These methods are quite useful. By explaining what you read to someone you are able to recall the information and rearrange to make sense in your head. When you are able to connect those dots in way that makes sense in your head you are able to convert that information into long-term memory. Which is very useful for retaining information.

I really agree with this statement. Like it's always hard for me to stay focused on what we are talking when my mind is always wandering off

discrediting his talent as a musician

dangerous travels

Revenons plus en détail sur chacun des éléments OBLIGATOIREs d’une page web :

cf. code minimal dans le validateur ```html

<meta charset="utf-8"> <title></title>

coucou ```

La structure d’une page web :

I am not either, so this makes the author more relatable to me.

Is this the reason that we have such social problems in the United States? Hierarchies may benefit us as a whole, but somehow those at the bottom (along with a racist presumption that that's where they below) are hurt the most?

How do we turn this on it's head?

Though the persona is in doubt from all the things unsaid and unknown. The persona is still trying to trust the subject. It also may refer to him relieving someone of his own doubts.

regarding what my classmate Sama commented, I would say wars come first leading the creation of states later on. However, in the case of a war that is the out birth of a state, I would say that saying that states cause wars not accurate as I think that the deflection of the head of the state from the basic objective of the state formation and the deviation to meet the other superior powers interests (thus will be reflected on the interests of the special governing power) is the reason.

Quote:

Talks about the three-dimensional structure of the bacteriophage and a brief introduction on the receptor of the phage.

It's important to remember that the biggest thing has to do with the oath Jason and Medea made. As we read in Module 1, "Furthermore when men swear to do something for a woman--grant sanctuary, keep a secret, carry a letter--they also enter into a contract with the gods who function as the guarantors of the oath."

Oaths had a very important part in all of Greek culture, and I find that its in fact Aegus to warn Jason of this. Due to him protecting Medea, the latter aids Aegus and wards off Jason by her final punishment. As Karyn Greene writes, "Not only does Medea use oaths to punish Jason for everything that he has done against her, she also uses them to undo everything that she has done for him. While Jason may claim that he has never broken his oaths, it is clear that his punishments are justified because the gods, who play such an active role in the process, do not stop Medea from exacting her revenge. Medea systematically manipulates nearly every single character within the narrative to punish Jason for his betrayal and she is successful in doing so. Medea strips Jason of his reputation but she also takes so much more. By killing both her children and the princess, Medea makes it impossible for Jason’s line to continue. Never will Jason be able to increase the glory of his house, in fact, he will die a pathetic death by being hit over the head with piece of wood fallen from his own ship."

In the final moments, Jason has broken his oath, causing everything to be stripped from him; his hope to earn a crown, the Golden Fleece, his marriage, all gone. As the last testament he had, the Argo, crushed him, along with the weight of all his failures.

Greene, Karyn. Oath Making and Breaking in Euripides' Medea - Denison University. Denison University, 2012, https://digitalcommons.denison.edu/cgi/viewcontent.cgi?article=1007&context=ephemeris.

This is important to include. Without a look into how different students think and feel as a result of their experience with praise, the entire argument falls apart. It was a good choice by the author to write a hypothetical situation that teachers may be able to connect to one of their real students.

This is one of the main reasons qhy people immigrate to America. Ots very interesting to find out that in other countries make the same amount that American often take for granted America pays a lot more than other countries and its a major reason for many people immigrating to America. This quote makes me think about how America sometimes take for granted the resources they have supplied for them.

When we really think about it, we are extremely privileged. To us, fifty dollars really isn't a lot. There are many situations where I have said "oh wow, it's only fifty?" For people in other countries, the same amount of money is how much they earn in a whole month. This is one of the biggest reasons people immigrate to the US, and in turn, New York. They come here for better opportunities, such as finding good jobs with good wages. Many of the jobs that Americans think of as beneath them are taken up by immigrants because they are willing to do the work to earn the money. However, there is still so much talk about immigrants stealing jobs, even though these jobs had already existed but no one was working them. That talk of privilege comes back into play here. Because of the fact that we are privileged, we are able to think of these jobs as beneath us, but for immigrants coming into the country who need to find a job quickly and a job that is willing to take them, this is what they need to do. Even if they themselves believe of the job as beneath them, they essentially have no choice; they need to work to survive.

Reviewer #2 (Public Review):

This study reports a novel role of thalamic activity in the late components of a cortical event-related potential (ERP). To show this association, the authors used high-density EEG together with multiple deep electrophysiological recordings combined with electrical stimulation of superficial and deep cortical layers. Stimulation of deep layers elicits a late ERP component that is closely related to bursts of thalamic activity during quiet wakefulness. This relationship is quite noticeable when deep layers of the cortex are stimulated, and it does depend on the arousal state, being maximal during quiet wakefulness, diminished during active wakefulness, and absent during anesthesia.

The study is very well performed, with a high number of subjects and appropriate methodology. Performing simultaneous recording of EEG and several neuropixels probes together with cortical microstimulation is no small feat considering the size of the mouse head and the fact that mice are freely behaving in many of the experiments. It is also noticeable how the authors use a seemingly outdated technique (electrical microstimulation) to produce compelling and significant research. The conclusions regarding the thalamic contributions to the ERP components are strongly supported by the data.

The spatiotemporal complexity is almost a side point compared to what seems to be the most important point of the paper: showing the contribution of thalamic activity to some components of the cortical ERP. Scalp ERPs have long been regarded as purely cortical phenomena, just like most EEGs, and this study shows convincing evidence to the contrary.

The data presented seemingly contradicts the results presented by Histed et al. (2009), who assert that cortical microstimulation only affects passing fibers near the tip of the electrodes, and results in distant, sparse, and somewhat random neural activation. In this study, it is clear that the maximum effect happens near the electrodes, decays with distance, and is not sparse at all, suggesting that not only passing fibers are activated but that also neuronal elements might be activated by antidromic propagation from the axonal hillock. This appears to offer proof that microstimulation might be much more effective than it was thought after the publication of Histed 2009, as the uber-successful use of DBS to treat Parkinson's disease has also shown.

Sometimes it feels like the only parts of the paper that actually matter are the abstract, figures, and conclusion. This is a good reminder that the rest of the paper is also important--if you're interested in the results, then you'll want to be able to figure out how the writer got them.

A common theme is everyone reads the actual paper part of the paper last

Her awareness to the fact that Beau will never be satisfied and she will never be enough for him and any from is mind boggling because it's so ridiculous. In her head the thinks sleeping with someone is enough the make them want you forever but neither of her characters got emotionally intimate with him, yes she fell for him because she was naive but her knows better and experienced better; experience a lot of woman and most if not all of them has only offered him sex, a momentous passion, not has really connected with him beyond. How can she expect him to be faithful to a fleeting moment? She is honestly obsessed with him and is so lost in her deception that she is now Irreversible.

She wants to leave him with mystery so that there will be a second time of them seeing each other therefore she still has that power.

The connection between Chris and the deer is very interesting

I think this is interesting because this is about perspective. To Chinese people the Manchu seemed barbaric because of their modernity in China.

This is exactly how I felt about a lot of my classes. I know that in the end I will be happy I took some of these courses but this was definitely a recurring thought in my head during some of these classes.

正如获奖不代表高票房，不获奖不代表不会受到观众喜爱。以aftersun为例子，出发点和内容很好，也获了奖，但是并不卖座。但是对于电影工业来说，有了钱才有可能保持创造力（需要佐证这句话）。

the enlightenment allows change + progress but not individuality. individulity is lost due to conformity + custos

Extreme foreshadowing, but can even can ponder the question to the audience if Mrs.Mallard was dead since the news of her husbands death in the very beginning paragraph the story. Chopin even portrays the use of the word of "motionless" , which can be a significant symbolism of death or shock.

Was it all in her head or was it thought the husband had died?

I can relate to this because I often refer back to my journal when I think of any event or situation that came across my head. When I write something in my journal or planner, I tend to write it as if I'm talking and telling a story to my future self.

Very impressive, but if this is what is required, I don't think most students feel they have time to do this. To me, this seems almost like the academic equivalent of hitting your head against a wall until you figure something out.

• Comparing = biophyiscal boundaries with = socio-economic boundaries provides an important : insight
• these : metrics - can be considered to be =downscaled doughnut economics - metrics
• All these cities : violate = doughnut economics
• the : highly industrialized cities have = high carbon emissions - but have good = socio-economic boundaries while
• the : poorly industrialized cities have = low carbon emissions - but poor = socio-economic boundaries
• to : balance out, each category must head in : opposite directions

Clearly at that time, people were understaffed, underpreparred, and overworked.

pathos, need to come back to this bit several times with a level head. huge eye roll.

This describes where you are starting from and where you are about to head.

He is describing both the temple, and the throne, of Zeus.

Description of the Throne of Zeus

temple / statue of Zeus

Explaining the Temple of Zeus.

description of the statue and throne of Zeus by Pheidias located in the Temple of Zeus

Statue of Zeus described here

Descriptive of the statue of Zeus and how it was decorated.

Statue of zeus description

This helped me out in my current life. At my job my coworker has problem with the nurses and their lazy ways. She said she wouldn't tell our supervisor because it wont help out the situation at hand. She wanted me to join her in going over my supervisor's head. I told her that wouldn't be a good idea and consequences could be handed down. She did any way and now she has Heat on her back. My supervisor is secretly watching her like a hawk waiting for her to slip up. Lesson learned stay in your lane and go by the chain of command.

I really like how he used the fish story to make this point. I find this to be a very true statement and it reflects how as people we do not like to face our problems head on (at least I know I personally don't).

This puts a very vivid picture in your head and brings out a lot of emotion to the reader

comparable attributes to other religious stories

It is interesting how similar the story is to the Christian creation belief, and many others.

description of temple of Hera

Explaining the in depths of the Temple of Hera.

Description of the inside of the Temple of Hera.

Further Description of the Temple of Hera

hard to read the descriptive text of this torture

stories about devout convert Christians- mostly children

This is so sad. It makes me wonder Why the Iroqious were so brutal.

...coming back to this, now. ...

It is like every form of media that enters a fan's head is an ingredient with a fixed taste, but then in the creative process of storytelling a fan uses those ingredients in a distinct way to create a dish with entirely new flavors.

