyond its preferred motif, possibly in cooperation with other TFs. This is consistent with many studies that in mESCs have shown that SALL4 binds at OCT4/NANOG/SOX2 target motifs. This is now discussed in a dedicated paragraph in the discussion.

Figure 4G. How was the ATAC-seq data normalized for the WT and SALL4-het-KO lines for this comparison? The background levels of accessibility seem quite different in Replicate 1.

The bigwigs used to make the heatmaps are normalized by sequencing depth using the Deeptools Suite (normalization by RPKM).

Figures 5B-C could be exchanged to flow better with the text. A Venn diagram could be included to show the overlap between the sites losing BRG1 in SALL4-het-KO (13,505 sites) and the Day5-specific SALL4 sites (17,137 sites).

Done, thank you.

At Day 5, the authors suggest a shift towards neural differentiation. It could be interesting for the authors to perform qRT-PCR at Day 5 for some neural markers or look in the Day 14 data for markers of neural differentiation at the expense of CNCC markers.

See updated Supplementary Fig. S8, where we show timecourse expression of several genes, including neural markers.

Is the data used to plot Figure 5D the same as Figure 4G. If so, why is only one replicate shown in Figure 5D?

Only one replicate was shown in the main figure purely for lack of space, but the experiment was replicated twice (with the two different clones), and the results were exactly the same. See plots below for your convenience:

Figure 6A. How many replicates are shown? If n=2, boxplots are not an appropriate to represent the distribution of the data. Please include n= X in the figure legend and plot the raw data points also.

Done, thank you, and as suggested we are no longer using boxplots for this panel.

Figure 6B. What is the significance of CD99 for CNCC differentiation?

Figure 6F. No error bars are shown, how many replicates were performed for this time couse? The linear regression line does not appear to add much value and could be removed.

As suggested, we have removed these plots and replaced them with individual genes plots, which include error bars. See updated Supplementary Figure S8.

At line 304, the authors state "while SALL4-het-KO showed a significant downregulation of these genes". Perhaps 'failed to induce these genes' may be more accurate unless they were expressed at Day 5 and downregulated at Day 14.

Done, thank you.

Lines 332-335. The genes selected for pluripotency, neural plate border, CNCC specification could be plotted separately in the Supplement to show individual gene expression dynamics.

Done, thank you, see point 24.

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

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Summary

In this manuscript, the authors build on their previous work (Pagliaroli et al., 2021) where they identified an interaction between the transcription factor SALL4 and the BAF chromatin remodeling complex at Day-5 of an iPSC to CNCC differentiation protocol. In their current work, the authors begin by exploring this interaction further, leveraging AlphaFold to predict interaction surfaces between SALL4 and BAF complex members, considering both SALL4 splice isoforms: a longer SALL4A (associated with developmental processes) and a shorter SALL4B (associated with pluripotency). They propose that SALL4A may interact with DPF2, a BAF complex member, in an isoform-dependent manner. The authors next explore the role of SALL4 in craniofacial development, motivated by patient heterozygous loss of function mutations, leveraging iPSC cells with an engineered SALL4 frameshift mutation (SALL4-het-KO). Using this model, the authors first demonstrate that a reduced expression of SALL4 does not impact the iPSC identity, perhaps due to compensation via upregulation of SALL1. Upon differentiation to neuroectoderm, SALL4 haploinsufficiency causes a reduction in newly accessible sites which are associated with a reduction in SALL4 binding and therefore a loss of BAF complex recruitment. Interestingly, however, there were few transcriptional changes at this stage. Later in the CNCC differentiation at Day-14 when the wildtype cells have switched expression of CNCC markers, the SALL4-het-KO cells fail to switch cadherin expression associated with a transition from epithelial to mesenchymal state, and fail to induce CNCC specification and post-migratory markers. Together the authors propose that SALL4 recruits BAF to CNCC enhancers as early as the neuroectodermal stage, and failure of BAF recruitment in SALL4-het-KO lines results in a loss of open chromatin at regulatory regions required later for induction of the CNCC programme. The failure of the later differentiation is compelling in the light of the early stages of the differentiation progressing normally, and the authors outline an interesting proposed mechanism whereby SALL4 recruits BAF to remodel chromatin ahead of CNCC enhancer activation, a model that can be tested further in future work.