!- COP28 President : is head of UAE ‘s largest oil company - putting the Fox in charge of the hen house

Up to "So, you refuse to shake hands with me, eh? (g Slaps T with glove; T: This means war

here is one way to work onthe first draft

It is jarring seeing this much trust and belief in a plan that has never been anywhere outside of Putin's mind. The people and other politicians were just totally on board with a plan that isn't a plan but is a plan in Putin's head.

Reading this gave me the chills. The thought that went through my head was "why would she do that?" I thought was it due to stress? She was attending an ivy league so there was definitely challenges there. Her actions aren't justified though no matter what race or how successful she is.

This I feel is a great way to leave the students thinking about the information and to make sure the ideas stay in their head.

Funn

A quque is used, we always choose from the head of the list to update the arcs. It means visiting the parent vertices on the tree before visiting the children vertices on the BFS tree.

• It gives the shortest path (In terms lf the number of vertices) to go to each of the leaf nodes in the graph.

you must allow it to exist if it will ever grow and become. if you never rpactice at it it will enveer be as good as you like if you never allow it to live it will froever be stifled trapped in your head write adn the words will come forth unbidden uninhibited paint and hte lines will simply blur draw and the ink will come out line aby line uhntil you have formed the oerfect form draw and rub and smudge and you go from having a sheet of paper to a fascimile of a person leaving you feeling as if you ar ebeing stared at through the screen build and you will have the object of your beliefs question and search and learn until you have all the tools you need and allow it to exist build it up and let it be you want to be the successful entrepenuer with everything of which they have evr dreamed? okay then let it be find out what you have to do what must be done and then do it or you will never achieve your dreams or have all taht you desire. Creativitiy is in the very air we breathe and why we surive at all it is how new surgeries are created how we get too and from school how we learn and have things to learn it is the lifeblood of life and to exist is to create in tandem with the world

ffs

I like how the text helped give a better image of what the octopus looks like and the different things that also come with the octopus. Before I didn't really have and understanding but now I completely do.

Amen

Reviewer #2 (Public Review):

The manuscript describes a novel transparent electrode array and demonstrates its combination with two-photon calcium imaging in mouse neocortex. Using a computational model, the authors propose that surface multi-unit activity mainly reflects L1 axonal activity and they find a small population of L2/3 neurons that correlates with this activity. While the multi-modal approach with the innovative device in our view is interesting and potentially useful, we have several technical and scientific concerns that should be addressed by the authors.

Strengths:<br /> We find the general scope of this manuscript, to establish a hybrid electrophysiological and optical approach for studying neocortical activity, very interesting and relevant. The authors provide a compelling use case for combined ECoG and two-photon imaging. While extracellular action potentials have been recorded from the cortical surface, the underlying source is unknown and the device and techniques introduced by the authors are appropriate to address this question. The introduced device can be implanted chronically and has good long-term stability, providing longitudinal optical and electrical recordings from the cortex. The authors perform recordings in awake, head-fixed animals which provides the opportunity to relate ECoG and single-cell data to the animal's behavioral state. The combination of empirical data and biophysical modelling is a powerful means by which to answer such questions.

Weaknesses:<br /> The central claim of the paper relies heavily on the computational model and the physiological data could be more completely analyzed. Based on a sample of 136 L2/3 neurons the authors find a small proportion (13%) that correlates with the ECoG MUA (eMUA). Based on this, they use a model to show that ECoG MUA likely reflects axonal spikes. They then posit that these layer 2/3 neurons are tightly correlated to the layer 1 input. The presentation of their data and the specifics of their model makes it difficult to assess the validity of this claim. They do not sufficiently discuss possible confounds in the data, caveats of their model, or alternative explanations of the observed low proportion of L2/3 neurons that correlate with the ECoG MUA.

Most relevantly, the authors do not measure single units with their ECoG. The eMUA is a complex mixture of many neuronal sources, and interpretation is therefore difficult. They relate the calcium transients of small populations of single L2/3 neurons with the aggregate measure of population activity reflected in eMUA. It is possible that the eMUA reflects population activity in the local circuit and might therefore have a low correlation with individual single units. Critically, there is no information on the sensitivity of calcium recordings. Do the imaging data detect single action potentials, or are they biased to bursts of more than 1 AP?

The analysis pipeline and values used for computing the correlation coefficients are counterintuitive. The fluorescence data are first interpolated from 15 Hz to 4 kHz and then both eMUA and imaging data are effectively down-sampled to 2 Hz. A single correlation coefficient is then estimated for each neuron, regardless of behavioral state, even though the authors themselves show that the activity of single neurons and the ECoG signal depend on the state of the animal.

There is also insufficient information on the weight of the implant and its effect on mouse behavior. How does the movement of implanted and non-implanted mice differ? Must mice be singly housed? Finally, the modeling parameters are highly specific, using independently driving spikes, while the activity of neurons can be highly correlated. Likewise, the contribution of tangentially oriented axons that could relate to long-range connections conveying information related to the animal's motion or level of arousal is not considered. The manuscript would benefit from further analysis of the physiological data, consideration of alternative explanations and forthright discussion of limitations and caveats of their device and approach.

Again...my head is exploding with the feminine series.

I would definitely search for "relics" myself if I was on this journey.

Physical attributes of bacteriophage.

Bacteriophages are three-dimensional structures that are responsible to recognize surface molecules on the host bacterium.

Bacteriophages are found in different enviroments.

This shows where they bacteriophages are found in the environment, while also giving a describive overview as to what bacteriophages look like.

She seems upset about getting exposed for her views and facing the consequences, she thinks this is a result of "cancel culture"

the "voyage to discovery" could refer to the idea of learning new things. When Marcel Proust states that the way to learn is "not in seeking new landscapes but in having new eyes" we could conclude that what he is saying is that the most effective way gain knowledge is to observe different perspectives of the same thing or subject, not to fill your head with a variety of subjects.

putin not as tough as the masculine image he projects

avoiding journalists

this likely means that the system nearly fully recognized that wound site as the anterior end instead of posterior now.

interesting that APC is used to prevent head formation by degrading b-catenin, is it expressing everywhere except for the head? how exactly does it differentiate the cells

I know they say that the new head move autonomously from the rest of the body, I wonder how much control this new brain has over the body and if it is fully/properly developed.

This is very odd, I wonder if these spots may have been accidentally wounded and so the body thinks it needs to grow a head there (if maybe wounds are also regulated by catenin and ACP) or if its something similar to how mutation in these genes in humans can cause cancerous growths

This may not be a great question but I wonder if this is transient or stable. If the worms that grew a head where its tail should be were amputated again would it grow back wrong again?

Ist it really that interesting? to me it seems that an upstream component would not affect the specification because it does not matter yet, the pathways start out the same and then diverge based on ACP/beta-catenin

figure 1 seems to support their hypothesis as the control group regenerated head and tail as expected, the b-catenin group did not regenerate a tail and a second head was regenerated instead, Dvl-a/b did not regenerate at all, and APC regenerated tails at both ends with no head.

Clasificación macroscópica de los tumores pancreáticos mucinosos intraductales (TPMI): - De la vía principal (Más degeneración maligna 60%) - De rama ductal (Menor degeneración maligna 5%)

Is there any symbolic meaning behind these two figures?

This was the most refreshing sentence to read. It sums up evidence that proficiency equates exposure to language in addition to the factors of instruction quality, motivation, and opportunities. Kept nodding my head. If a child has lots of exposure and practice versus not... then more practice would mean efficiency and proficiency.

eLife assessment

This study provides fundamental insight into the functional impact of CDK4 inhibition on cells in the tumor microenvironment, which is of high importance and interest to the field. The compelling conclusion that proliferative exhausted T cells are associated with response in HPV+ head and neck cancer is supported by the cohort of 14 patients with paired tumor and adjacent normal tissue and rigorous bioinformatic analysis of nearly 50,000 single CD3+ T cell transcriptomes. This work will be of interest to researchers across tumor types and in other immunological fields of study.

Joint Public Review:

In this study, the authors transcriptomically characterize TIL from head and neck cancers and associate their transcriptional programs with overall survival as a function of HPV positivity. Specifically, they study the impact of CDK4 inhibition on TIL from these tumors. They find an exhausted T cell subset that preferentially expresses CDK4. They then perform some in vitro studies to test the function of exhausted T cells and the impact of CDK4 inhibition on different TIL subsets from head and neck tumors. Understanding the functional impact of different cancer therapies on cells in the TME is of high importance and interest to the field.

1. Line 215: The authors state that pairing TCRseq with RNAseq reflects the magnitude of TCR signaling. This is absolutely not the case. TCR sequencing does not reflect TCR signaling strength.<br /> 2. A lot of discussion around "activation" is presented, but there is no evidence to support which genes or gene programs are associated with "activation".<br /> 3. Line 249: It is unclear why the authors are indicating that TCRseq was used in pseudotime analysis. This type of analysis does not take TCRs into account but rather looks at the proportion of spliced mRNA of individual genes from the DGE data.<br /> 4. There is no way to know if the differences in proliferation and cell viability shown in Figs. 4a and b, respectively, are meaningful or not. Proper controls or replicates should be provided to fully understand if this difference is biologically meaningful. Likewise, what is the evidence that P-Tex cells are self-renewing rather than expanding?

Example of someone getting the crap and worries out so that their writing can begin apace. Its sort of like writers' therapy and closely akin to those who talk about morning pages.

Also similar to teachers of young children who encourage their students to get their "wiggles out" so that they can focus on the classwork at hand.

devastating picture to paint

yes

How painfully true this may have been in 1899, it's now much worse in 2023!

Specialization of knowledge tends to fit the lifeways of the people who hold and maintain it. Changing lifeways means one must lose one or more domains and begin using or curating different domains of knowledge.

In a global world of specialization, humans who specialize are forced to rely more heavily on the experience and veracity of those around them who have also specialized. One may be able to have a Ph.D. in astrophysics, but their knowledge of the state of the art in anthropology or economic policy may be therefore utterly undeveloped. As a result they will need to rely on the knowledge and help of others in maintaining those domains.

This knowledge specialization means that politicians will need to be more open about what they think and say, yet instead politicians seem to be some of the least knowledge about almost anything.

This is just the start of a somewhat well-formed thesis I've developed elsewhere, but not previously written out... more to come...

THE MONOLOGUE CONTINUES, UNDERSTAND ME.

It doesn’t take much “thought” to see these star charts–our Astrological road maps to ‘wisdom of the Ancients’ might actually be something closer to road maps than I could have previously fathomed–let alone imagined. I’m staring at “Monoceros” and seeing it’s definately connected to “the kissing disease” and to Eros and to Cupid–and seeing … this one not for the first time that character linked to Orion and to the “Speare” of Sagittarius.

I’ve commented … ‘on the show in my head’ that it seems the entirety of the Milky Way might be something like our world … it could be a microcosmic map to something much larger–it could be the seed of “galaxies” in this place that might very well be the “thing” that connects the end and the beginning; rather than the beginning and the end as I once … commented was the original “glyph” i read in the letter “H.”

--

someone commented on the site, they posted a picture from my earlier work ... the "WHY?" one that depicted starvation and crucifixion and no press.

in related news the LA Times spoke, it echoed "and he's thinking about his own mortality" seconds after the event ... the self questioning of whether or not I have any "kind of divinity" in me at all. Dana too, has echoed back that there's a message I am missing.

I forgot to mention the press junket's every day, that was a kind of speech that you can't really "feel" in the rest of the articles that talk about things like walls and "something missing." Acosta may have written more on the reason, but I wasn't able to find out exactly what it was they were saying.

Lately science has started talking about things ... "going haywire" I'm here with "IGNITION" and LLNL on my mind, and also the power of star creation and destruction connecting to the Pentagon and Deuteronomy and ... deuterium and fusion and fission and the Vooshan Young.

I imagine some people read through this feed, the one I'm posting to. I've just posted this:

This is what I have to do to ensure things "aren't vanished upon death" or worse, while I'm still typing about them in the very same day. The post was "vanished" from github, and I mean; it's here for the protection of not just veracity and Americana, but Hypothesis itself.

• https://www.publishthis.email/fw-i-am-not-sure-how-to-break-this-to-you-...-i-feel-in-all-honesty-that-i-am-a-true-ameri-Hkm-2tuos

• https://ipfs.2read.net/ipfs/QmXCxWmuGF48LezyiGLFRzcRjjMzLCDZnRaYajFajvndGv/

Edit: the Github posting didn't disappear it was just marked as closed; along with an explanation "about hearing and answering before."

There's a fortune in building the thing; and putting it together; it's basically "the next big thing" a news and "what's popular on the web" aggregator that has advanced search and friendship capabilities. Integrating with LinkedIn and Facebook and Twitter and ... "most of all abstracting those things with an identity system that ties directly to IPFS and strong identity validation and authentication--

Out of the Ether ..

PS: noting that this link ties to the root directory of fromthemachine dot org and it's already an aged post about this very thing, building something with Ethereum that "is glaringly missing" .. including "find your friends" integration that doesn't require them to send you a long random string of letters and numbers--just being "already connected". Creating a Wallet/Address system that ties together the social networks and "crypto trading" is glaringly missing, and we can already see applications like Snapchat and Tik-Tok that have done it in a way that creates a significant growth factor ... I mean it almost instantly made Tik Tok as big as Instagram.

This tool is a key; being able to see "what everyone is saying about the front page news on the outlets you read every day" in a news feed and interface similar to Facebook's ... "I think that's a game changer for me; I would use it."

On the Jokerzettel

9/8j Im Zettelkasten ist ein Zettel, der das Argument enthält, das die Behauptungen auf allen anderen Zetteln widerlegt.

Aber dieser Zettel verschwindet, sobald man den Zettelkasten aufzieht.

D.h. er nimmt eine andere Nummer an, verstellt sich und ist dann nicht zu finden.

Ein Joker.

—Niklas Luhmann, ZK II: Zettel 9/8j

Translation:

9/8j In the slip box is a slip containing the argument that refutes the claims on all the other slips. But this slip disappears as soon as you open the slip box. That is, he assumes a different number, disguises himself and then cannot be found. A joker.

Many have asked about the meaning of this jokerzettel over the past several years. Here's my slightly extended interpretation, based on my own practice with thousands of cards, about what Luhmann meant:

Imagine you've spent your life making and collecting notes and ideas and placing them lovingly on index cards. You've made tens of thousands and they're a major part of your daily workflow and support your life's work. They define you and how you think. You agree with Friedrich Nietzsche's concession to Heinrich Köselitz that “You are right — our writing tools take part in the forming of our thoughts.” Your time is alive with McLuhan's idea that "The medium is the message." or in which his friend John Culkin said, "We shape our tools and thereafter they shape us."

Eventually you're going to worry about accidentally throwing your cards away, people stealing or copying them, fires (oh! the fires), floods, or other natural disasters. You don't have the ability to do digital back ups yet. You ask yourself, can I truly trust my spouse not to destroy them?,What about accidents like dropping them all over the floor and needing to reorganize them or worse, the ghost in the machine should rear its head?

You'll fear the worst, but the worst only grows logarithmically in proportion to your collection.

Eventually you pass on opportunities elsewhere because you're worried about moving your ever-growing collection. What if the war should obliterate your work? Maybe you should take them into the war with you, because you can't bear to be apart?

If you grow up at a time when Schrodinger's cat is in the zeitgeist, you're definitely going to have nightmares that what's written on your cards could horrifyingly change every time you look at them. Worse, knowing about the Heisenberg Uncertainly Principle, you're deathly afraid that there might be cards, like electrons, which are always changing position in ways you'll never be able to know or predict.

As a systems theorist, you view your own note taking system as a input/output machine. Then you see Claude Shannon's "useless machine" (based on an idea of Marvin Minsky) whose only function is to switch itself off. You become horrified with the idea that the knowledge machine you've painstakingly built and have documented the ways it acts as an independent thought partner may somehow become self-aware and shut itself off!?!

https://www.youtube.com/watch?v=gNa9v8Z7Rac

And worst of all, on top of all this, all your hard work, effort, and untold hours of sweat creating thousands of cards will be wiped away by a potential unknowable single bit of information on a lone, malicious card and your only recourse is suicide, the unfortunate victim of dataism.

Of course, if you somehow manage to overcome the hurdle of suicidal thoughts, and your collection keeps growing without bound, then you're sure to die in a torrential whirlwind avalanche of information and cards, literally done in by information overload.

But, not wishing to admit any of this, much less all of this, you imagine a simple trickster, a joker, something silly. You write it down on yet another card and you file it away into the box, linked only to the card in front of it, the end of a short line of cards with nothing following it, because what could follow it? Put it out of your mind and hope your fears disappear away with it, lost in your box like the jokerzettel you imagined. You do this with a self-assured confidence that this way of making sense of the world works well for you, and you settle back into the methodical work of reading and writing, intent on making your next thousands of cards.

Hamlet may love Ophelia, but he is the heir and one day he will be king, therefore he'll have the responsibility of the whole state on his shoulders and he won't be able to do as he wants.

think about this more

what does that mean exactly?

True. I might still think "Wait a minute! I didn't know you had a cat!" but it would be inside my head, probably I wouldn't say it out loud in the discourse

North Americas are politically more diverse than britian

Something about the phrasing of this seems to flip the framing of the pandemic that we normally think through on its head. It really emphasizes how much of a role we play in it just by virtue of existing as human beings. The pandemic is a hindrance on society, and yet, could not exist without society.

This reminds me of my experience reading fragmented work, such as that of Sappho. The reader is forced to fill out certain parts in their head, collaborating on something that is eventually unique and more particular to the individual.

The seat of feminine justice with little mercy for the men-folk

Arthur introduces the topic of Robert Pirsig and slips into the GTD conversation on 2004-05-19.

Was this a precursor link to the Pile of Index Cards in 2006?

Note that there doesn't seem to be any discussion of any of the methods with respect to direct knowledge management until the very end in which arthur returns almost four months later to describe a 4 x 6" card index with various topics he's using for filing away his knowledge on cards. He's essentially recreated the index card based commonplace book suggested by Robert Pirsig in Lila.

BYPRIYANKA RUNWAL PUBLISHED DECEMBER 22, 2022 • 9 MIN READ Typically, vaccines help protect us against diseases. But cancer vaccines are different; they are potential therapies for treating people who already have cancer. These treatments have been years in the making, and failures have been frequent, but they’re now starting to show some promise.

In the last decade, technological innovations like genome sequencing have allowed scientists to take a closer look at tumor cells and their genetic abnormalities. This is helping them design vaccines aimed at much more specific targets. At the same time, we’ve been learning a lot more about the immune system and how it recognizes and destroys a patient’s tumor, says cellular immunologist Stephen Schoenberger at the La Jolla Institute for Immunology in San Diego.

Cancer vaccine research is still in the nascent stages, says Nina Bhardwaj, a hematology and medical oncology expert at the Icahn School of Medicine at Mount Sinai in New York. But early results from clinical trials testing dozens of vaccine candidates against a variety of cancers look encouraging, she says.

The goal is to roll out vaccines that destroy cancer cells, but some scientists are also testing vaccines that might one day prevent a high-risk individual from developing cancer.

What are cancer vaccines? The purpose of all vaccines, be it a cancer vaccine or COVID-19 vaccine, is to educate the immune system and provide a preview of the target that needs to be identified and destroyed to keep the body safe. The COVID-19 vaccine teaches the immune system what the SARS-CoV-2 virus looks like so when the pathogen infects, immune cells can quickly locate the virus and kill it. Similarly, a cancer vaccine educates immune cells about what a tumor cell “looks like,” enabling them to seek and destroy these cancer cells.

The ability of a cancer vaccine to teach the immune system is what distinguishes it from other immunotherapies that utilize therapeutic agents such as cytokine proteins and antibodies and include strategies like genetically engineering a patient’s immune cells to fight cancer.

Experts says that cancer vaccines can potentially destroy cancer cells that might have survived other treatments, stop the tumor from growing or spreading, or stop the cancer from coming back.

Some therapeutic cancer vaccines rely on removing immune cells called dendritic cells from a patient’s blood sample and exposing them in the laboratory to the key proteins obtained from the individual’s cancer cells. These educated cells are then returned to the patient with the expectation they will stimulate and train other immune cells, such as T cells, to detect and destroy the cancer.