Major comments

The link between SALL4 DNA binding and BAF recruitment is nicely argued, and very interesting as altered chromatin accessibility at Day 5 in the neuroectodermal stage is associated with only few changes in gene expression, while gene expression is greatly impacted later in the CNCC stage at Day 14. The in silico predictions of SALL4-BAF interaction interfaces are perhaps less convincing, requiring experimental follow-up outside the scope of this paper. Some of the associated figures could perhaps be moved to the supplement to enhance the focus on the later functional genomics experiments.

1. A lot of emphasis is placed on the AlphaFold predictions in Figure 1, however the predictions in Figure 1B appear to be mostly low or very low confidence scores (coloured yellow and orange). It is unclear how much weight can be placed on these predictions without functional follow-up, e.g. mutating certain residues and showing impact on the interaction by co-IP. The latter parts of the manuscript are much better supported experimentally, and therefore perhaps some of the Figure 1 could move to a Supplemental Figure (e.g. the right-hand part of 1B, and the lower part of Figure 1C showing SALL4B predicted interactions). The limitations of AlphaFold predictions should be acknowledged and the authors should discuss how these predicted interactions could be experimentally explored further in the future.
2. The authors only show data for one heterozygous knockout clone for SALL4. It is usual to have more than one clone to mitigate potential clonal effects. The authors should comment why they only have one clone and include any data for a second clone for key experiments if they already have this. Alternatively, the authors could provide any quality control information generated during production of this line, for example if any additional genotyping was performed.
3. The authors show all genomics data (ATAC-seq, CUT&RUN and ChIP-seq) as heatmaps and average profiles. It would be valuable to see some representative loci for the ATAC seq (perhaps along with SALL4 and BRG1 recruitment) at some representative and interesting loci.
4. Figure 4A. The schematic could be improved by including brightfield or immunofluorescent images at the three stages of the differentiation. Are the iPS cells seeded as single cells, or passaged as colonies before starting the differentiation. Further details are required in the methods to clarify how the differentiation is performed, for example at what Day are the differentiating cells passaged, this is not shown on the schematic in Figure 4A.
5. There is likely some heterogeneity of cell types in the differentiation at Day 5 and Day 14. Can the authors comment on this from previous publications or perhaps conduct some IF for markers to demonstrate what proportions of cells are neuroectoderm at Day 5 and CNCCs at Day 14.
6. For the motif analysis for Day 5-specific SALL4 binding sites (Figure 4E), was de novo motif calling performed? Were any binding sites reminiscent of a SALL4 binding site observed (e.g. an AT-rich motif)? Could the authors comment on this in the text - if there is no SALL4 binding motif, does this suggest SALL4 is recruited indirectly to these sites via interaction with another transcription factor for example?
7. Does SALL1 remain upregulated at Day-5 and Day-14 of the differentiation for the SALL4-het-KO line? Are binding sites known for this TF and were they detected in the motif analysis performed? Further discussion of the impact of the overexpression of SALL1 on the phenotypes observed is warranted - e.g. for Figure 5F, could the sites associated with a gain of BRG1 peaks upon loss of SALL4 be associated with SALL1 being upregulated and 'hijacking' BAF recruitment to distinct sites associated with nervous system development? Is SALL1 still upregulated at Day 5?
8. Related to the point above, SALL4A is proposed to have an isoform-specific interaction with the BAF complex. It would be valuable to plot SALL4A and SALL4B expression from the available RNA-seq data at Day 0, 5 and 14 to explore whether stage-specific isoform expression matches with the proposed role of SALL4A to interact with BAF at Day 5. It could be valuable to also look at expression of SALL1, 2 and 3 across the time course to see whether additional compensation mechanisms are at play during the differentiation.
9. At line 264, The authors state "SALL4 recruits the BAF complex at CNCC developmental enhancers to increase chromatin accessibility". Given that this analysis is performed at Day 5 of the differentiation, which is labelled as neuroectoderm what evidence do the authors have that these are specifically CNCC enhancers? Statements relating to enhancers should generally be re-phrased to putative enhancers (as no functional evidence is provided for enhancer activity), and further evidence could be provided to support that these are CNCC-specific regulatory elements, e.g. showing representative gene loci from CNCC-specific genes. Discussion of the RNA-seq presented in Supplementary Figure 2B may also be appropriate to introduce here given that large numbers of accessible chromatin sites are detected while the expression of very few genes is impacted, suggesting these sites may become active enhancers at a later developmental stage.
10. Do any of the putative CNCC enhancers detected at Day 5 as being sensitive to SALL4 downregulation and loss of BAF recruitment overlap with previously tested VISTA enhancers (https://enhancer.lbl.gov/vista/)?