T cells can do one of the most amazing tricks in biology, says medical oncologist Christopher Klebanoff at New York’s Memorial Sloan Kettering Cancer Center. They carry a receptor that can recognize and bind to proteins present on tumor cell surfaces—as a lock fits a key. Once bound, the T-cells use mechanical force to punch a hole through the tumor cell and destroy it, he says.

But “vaccines haven’t been very good at generating the quality and quantity of T cells necessary to eliminate large tumors,” Bhardwaj says. It’s ideal to vaccinate when the tumor is small, she says.

To boost the power of the vaccine, researchers often combine it with drugs that enhance this antitumor immune response.

Vaccine-makers now are increasingly relying on mRNA technology—also used to create COVID-19 vaccines—to instruct dendritic cells in a patient’s body to produce the tumor-specific proteins or peptides that will generate an immune response.

A few vaccines are preventative as they teach the body to kill a cancer-causing viruses like hepatitis B and human papillomavirus, thus averting an infection that could otherwise lead to a tumor.

How do scientists create cancer vaccines? All cancer-treating vaccines rely on proteins, called tumor-associated antigens—a molecule that triggers an immune response when it’s either more plentiful on the surface of cancer cells compared to healthy ones, or exists in an abnormal or mutated form. Once T cells “see” these antigens they recognize the cells as cancerous and kill them.

Cancer biologists identify these tumor antigens with sophisticated sequencing technology that spots specific differences between the DNA or RNA of a healthy cell versus a cancer cell. The trick is to understand which mutations will generate a T cell response and would make a good target for a vaccine, Schoenberger says.

His research group selects antigens based on a patient’s response to the cancer. By studying the T cells in their blood samples, “we’re looking at what the patient’s own immune system has selected among the tumor-expressed mutations to target,” Schoenberger says. He identifies antigens that are unique to an individual’s tumor cells and uses a combination of tumor-specific antigens from different patients to create vaccines. Other researchers look for antigens that are shared between individuals who have a certain cancer, or between different cancer types.

Vaccines designed to target molecules that are overproduced by cancer cells but also present in smaller amounts in healthy cells tend to have limitations and may not initiate an effective immune response. “That’s been a huge hurdle,” says cancer immunologist Lisa Butterfield at the University of California, San Francisco. There’s also the danger of inducing autoimmunity, in which the immune system ends up attacking healthy cells, resulting in bodily disorders that are hard to treat. More efforts are now focused on finding target molecules called neoantigens that are specific to tumors.

Are there approved vaccines to treat cancer and how they do work? In 2010, the U.S. Food and Drug Administration approved the first therapeutic cancer vaccine, called Sipuleucel-T, to treat advanced prostate cancer. Its target is an antigen called prostatic acid phosphatase. It’s present in normal prostate cells but is found in higher amounts in cancerous ones. Clinical trials showed patients vaccinated with Sipuleucel-T lived about four months longer, although their tumors remained the same size.

Other vaccines that have been approved against viruses like hepatitis B and human papillomavirus are also considered cancer vaccines because they prevent viral infections that could one day lead to liver, cervical, head, and neck cancers.

These preventative vaccines work by generating antibodies against the virus, and to the best of our knowledge, not a very effective T cell response, Klebanoff says. “That’s why they can’t be used as a therapy against cancer.”

What cancer vaccines are in the pipeline? Scientists are testing dozens of cancer vaccines, often in combination with other immunotherapies. They’re targeting various types of cancer, including skin, breast, bladder, prostate, and pancreatic cancer.

Last week, vaccine-maker Moderna announced that its candidate mRNA vaccine against stage three or four melanoma showed a 44 percent reduction in risks of skin cancer recurrence or death among patients who received both the vaccine and a Merck drug called Keytruda, which boosts the immune response against cancer cells, compared to those who took only Keytruda. Moderna’s personalized mRNA vaccine trains the immune system to produce T cells against 34 cancer-specific antigens. Although the results of this phase two clinical trial are yet to be peer-reviewed, the company, along with Merck, is planning a larger phase three trial in 2023 to test the vaccine’s safety and efficacy.

Cancer immunologist Olivera Finn at the University of Pittsburg is testing a preventative vaccine that can be given at a pre-cancer stage, when an individual develops benign growths called polyps—which are not dangerous but can turn malignant—inside their colon. The vaccine targets an abnormal form of a protein called MUC1 produced by some non-malignant colon polyp cells. It led to a 38 percent reduction in recurrence within three years of vaccinating nearly 50 individuals with advanced polyps. “If you don’t get a new polyp, you’re not going to be at an increased risk of a colon cancer,” Finn says.

An important next step for scientists is to figure out why some people respond better to the vaccines than others and how long they’ll be protected. Until then, the hope is to see more vaccine candidates progress to phase three randomized clinical trials where their safety and effectiveness will be evaluated in a large group of patients.

What challenges do scientists foresee? Despite the renewed excitement in developing and testing cancer vaccines, given technological advances, some scientists like Klebanoff remain skeptical. He wonders if the vaccines will ever be potent enough to cause tumor shrinkage in a way that is clinically meaningful and whether alternatives like engineering a patient’s T cells—so called CAR-T cell therapy—so they can better recognize tumor cells will be a more effective strategy. His research group uses the latter approach. But he remains eager to see what the data from ongoing vaccine clinical trials reveals.

Since therapeutic vaccines are often tested in patients with advanced cancer who have had their tumor surgically removed and have been through chemotherapy or radiation, their immune systems are really beat up, Schoenberger says. It’s likely that the vaccines may not perform as well at this late stage of disease. We’ll need to find the patients and the specific clinical settings in which cancer vaccines are most effective, he says.

Cancer vaccines are still in the early phases of testing and refining, Butterfield cautions. There’s a lot of work to be done, both on the preventative and therapeutic vaccines front.

Voting by head vs voting by order - to ensure liberty it is necessary for the representational bodies to be equally distributed so that the majority in the Third Estate receive equal vote without being overturned by the nobility.

could mean quite literally that the ride with no head lights a midnight could end in burning flames or a fun trip

This makes me wonder what we would have uncovered if people had the right to truly voice what they were feeling. We can interpret as best as we can but I would have loved to have known what was going on inside the everyday persons head.

I personally believe everyone has right to chase dreams and pursue success in their life. However, my opinion is built based on human equality. But unfortunately there is no such equality in the society, even if we are talking about relatively equality here. I strongly agree with the statement that the higher SES, the greater chance to pursue success.

This is an idea that I think is very important, and it is one that had come up often in my education courses throughout the past few years. It is true that the education system in the United States has some serious issues that need to be addressed, especially in regards to providing equal, and high quality education for all children. Obviously, students who's parents are wealthier are going to have an advantage over their peers who's parents do not have as many resources available to them.

Last semester I took an Urban US history course, and we discussed the impact that Ferdal Redlining has had on our communities, specifically on schools and public services. Decades ago, the government essentially put neighborhoods in different categories from A to D, and these categories were influenced by things like household income, overall safety, and race. Ethnically diverse areas were often classified as C or D which were viewed as undesirable areas to live, and federal funding was often allocated to A and B areas, promoting better public services in these areas. It is interesting to see how a policy from decades ago continues to impact our communities to this day.

I think this idea is very important, and it is something hat repeatedly comes up in all of my education courses. It is true that there are many issues within the education system in the United States, especially in regards to providing equal and high-quality education for all children. It seems unfair that certain students have a natural advantage over their peers simply because their parents can afford to send them to a better school or provide them with extra resources to succeed academically.

In analyzing the authors work, from the surface it did sound negative. As though he had set unrealistic expectations of what true beauty is, as he compares his mistress eyes to the sun, corals to her lips, the color of her breasts to that of snow, her cheeks to that of red and white roses, etc. However, from the analysis it could be interpreted as him loving his mistress in spite of her flaws; that beauty is not just about one's physical appearance.

frustrating that this is now seen as a kooky whiner’s view and not the view of the guy who got to see the experiment from the inside

I sometimes feel the need to find all of my mistakes in my first revision. Getting it into my head that I have unlimited revisions would be good.

To be honest, I feel like the idea in my head is that these processes occur almost linearly most of the time. It makes each step of the process a big mountain to overcome. What if it in fact wasn't that way?

HEAD is cutting somewhere along the branch. Topping or trimming is cutting all the way to the joint.

Definitely a ghost story. This seems to be the main conflict in the story. Which of the ghost entities will be the main antagonist? he German doctor, Hessian troop, headless horseman or another.

per my comment earlier, this is one of the reasons why I struggle with native creation story vs christian creation story. In my head I believe both are true but it doesnt make sense at the same time.

Dont plagiarize

Related Guardian Article https://www.theguardian.com/science/head-quarters/2017/jul/20/the-power-of-framing-its-not-what-you-say-its-how-you-say-it

Reviewer #1 (Public Review):

Han and Eckstein asked human participants to follow the gaze of a person and to judge the presence/absence of a target person in videos. The videos contained a gazer and an additional person as gaze goal in present conditions. In absent conditions, this person was digitally removed from the video. The results show that participants use peripheral information about the most likely gaze goal to predictively execute a saccade towards the gaze goal before the gazer's head is oriented towards the goal. At the same time, foveal information about the head velocity of the gazer is processed, leading to more reverse saccades to the gazer when the head velocity of the gazer is low and/or when the head accelerates before the first saccade to the goal. Further control experiments show that the reverse saccades are effective in reducing the error of the following saccade because additional foveal information of the gazer's head direction is sampled. Predictive saccades are also observed when participants are not instructed to follow the gaze.

Strengths:

The study uses very clever experimental manipulations and analysis methods to understand when and where information is sampled for saccade programming. This is especially challenging because natural videos are used to investigate gaze control in an ecologically highly relevant scenario. Compared to previous studies on the sampling of information, in which mostly artificial and static targets were used, this is a large conceptual and methodological step forward and advances the state-of-the-art. The complex stimulus material is analysed using advanced AI techniques and traditional human annotations. Overall, the study contains a complex and rich data set that is created and analysed with innovative methods and it will certainly stimulate further research.

Weaknesses:

While the study uses clever and sophisticated manipulations to dissect the influence of different types of information on eye movement control, these manipulations inevitably lead to a few limitations of ecological validity, which might contribute to the findings:

1. Role of expectations: It seems that whenever there was a second person present in the video, it was always the gaze goal. This might influence the gaze dynamics of participants because participants can anticipate that the gazer will look towards the second person. This expectation might allow participants to infer the gaze goal with peripheral vision and reduce the necessity to rely on foveal information about the head direction of the gazer. Some or all of the differences between the present/absent conditions might actually reflect the effect of this expectation.

2. Absent videos: Absent videos were created by digitally removing the target/distractor person from the video. This is definitely useful to maximize the visual similarity of absent and present videos, but it also might lead to absent videos that do not contain a meaningful gaze goal in the scene. This can be seen in Figure 1e, where the gazer seems to look towards something that is outside of the video frame. This absence of a potential gaze goal might delay saccades and render them more variable, especially in terms of amplitude.

Reviewer #2 (Public Review):

As described in the manuscript, gaze following is a dynamic process that should be investigated with similarly dynamic stimuli (wherever possible). In this case, the authors used videos, rich with visual information, that could be deemed an appropriate example of such stimuli. By constructing scenarios where actors gazed toward 1) a target person, 2) distractor or 3) nothing, the authors were able to easily study observers' eye movements. First, they were able to determine a baseline for how observers follow gaze in each of the three aforementioned conditions which is an important reference for future studies of this nature. Further, they suggest that eye movements are affected by how gaze following interacts with peripheral information (i.e., processing gaze-related information from the actor is combined with peripheral information about the presence/absence of a target person). Second, the authors also determined that eye movement behavior is affected by gaze information (i.e., changes in the gaze of the principal actor), in an anticipatory manner. This was verified using a DNN approach (using only the gazer's head direction) and then, confirmed through human observers' ratings. Lastly, the authors noted the presence of subsequent, reverse saccades (in the direction of the gazer and then, toward the target), which were shown to play a role in correcting an initial inference based on a slow head velocity of the gazer (confirmed with an SVM approach). While these are important first inquiries related to understanding eye movement behavior elicited in response to gaze following, a few items remain to be further elucidated, including what additional, peripheral information (besides target/distractor absence and presence) drives eye movements during gaze following. Overall, the dynamic videos used by the authors, in combination with their investigations, provide an important first step toward studying gaze following in more realistic conditions.

What if our current education system was inspired by The Open Conspiracy!?!?!? Great aspirations but failure in practice

almost implies she can create a whole new story within her head, mocking a stereotype that goes with woman changing the story in order to fit their desires

Pre-frontal cortex develops faster in female brains

The cumulative effect on this is that girls get a head start once the societal imposed impact of gender inequality is removed. Girls are rewarded for this higher level of control and boys are now at a disadvantage at the same grade levels.

Confused on the neighbors statement, and may not want to continue with an explanation?

Neighbor trying to convince the other

change his opinion on the fence

The speaker isn't convinced and may not believe that's true.

wants to convince him that the wall is not needed, pleading

the neighbor prefers being alone while the writer wants to build a connection

No animals around, they mightve ran away and speakers questioning

Losing something whether that be a person, item, a property, a person is not, not normal. It's expected, and sometimes the knowledge of knowing that your going to lose that something is already engraved into your head so then once it actually happens you won't be too destroyed.

Reviewer #1 (Public Review):

This study provides further detailed analysis of recently published Fly Atlas datasets supplemented with newly generated single cell RNA-seq data obtained from 6,000 testis cells. Using these data, the authors define 43 germline cell clusters and 22 somatic cell clusters. This work confirms and extends previous observations regarding changing gene expression programs through the course of germ cell and somatic cell differentiation.

This study makes several interesting observations that will be of interest to the field. For example, the authors find that spermatocytes exhibit sex chromosome specific changes in gene expression. In addition, comparisons between the single nucleus and single cell data reveal differences in active transcription versus global mRNA levels. For example, previous results showed that (1) several mRNAs remain high in spermatids long after they are actively transcribed in spermatocytes and (2) defined a set of post-meiotic transcripts. The analysis presented here shows that these patterns of mRNA expression are shared by hundreds of genes in the developing germline. Moreover, variable patterns between the sn- and sc-RNAseq datasets reveals considerable complexity in the post-transcriptional regulation of gene expression.

Overall, this paper represents a significant contribution to the field. These findings will be of broad interest to developmental biologists and will establish an important foundation for future studies. However, several points should be addressed.

In figure 1, I am struck by the widespread expression of vasa outside of the germ cell lineage. Do the authors have a technical or biological explanation for this observation? This point should be addressed in the paper with new experiments or further explanation in the text.

The proposed bifurcation of the cyst cells into head and tail populations is interesting and worth further exploration/validation. While the presented in situ hybridization for Nep4, geko, and shg hint at differences between these populations, double fluorescent in situs or the use of additional markers would help make this point clearer. Higher magnification images would also help in this regard.

Hmmm the MIT Media lab, a prestigous institution that mines for elietes. I wonder if it actually enables them or uses them as pieces in a game!?!?!?

The president is basically a mixture of a governor (in this case of New York) and a king (in this case of Britain)

Evaluation 1

Ratings and predictions

Ratings (1-100)

• Overall assessment: 40 CI: 20-60
  • Comment: See main review
• Advancing knowledge and practice: 30 CI: 20-60
  • Comment: The paper itself makes an important argument about resilient foods, but I don’t know if the additional element of AGI risk adds much to Denkenberger & Pearce (2016)
• Methods: Justification, reasonableness, validity, robustness: 50 CI: 40-60
  • Comment: Very major limitations around the survey method, and implementation of certain parts of the parameter sensitivity analysis. However many elements of a high standard
• Logic & communication: 60 CI: 40-75
  • Comment: Major limitations around the logic and communication of the theoretical model of cost-effectiveness used in the paper. Minor limitations of readability and reporting which could have been addressed before publication (such as reporting 95% CIs without medians, and not reporting overall cost and benefit estimates)
• Open, collaborative, replicable: 70 CI: 40-75
  • Comment: Provided models are shared with any reader who asks, I couldn’t ask for more here. Limitations of survey replicability (particularly E model) prevent perfect score
• Relevance to global priorities: 90 CI: 60-95
  • Comment: I’d be surprised if I ever read a paper with more relevance to global priorities, although as mentioned there are a few version of this argument circulating such as Denkenberger & Pearce (2016)

Journal predictions (1-5)

• What ‘quality journal’ do you expect this work will be published in? 2 CI: 1-2
• On a ‘scale of journals’, what tier journal should this be published in? 2 CI: 1-2

Written report

This is a very interesting paper on an important and neglected topic. I’d be surprised if I ever again read a paper with such potential importance to global priorities. The authors motivate the discussion well, and should be highly commended for their clear presentation of the structural features of their model, and the thoughtful nature in which uncertainty was addressed head-on in the paper.

Overall, I suspect the biggest contribution this paper will make is contextualising the existing work done by the authors on resilient food into the broader literature of long-termist interventions. This is a significant achievement, and the authors should feel justifiably proud of having accomplished it. However, the paper unfortunately has a number of structural and technical issues which should significantly reduce a reader’s confidence in the quantitative conclusions which aim to go beyond this contextualisation.

In general, there are three broad areas where I think there are material issues with the paper:

1. The theoretical motivation for their specific philosophy of cost-effectiveness, and specifically whether this philosophy is consistent throughout the essay
2. The appropriateness of the survey methods, in the sense of applying the results of a highly uncertain survey to an already uncertain model
3. Some specific concerns with parameterisation

None of these concerns touch upon what I see at the main point of the authors, which I take to be that ‘fragile’ food networks should be contextualised alongside other sources of existential risk. I think this point is solidly made, and important. However, they do suggest that significant additional work may be needed to properly prove the headline claim of the paper, which is that in addition to being a source of existential risk the cost-effectiveness of investing in resilient food is amongst the highest benefit-per-cost of any existential risk mitigation.

Structure of cost-effectiveness argument

One significant highlight of the paper is the great ambition it shows in resolving a largely intractable question. Unfortunately, I feel this ambition is also something of a weakness of the paper, since it ends up difficult to follow the logic of the argument throughout.

• Structurally, the most challenging element of this paper in terms of argumentative flow is the decision to make the comparator for cost-effectiveness analysis ‘AGI Catastrophe’ rather than ‘do nothing’. My understanding is that the authors make this decision to clearly highlight the importance of resilient food – noting that, “if resilient foods were more cost effective than AGI safety, they could be the highest priority [for the existential risk community]” (since the existential risk community currently spends a lot on AGI Risk mitigation). So roughly, they start with the assumption that AI Risk must be cost-effective, and argue that anything more cost-effective than this must therefore also be cost-effective. The logic is sound, but this decision causes a number of problems with interpretability, since it requires the authors to compare an already highly uncertain model of food resilience against a second highly uncertain model of AGI risk.
• The biggest issue with interpretability this causes is that I struggle to understand what features of the analysis are making resilient food appear cost-effective because of some feature of resilient food, and which are making resilient food appear cost-effective because of some feature of AI. The methods used by the authors mean that a mediocre case for resilient food could be made to look highly cost-effective with an exceptionally poor case for AI, since their central result is the multiplier of value on a marginally invested dollar for resilient food vs AI. This is important, because the authors’ argument is that resilient food should be funded because it is more effective than AI Risk management, but this is motivated by AI Risk proponents agreeing AI Risk is important – in scenarios where AI Risk is not worth investing in then this assumption is broken and cost effectiveness analysis against a ’do nothing’ alternative is required. For example, the authors do not investigate scenarios where the benefit of the intervention in the future is negative because “negative impacts would be possible for both resilient foods and AGI safety and there is no obvious reason why either would be more affected”. While this is potentially reasonable on a mathematical level, it does mean that it would be perfectly possible for resilient foods to be net harmful and the paper not correctly identify that funding them is a bad idea – simply because funding AI Risk reduction is an even worse idea, and this is the only given alternative. If the authors want to compare AGI risk mitigation and resilient foods against each other without a ‘do nothing’ common comparator (which I do not think is a good idea), they must at the very least do more to establish that the results of their AI Risk model map closely to the results which cause the AI Risk community to fund AI Risk mitigation so much. As this is not done in the paper, a major issue of interpretability is generated.
• A second issue this causes is that the authors must make an awkward ‘assumption of independence’ between nuclear risk, food security risk and AI risk. Although the authors identify this as a limitation of their modelling approach, the assumption does not need to be made if AI risk is not included as a comparator in the model. I don’t think this is a major limitation of the work, but an example of how the choice of comparator has an impact on structural features of the model beyond just the comparator.
• More generally, this causes the authors to have to write up their results in a non-natural fashion. As an example of the sort of issues this causes, conclusions are expressed in entirely non-natural units in places (“Ratio of resilient foods mean cost effectiveness to AGI safety mean cost effectiveness” given $100m spend), rather than units which would be more natural (“Cost-effectiveness of funding resilient food development”). I cannot find expressed anywhere in the paper a simple table with the average costs and benefits of the two interventions, although a reference is made to Denkenberger & Pearce (2016) where these values were presented for near-term investment in resilient food. This makes it extremely hard for a reader to draw sensible policy conclusions from the paper unless they are already an expert in AGI risk and so have an intuitive sense of what an intervention which is ‘3-6 times more cost-effective than AGI risk reduction’ looks like. The paper might be improved by the authors communicating summary statistics in a more straightforward fashion. For example, I have spent some time looking for the probability the model assigns to no nuclear war before the time horizon (and hence the probability that the money spent on resilient food is ‘wasted’ with respect to the 100% shortfall scenario) but can’t find this – that seems to be quite an important summary statistic but it has to be derived indirectly from the model.