Minor comments

1. The authors are missing references in the introduction "a subpopulation of neural crest cells that migrate dorsolaterally to give rise to the cartilage and bones of the face and anterior skull, as well as cranial neurons and glia".
2. The discussion of congenital malformations associated with SALL4 haploinsufficiency is brief in the introduction. From OMIM, SALL4 heterozygous mutations are implicated with the condition Duane-radial ray syndrome (DRRS) with "upper limb anomalies, ocular anomalies, and, in some cases, renal anomalies... The ocular anomalies usually include Duane anomaly". That Duane anomaly is one phenotype among a number for patients with SALL4 haploinsufficiency could be clarified in the introduction. Of note, this is stated more clearly in the discussion but needs re-wording in the introduction.
3. The statements "show that the SALL4A isoform directly interacts with the BAF complex subunit DPF2 through its zinc-finger-3 domain" and "this interaction occurs between the zinc-finger-cluster-3 (ZFC3) domain of SALL4A and the plant homeodomains (PHDs) of DPF2" in the introduction appear overstated and should be toned down. To show this the authors would need to mutate or delete the proposed important zinc-finger domains from SALL4A, which is outside the scope of this work. Notably, this is less strongly-stated elsewhere in the manuscript, e.g "predict that this interaction is mediated by the BAF subunit DPF2", Line 162.
4. Could the authors clarify why 3 Alphafold output models are shown for SALL4B in Figure 1C, and only one output model for SALL4A?
5. Line 121. The authors state "DPF2, a broadly expressed BAF subunit,", but don't show expression during their CNCC differentiation. It would be good to include expression of DPF2 in Figure 1E.
6. The text states "a 11 bp deletion within the 3'-terminus of exon 1 of SALL4", while the figure legend states, "Sanger sequencing confirming the 19 bp deletion in one allele of SALL4 is displayed". The authors should clarify this disparity and experimentally confirm the deletion, e.g. by TA-cloning the two alleles and sequencing these separately to show that one allele is wildtype and the other has a frameshift deletion.
7. The authors generate an 11-bp (or 19-bp?) deletion in exon-1 - it would be valuable to include a discussion whether patients have been identified with deletions and frame-shift mutations in this region of SALL4 exon-1. And also clarify, if not clearly stated in the text, that both SALL4A and SALL4B will be impacted by this mutation. Are there examples of patient mutations which only impact SALL4A?
8. For the SALL4 blots in Figure 2B, is the antibody expected to detect both isoforms (SALL4A and SALL4B), and which isoform is shown? If two isoforms are detected, they should both be presented in the figure.
9. SALL4 expression should be shown for Figure 2C to see whether the >50% down-regulation of SALL4 at the protein level may be partially driven by transcriptional changes.
10. The number of experimental replicates should be indicated in all figure legends where relevant. Raw data points should be plotted visibly over the violin plots (e.g. Figure 2C).
11. For Figure 3A, the images of the DAPI and NANOG/OCT4 staining should be shown separately in addition to the overlay.
12. The metric 'Corrected Total Cell Fluorescence (CTCF)' should be described in the methods. The number of images used for the quantification in Figure 3A should be indicated in the legend, and error bars included if multiple images were quantified.