Fundamentally, I don’t understand why both approaches were not compared to a common scenario of ‘do nothing’ (relative to what we are already doing). The authors’ decision to compare AGI Risk mitigation to resilient foods directly would only be appropriate if the authors expect that increasing funding for resilient food decreased funding for AI safety (that is to say, the authors are claiming that there is a fixed budget for AI-safety-and-food-resilience, and so funding for one must come at the expense of the other). This might be what the authors have in mind as a practical consequence of their argument, as there is an implication that funding for resilient foods might come from existing funding deployed to AGI Risk. But it is not logically necessary that this is the case, and so it creates great conceptual conclusion to include it in a cost-effectiveness framework that requires AI funding and resilient food funding to be strictly alternatives. To be clear, the ‘AI subunit’ is interesting and publishable in its own right, but in my opinion simply adds complexity and uncertainty to an already complex paper.

Continuing on from this point, I don’t understand the conceptual framework that has the authors consider the value of invested dollars in resilient food at the margin. The authors’ model of the value of an invested dollar is an assumption that it is distributed logarithmically. Since the entire premise of the paper hinges on the reasonability of this argument, it is very surprising there is no sensitivity analysis considering different distributions of the relationship between intervention funding and value. Nevertheless, I am also confused as to the model even on the terms the authors describe; the authors’ model appears to be that there is some sort of ‘invention’ step where the resilient food is created and discovered (this is mostly consistent with Denkenberger & Pearce (2016), and is the only interpretation consistent with the question asked in the survey). In which case, the marginal value of the first invested dollar is zero because the ’invention’ of the food is almost a discrete and binary step. The marginal value per dollar continues to be zero until the 86 millionth dollar, where the marginal value is the entire value of the resilient food in its entirety. There seems to be no reason to consider the marginal dollar value of investment when a structural assumption made by the authors is that there is a specific level of funding which entirely saturates the field, and this would make presenting results significantly more straightforward – it is highly nonstandard to use marginal dollars as the unit of cost in a cost-effectiveness analysis, and indeed is so nonstandard I’m not certain fundamental assumptions of cost-effectiveness analysis still hold. I can see why the authors have chosen to bite this bullet for AI risk given the existing literature on the cost of preventing AI Catastrophe, but there seems to be no reason for it when modelling resilient food and it departs sharply from the norm in cost-effectiveness analysis.

Finally, I don’t understand the structural assumptions motivating the cost-effectiveness of the 10% decline analysis. The authors claim that the mechanism by which resilient foods save lives in the 10% decline analysis is that “the prices [of non-resilient food] would go so high that those in poverty may not be able to afford food” with the implication that resilient foods would be affordable to those in poverty and hence prevent starvation. However, the economic logic of this statement is unclear. It necessitates that the production costs of resilient food is less than the production costs of substitute non-resilient food at the margin, which further implies that producers of resilient food can command supernormal profits during the crisis, which is to say the authors are arguing that resilient foods represent potentially billions of dollars of value to their inventor within the inventor’s lifetime. It is not clear to me why a market-based solution would not emerge for the ‘do nothing’ scenario, which would be a critical issue with the authors’ case since it would remove the assumption that ‘resilient food’ and ‘AGI risk’ are alternative uses of the same money in the 10% scenario, which is necessary for their analysis to function. The authors make the further assumption that preparation for the 100% decline scenario is highly correlated with preparation for the 10% decline scenario, which would mean that a market-based solution emerging prior to nuclear exchange would remove the assumption that ‘resilient food’ and ‘AGI risk’ are alternative uses of the same money in the 100% decline scenario. A supply and demand model might have been a more appropriate model for investigating this effect. Once again, I note that the supply and demand model alone would have been an interesting and publishable piece of work in its own right.

Overall, I think the paper would have benefitted from more attention being paid to the underlying theory of cost-effectiveness motivating the investigation. Decisions made in places seem to have multiplied uncertainty which could have been resolved with a more consistent approach to analysis. As I highlighted earlier, the issues only stem from the incredible ambition of the paper and the authors should be commended for managing to find a route to connect two separate microsimulations, an analysis of funding at the margin and a supply-and-demand model. Nevertheless, the combination of these three approaches weakens the ability to draw strong conclusions from each of these approaches individually.

Methods

With respect to methods, the authors use a Monte Carlo simulation with distributions drawn from a survey of field experts. The use of a Monte Carlo technique here is an appropriate choice given the significant level of uncertainty over parameters. The model appears appropriately described in the paper, and functions well (I have only checked the models in Guesstimate, as I could not make the secondary models in Analytica function). A particular highlight of the paper is the figures clearly laying out the logical interrelationship of elements of the model, which made it significantly easier to follow the flow of the argument. I note the authors use ‘probability more effective than’ as a key result, which I think is a natural unit when working in Guesstimate. This is entirely appropriate, but a known weakness of the approach is that it can bias in favour of poor interventions with high uncertainty. The authors could also have presented a SUCRA analysis which does not have this issue, but they may have considered and rejected this approach as unnecessary given the entirely one-sided nature of the results which a SUCRA would not have reversed.

The presentation of the sensitivity analysis as ‘number of parameters needed to flip’ is nonstandard, but a clever way to intuitively express the level of confidence the authors have in their conclusions. Although clever, I am uncertain if the approach is appropriately implemented; the authors limit themselves to the 95% CI for their definition of an ‘unfavourable’ parameter, and I think this approach hides massive structural uncertainty with the model. For example, in Table 5 the authors suggest their results would only change if the probability of nuclear war per year was 4.8x10^-5 (plus some other variables changing) rather than their estimated of 7x10^-3 (incidentally, I think the values for S model and E model are switched in Table 5 – the value for pr(nuclear war) in the table’s S model column corresponds to the probability given in the E model). But it is significantly overconfident to say that risk of nuclear war per year could not possibly be below 4.8x10^-5, so I think the authors overstate their certainty when they say “reverting [reversing?] the conclusion required simultaneously changing the 3-5 most important parameters to the pessimistic ends”; in fact it merely requires that the authors have not correctly identified the ‘pessimistic end’ of any one of the five parameters, which seems likely given the limitations in their data which I will discuss momentarily. I personally would have found one- and two-dimensional threshold analysis a more intuitive way to present the results, but I think the authors have a reasonable argument for their approach. As described earlier, I have some concerns that an appropriate amount of structural sensitivity analysis was undertaken, but the presentation of uncertainty analysis is appropriate in its own terms (if somewhat nonstandard).

Overall, I have no major concerns about the theory or application of the modelling approach. However, I have a number of concerns with the use of the survey instrument:

First, the authors could have done more to explain the level of uncertainty their survey instrument contains. They received eight responses, which is already a very low number of responses for a quantitative survey. In addition, two of the eight responses were from authors of the paper. The authors discuss ‘response bias’ and ‘demand characteristic bias’ which would not typically be applied to data generated by an approximately autoethnographic process – it is obvious that the authors of a survey instrument know what purpose the instrument is to be used for, and have incentives to make the survey generate novel and interesting findings. It might have been a good sensitivity analysis to exclude responses from the authors and other researchers associated with ALLFED since there is a clear conflict of interest that could bias results here.

Second, issues with survey data collection are compounded by the fact that some estimates which are given in the S Model are actually not elicited with the survey technique – they are instead cited to Denkenberger & Pearce (2016) and Denkenberger & Pearce (2017). This is described appropriately in the text, but not clearly marked in the summary Table 1 where I would expect to see it, and the limitation this presents is not described clearly. To be explicit, the limitation is that at least two key parameters in the model are based on a sample of the opinions of two of the eight survey respondents, rather than the full set of eight respondents. As an aside on presentation, the decision to present lower and upper credible intervals in Table 1 rather than median is non-standard for an economics paper, although perhaps this is a discipline-specific convention I am unaware of. Regardless, I’m not sure it is appropriate to present the lowest of eight survey responses as the ‘5th percentile’, as it is actually the 13th percentile and giving 95% confidence intervals implies a level of accuracy the survey instrument cannot reach. While I appreciate the 13th percentile of 8 responses will be the same as the 5th centile of 100 samples drawn from those responses, this is not going to be clear to a casual reader of the paper. ‘Median (range)’ might be a better presentation of the survey data in this table, with better clarity on where each estimate comes from. Alternatively, the authors could look at fitting a lognormal distribution to the survey results using e.g. method of moments, and then resample from the new distribution to create a genuine 95% CI. Regardless, given the low number of responses, it might have been appropriate simply to present all eight estimates for each relevant parameter in a table.