13. Figure 3C - what are the 114 differentially expressed genes? Some interesting genes could be labelled on the plot and the data used to generate this plot should be included as a Supplementary Table. Supplementary Tables should similarly be provided for Figure 6C, Day 14 and Supplementary Figure 2B, Day 5.
14. Figure 4B. The shared peaks are not shown. For completeness, it would be ideal to show these sites also.
15. Figure 4C is difficult to interpret. Why is the plot asymmetric to the left versus right? What does the axis represent - % of binding sites?
16. Line 224-225. What do n= 3,729 and n= 6,860 refer to? There appear to be many more binding sites indicated in Figure 4B, therefore these numbers cannot represent 86% and 97% of sites?
17. Figure 4E. Several TFs mentioned in the text (Line 243) are not shown in the figure, it would be good to show all TFs motifs mentioned in the text in this figure. Again, there is no mention of whether a sequence-specific motif is detected for SALL4 (e.g. an AT-rich sequence) from this motif analysis.
18. Figure 4G. How was the ATAC-seq data normalized for the WT and SALL4-het-KO lines for this comparison? The background levels of accessibility seem quite different in Replicate 1.
19. Figures 5B-C could be exchanged to flow better with the text. A Venn diagram could be included to show the overlap between the sites losing BRG1 in SALL4-het-KO (13,505 sites) and the Day5-specific SALL4 sites (17,137 sites).
20. At Day 5, the authors suggest a shift towards neural differentiation. It could be interesting for the authors to perform qRT-PCR at Day 5 for some neural markers or look in the Day 14 data for markers of neural differentiation at the expense of CNCC markers.
21. Is the data used to plot Figure 5D the same as Figure 4G. If so, why is only one replicate shown in Figure 5D?
22. Figure 6A. How many replicates are shown? If n=2, boxplots are not an appropriate to represent the distribution of the data. Please include n= X in the figure legend and plot the raw data points also.
23. Figure 6B. What is the significance of CD99 for CNCC differentiation?
24. Figure 6F. No error bars are shown, how many replicates were performed for this time couse? The linear regression line does not appear to add much value and could be removed.
25. At line 304, the authors state "while SALL4-het-KO showed a significant downregulation of these genes". Perhaps 'failed to induce these genes' may be more accurate unless they were expressed at Day 5 and downregulated at Day 14.
26. Lines 332-335. The genes selected for pluripotency, neural plate border, CNCC specification could be plotted separately in the Supplement to show individual gene expression dynamics.

Significance

This work provides a conceptual advance in understanding the aetiology of human SALL4-mediated craniofacial malformations in a cell-type specific manner. Leveraging an in vitro differentiation system, the authors define development timepoints and cell types impacted by altered SALL4 dosage. Additionally, the authors provide interesting mechanistic insights how the teratogen thalidomide may impact craniofacial development through proteasomal targeting and degradation of SALL4, and subsequent impact on neural crest differentiation progression.

Several audiences will be interested in this work: stem cell and developmental biologists (especially those interested in neural crest and facial development), and researchers interested in enhancer regulation, chromatin biology or gene regulatory mechanisms. Clinician scientists and geneticists will be interested in the proposed implications for mechanisms of disease.

Field of expertise: We have expertise in mechanisms of gene regulation and in vitro models of early development. We are not experts in modeling protein interactions in silico.