Third, the authors could have done more to make it clear that the ‘Expert Model’ was effectively just another survey with an n of 1. Professor Sandburg, who populated the Expert Model, is also an author on this paper and so it is unclear what if any validation of the Expert Model could reasonably have been undertaken – the E model is therefore likely to suffer from the same drawbacks as the S model. It is also unclear if Professor Sandburg knew the results of the S Model before parameterising his E Model – although this seems highly likely given that 25% of the survey’s respondents were Professor Sandburg’s co-authors. This could be a major source of bias, since presumably the authors would prefer the two models to agree and the expert parameterising the model is a co-author. I also think more work is needed to be done establishing the Expert’s credentials in the field of agricultural R&D (necessary for at least some of the parameter estimates); although I happily accept Professor Sandburg is a world expert on existential risk and a clear choice to act as the parameterising ‘expert’ for most parameters, I think there may have been alternative choices (such as agricultural economists) who may have been more obviously suited to giving some estimates. There is no methodological reason why one expert had to be selected to populate the whole table, and no defence given in the text for why one expert was selected - the paper is highly multidisciplinary and it would be surprising if any one individual had expert knowledge of every relevant element. Overall, this limitation makes me extremely hesitant to accept the authors’ argument that the fact that S model and E model are both robust means the conclusion is equally robust

Generally, I am sympathetic to the authors’ claim that there is unavoidable uncertainty in the investigation of the far future. However, the survey is a very major source of avoidable uncertainty, and it is not a reasonable decision of the authors to present the uncertainty due to their application of survey methods as the same kind of thing as uncertainty about the future potential of humanity. There are a number of steps the authors could have taken to improve the validity and reliability of their survey results, some of which would not even have required rerunning the survey (to be clear however, I think there is a good case for rerunning the survey to ensure a broader panel of responses). With the exception of the survey, however, methods were generally appropriate and valid.

Parameter estimates

Notwithstanding my concerns about the use of the survey instrument, I have some object level concerns with specific parameters described in the model.

• The discount rate for both costs and benefits appears to be zero, which is very nonstandard in economic evaluation. Although the authors make reference to “long termism, the view that the future should have a near zero discount rate”, the reference for this position leads to a claim that a zero rate of pure time preference is common, and a footnote observing that “the consensus against discounting future well-being is not universal”. To be clear, pure time preference is only one component of a well-constructed discount rate and therefore a discount rate should still be applied for costs, and probably for future benefits too. Even notwithstanding that I think this is an error of understanding, it is a limitation of the paper that discount rates were not explored, given they seem very likely to have a major impact on conclusions.
• A second concern I have relating to parameterisation is the conceptual model leading to the authors’ proposed costing for the intervention. The authors explain their conceptual model linking nuclear war risk to agricultural decline commendably clearly, and this expands on the already strong argument in Denkenberger & Pearce (2016). However, I am less clear on their conceptual model linking approximately $86m of research to the widescale post-nuclear deployment of resilient foods. The assumption seems to be (and I stress this is my assumption based on Denkenberger & Pearce (2016) – it would help if the authors could make it explicit) that $86m purchases the ‘invention’ of the resilient food, and once the food is ‘invented’ then it can be deployed when needed with only a little bit of ongoing training (covered by the $86m). This seems to me to be an optimistic assumption; there seems to be no cost associated with disseminating the knowledge, or any raw materials necessary to culture the resilient food. Moreover, the model seems to structurally assume that distribution chains survive the nuclear exchange with 100% certainty (or that the materials are disseminated to every household which would increase costs), and that an existing resilient food pipeline exists at the moment of nuclear exchange which can smoothly take over from the non-resilient food pipeline.

I have extremely serious reservations about these points. I think it is fair to say that an economics paper which projected benefits as far into the future as the authors do here without an exploration of discount rates would be automatically rejected by most editors, and it is not clear why the standard should be so different for existential risk analysis. A cost of $86m to mitigate approximately 40% of the impact of a full-scale nuclear war between the US and a peer country seems prima facie absurd, and the level of exploration of such an important parameter is simply not in line with best practice in a cost-effectiveness analysis (especially since this is the parameter on which we might expect the authors to be least expert). I wouldn’t want my reservations about these two points to detract from the very good and careful scholarship elsewhere in the paper, but neither do I want to give the impression that these are just minor technical details – these issues could potentially reverse the authors’ conclusions, and should have been substantially defended in the text.

Conclusions

Overall, this is a novel and insightful paper which is unfortunately burdened with some fairly serious conceptual issues. The authors should be commended for their clear-sighted contextualisation of resilient foods as an issue for discussion in existential risk, and for the scope of their ambition in modelling. Academia would be in a significantly better place if more authors tried to answer far-reaching questions with robust approaches, rather than making incremental contributions to unimportant topics.

Where the issues of the paper lie are structural weaknesses with the cost-effectiveness philosophy deployed, methodological weaknesses with the survey instrument and two potentially conclusion-reversing issues with parameterisation which should have been given substantially more discussion in the text. I am not convinced that the elements of the paper which are robust are sufficiently robust to overcome these weaknesses – my view is that it would be premature to reallocate funding from AI Risk reduction to resilient food on the basis of this paper alone. The most serious conceptual issue which I think needs to be resolved before this can happen is to demonstrate that ‘do nothing’ would be less cost-effective than investing $86m in resilient foods, given that the ‘do nothing’ approach would potentially include strong market dynamics leaning towards resilient foods. I agree with the authors that an agent-based model might be appropriate for this, although a conventional supply-and-demand model might be simpler.

I really hope the authors are interested in publishing follow-on work, looking at elements which I have highlighted in this review as being potentially misaligned to the paper that was actually published but which are nevertheless potentially important contributions to knowledge. In particular, the AI subunit is novel and important enough for its own publication.

Evaluator details

1. Name: Alex Bates
2. How long have you been in this field? In the field of cost-effectiveness analysis, 10 years. I wouldn’t consider myself to be in the field of x-risk
3. How many proposals and papers have you evaluated? I’ve lost count, but probably mid double figures - perhaps 50?

eLife assessment

This study demonstrates ultrafast real-time decoding of place fields in the hippocampus thanks to a head-mounted microscope for calcium imaging and to a novel data processing pipeline. This is a useful tool that aims at obtaining real-time capabilities that will enable closed-loop experiments that include decoding of a wide neuronal population, which could be applied in a variety of neuroscience fields. This will be of interest to anyone studying behaviors or functions that involve the hippocampus.

Author Response

Reviewer #1 (Public Review):

The authors sought to define the molecular mechanism of activation of the thrombopoietin receptor (TpoR), a very important cytokine receptor that regulates megakaryocyte differentiation and platelet production. They conducted a thorough series of experiments combining mutagenesis experiments with sophistical biological assays and that also includes solid-state NMR structural measurements. This work builds on a body of previous studies of TpoR from this group and from others. They focused both on (1) the role and impact of W515 located in the juxtamembrane cytosolic domain and (2) the impact of introducing either Asn at sites in the transmembrane domain to induce various dimerization modes, or insertion of pairs of Ala residues to induce helical rotation to the TM domain. There is a lot of nice data in this paper, which is fairly intricate - a tough read, but that's because it's a complicated system. The writing is excellent.

This paper presents a model for receptor activation in which the inactive receptor is the monomeric form of the receptor in which the juxtamembrane domain, including W515, maintains a helical structure. Activation of the receptor triggers dimerization of the transmembrane domain and loss of helicity of the juxtamembrane segment, which facilitates optimal interactions of the kinase domains with their JACK2 domain phosphorylation substrates.

There is a lot to like in this careful work and the resulting manuscript. There is one major shortcoming in this manuscript, which concerns W515. It is known that mutation of W515 to any of 17 of the canonical amino acids, including Phe, is sufficient to trigger homodimerization and receptor activation. The authors present some evidence that the phenomenon behind this is that mutation of W515 to almost any other residues disrupts the helical secondary structure of the critical juxtamembrane segment, which promotes dimerization and receptor activation. What I find puzzling is why a Trp at site 515 promotes helix formation, but nearly all other amino acids at this site disrupt helix formation. This strongly suggests the side chain of W515 must be interacting with another domain of the protein in the inactive state, in a manner that is responsible for how Trp stabilizes the juxtamembrane helix, which is a central feature that helps define that state. I think that for this paper, this dangling missing piece of their mechanistic model should be resolved.

We agree with the reviewers that the mechanism by which Trp515 stabilizes the TM helix is central to the mechanism of activation. More broadly, our studies over the past decade have sought to address the importance of the entire RWQFP insert in the TM domain. Our working model for this sequence has been that cation-π interactions are central to the role of the Trp and the accompanying amino acids.

Arginine and tryptophan both are over-represented at the cytoplasmic TM-JM boundaries of membrane proteins. Arginine is positively charged and part of the “positive-inside” rule for membrane protein insertion. Arginine and lysine define the cytoplasmic ends of TM helices and prefer to be accessible to the water-exposed membrane surface. In contrast, tryptophan residues prefer hydrophobic head-group or membrane interior locations. A revealing aspect of the RWQFP motif is that the arginine and tryptophan are located at the membrane to cytosolic border. As a result, in order to accommodate arginine in a more water-inaccessible membrane environment, it interacts with the surface of the tryptophan indole ring. Partitioning of the RWQF sequence in a more water-inaccessible environment also drives the formation of helical secondary structure as an unpaired backbone C=O...NH in a hydrophobic environment is estimated to cost 3-6 kcal/mol of energy.

We have taken two approaches in respond to this essential criticism of the reviewers: one structural and one computational. Additional NMR data (structural approach) has been included in the supporting information (see response to point 2 below). Computational approaches provide a second way to address whether a cation– interaction between Trp515 and the positively charged Arg514 is responsible for stabilizing the C-terminal TM helix. We have included a new supporting figure using Alpha-Fold 2.0 that probes the structural changes upon mutation of Trp515. In the wild-type receptor, Arg514 is predicted to form a cation– interaction with Trp515. In the W515K mutant, the helical secondary structure in the RKQFP sequence is disrupted and Arg514 forms a new cation– interaction with Trp529. Similar changes occur in other Trp515 mutants (e.g. W515A) highlighting the ability of Alpha-Fold to predict such interactions and the consequences of mutation. Overall, 15 out of 19 W515X mutants are predicted to be unfolded. Experimentally, 17 out of 19 mutations lead to activation. Importantly, W515C and W515P are the only two amino acid substitutions that do not cause constitutive activity experimentally (Defour, Chachoua, Pecquet, & Constantinescu, 2016). Computationally, these two sites do not predict helix unraveling. In short, the overall the predictions of Alpha-Fold agree with the unique nature of tryptophan at position 515.

In addition, we have expanded the arguments supporting the potential role of cation–π interactions by adding a new section entitled “Unfolding of the RWQF -helical motif is a common mechanism of receptor activation”.

These modifications are now in the revised manuscript starting with line 213:

Our working model for the mechanism of activation in the wild-type or mutant receptors is that the RWQF motif is stabilized in the inactive state as an -helix as a result of a cation- interaction between R514 and W515. This interaction allows the RWQF sequence to partition into the more hydrophobic head-group region of the bilayer. Both Arg and Trp are over-represented at the cytoplasmic ends of TM helices (von Heijne, 1992), but whereas Arg prefers a water-accessible environment, Trp prefers to be buried in a more hydrophobic environment (Yau, Wimley, Gawrisch, & White, 1998). Since Arg and Trp are located at the border between membrane and cytosolic domains and Arg precedes Trp in the sequence, partitioning into the membrane head-group region results in a favorable interaction of the positive charge associated with the guanidinium group of the R514 side chain with the partial negative charge associated with the aromatic surface of the W515 side chain. Partitioning of the RWQF sequence into the more water-inaccessible environment drives the formation of helical secondary structure as an unpaired backbone C=O...NH in a hydrophobic environment is estimated to cost 6 kcal/mol of energy (Engelman, Steitz, & Goldman, 1986). In this model, activation of the receptor results in or is caused by disruption of the R514-W515 cation-π interaction. In the W515 mutants, R514 is no longer stabilized in a membrane environment and the helix containing the RWQFP sequence unravels to allow the positively charged side chain to reach outside of the membrane. In the case of the Asn mutants and in the wild-type receptor with bound Tpo, dimerization of hTpoR (or rotation of the TM helices in mTpoR dimer), places W515 in the center of the helix-helix interface. The data suggest that a steric clash of the W515 side chains results in unraveling of the cytoplasmic end of the TM helix.<br /> Computational and additional NMR data are provided in the supplementary figures to support the model of helix unraveling suggested by the solid-state NMR studies. Computationally, we used AlphaFold 2.0 (Jumper et al., 2021) calculations of hTpoR TM-JM peptides to predict the influence of all possible mutations at position 515 on the TM-JM helix structure. Remarkably, -helix unraveling was predicted for 15 out of 20 possible amino acids at 515 (supplement 2 to Figure 3). Importantly, two of the mutations that are not predicted to cause helix unraveling are W515C and W515P. Experimentally, these two amino acid substitutions are the only ones that do not induce constitutive activity among all possible amin oacid substitutions at W515 (Defour et al., 2016). Introducing a Trp at the preceding position 514 instead of R/K in W515K/R mutants reverses helix unfolding in AlphaFold simulations (supplement 3 to Figure 3). This result agrees with our previous data that the WRQFP mutant is inactive and is essentially monomeric (J. P. Defour et al., 2013). Structurally, we have undertaken solution-NMR studies of the wild-type hTpoR TM-JM peptide and its W515K mutant. Relaxation measurements of the backbone 15N resonances show that W515K mutation leads to association of the TM helices, and that it induces upfield chemical shift changes in the RWQF sequence consistent with helix unraveling (supplement 1 to Figure 3).

Reviewer #2 (Public Review):

The thrombopoietin receptor (TpoR) regulates stem cell proliferation, platelet production, and megakaryocyte differentiation. Past cell biology and biophysical studies have established that ligand-induced dimerization constitutes the mechanism of activation of TpoR. Specifically, ligands bind to the extracellular domain of TpoR and generate an allosteric response that is transmitted to the transmembrane domain, activating downstream signaling. However, up to now the molecular details of how the allosteric signals are transmitted to the intramembrane domains have been elusive. In this manuscript, Constantinescu and co-workers combined NMR, in vitro, and in vivo assays to investigate the activation and oncogenicity of TpoR. The authors concluded that the unwinding of the juxtamembrane domain is the main structural event that determines TpoR activation and regulates oncogenicity. The solid-state NMR studies were carried out in lipid membranes with polypeptides spanning the juxtamembrane and transmembrane residues. The authors show a series of spectra of 13CO resonances that encompass the juxtamembrane domain that is diagnostic of a structural transition from a helical conformation to a partially disordered state. The unwinding of the helical juxtamembrane domain was confirmed by site-specific mutations in this region. The chemical shift changes clearly indicate the transition from order to disorder (and vice versa) for selected sites. These conclusions are compounded by INEPT-type experiments that detect the most dynamic region of polypeptides. To rationalize the molecular mechanism for activation, the authors also used Ala-Ala insertions at strategic positions along the transmembrane domain. These experiments showed that the specific orientation of the transmembrane residues is central for TpoR activation, and a slight rotation of the helix is critical for activation of the receptor. Transcriptional activity assays confirm the importance of the proper orientation of the transmembrane domain for receptor activation.

Overall, I believe the data are solid, and both biophysical and cell biology studies support the conclusions of the authors. These new findings represent a significant advancement in understanding cytokine receptor activation.

We thank the reviewer for these comments.

Reviewer #3 (Public Review):

The authors sought to propose a mechanism by which cancer-causing mutations in the thrombopoietin receptor (TpoR) activate the receptor. To do so, they used a systematic approach of introducing non-native and naturally occurring mutations into the receptor and use a combination of in-vivo and cell-based assays and solid-state NMR spectroscopy. They propose that the proximity of the asparagine mutations to the cytosolic boundary influences the secondary structure of the receptor and suggests that this structural change induces receptor activation.

The strengths of this work are the importance of the system being studied and tackling a problem that is not yet fully resolved. The authors acquired a large and convincing set of biological data, including in vivo experiments that support the gain-of-function/activating role of the mutations studied. The solid-state NMR data are of high quality as well. In particular, the INEPT data in figure 6a display very clear differences within one region of the wild-type compared to the mutants.

One significant weakness is the validity of the conclusions given the limited atomistic measurements presented. Namely, the authors make rather specific conclusions about protein folding based on a single set of 13C alanine carbonyl chemical shifts in the wild-type and mutant TM peptides. Essentially, the authors observe chemical shift perturbations at this carbonyl carbon when mutations are introduced into a protein and use this information to make conclusions about secondary structure. I am not convinced that the authors have presented sufficient evidence to justify the conclusion that the helix unwinds and that this is responsible for the mechanism of activation. While the other cell-based experiments in mutations are interesting, deciphering such a specific folding mechanism with limited atomistic data is not justified.

We added both computational data and solution NMR to support our conclusion.

Author Response

Reviewer #1 (Public Review):

Voltage-clamp fluorometry combines electrophysiology, reporting on channel opening, with a fluorescence signal reporting on local conformational changes. Classically, fluorescence changes are reported by an organic fluoropohore tethered to the receptor thanks to the cysteine chemistry. However, this classical approach does not allow fluorescent labeling of solvent-inaccessible regions or cytoplasmic regions. Incorporation of the fluorescent unnatural amino acid ANAP directly in the sequence of the protein allows counteracting these limitations. However, expression of ANAP-containing receptors is usually weak, leading to very small ANAP-related fluorescence changes (ΔFs).

In this paper, the authors developed an improved method for expression of full-length, ANAP-mutated proteins in Xenopus oocytes. In particular, they managed to increase the ratio of full-length over truncated proteins for C-terminal ANAP incorporation sites. Since C-terminally truncated P2X receptors are usually functional, it is important to maximize the full-length over truncated protein ratio to have a good correspondence between the observed current and fluorescence. Using their improved strategy, they screened for ANAP incorporation sites and ATP-mediated ANAP ΔFs along the whole structure of the P2X7 receptor: extracellular ligand binding domain (head domain), M2 transmembrane segment (gate), as well as a large extracellular domain specific for the P2X7 subtype, the "ballast" domain. The functional role of this domain and its motions following ATP application are indeed unknown. Monitoring ANAP fluorescence changes in this region following ATP binding provides a unique way to study those questions. By analyzing ATP-induced ΔFs from different parts of the receptors, the authors conclude that the ATP-binding domain mainly follows gating, while intracellular "ballast" motions are largely decoupled from ATP-binding

Strengths of the paper:

This paper provides an improved method for efficient unnatural amino acid incorporation in Xenopus oocytes. Thanks to this technique, they managed to enhance membrane expression of ANAP-mutated P2X7 receptors and observed strong fluorescent changes upon ATP application. The paper furthermore describes an impressive screen of ANAP-incorporation sites along the whole protein sequence, which allows them to monitor conformational changes of solvent-inaccessible regions (transmembrane domains) and cytoplasmic regions that were not accessible to cysteine-reactive fluorophores. This screen was performed in a very thorough manner, each ANAP mutant being characterized biochemically for membrane expression, as well as in term of fluorescence changes. The limitations of the approach -small ΔF upon ATP application on wt receptors, problem of baseline fluorescence variations in presence of calcium- are well explained. Overall, this study should thus not only serve as a guide to anyone willing to perform VCF on P2X7 receptors but it should be useful to the whole community of researchers using unnatural amino acids. Thanks to orthogonal labeling with TMRM and ANAP, the authors managed to simultaneously monitor the motions of the extracellular and intracellular domains of P2X7. Finally, they propose methods to simultaneously monitor intracellular domain motion and downstream signaling.

Weaknesses:

Although the fluorescence screen is impressive and well conducted, the biological conclusions remain superficial at this stage. The paper furthermore lacks quantitative analysis. Finally, the title only reflects a minor part of the paper and is therefore not representative of the paper content.

Quantitative analyses (DRCs and current rise times) were now added for the key mutations. In addition, we performed a variety of experiments to address the challenging question of mechanistic insight (mutants that track facilitation) and effects of intracellular factors (mutation of calmodulin binding site, FRET experiments with calmodulin). These data confirmed that deletion of a cysteine-rich intracellular region eliminates current facilitation (Roger et al., 2010) and that some of our mutants indeed track facilitation. However, mutation of the CaM binding site and FRET experiments did not support an effect of calmodulin or were inconclusive. As pointed out above, we think that VCF has limited capacity to identify novel biologically relevant consequences of receptor activation but is more suited to determine the sites and dynamics of already defined interactions.

The title was changed to: "Improved ANAP incorporation and VCF analysis reveals details of P2X7 current facilitation and a limited conformational interplay between ATP binding and the intracellular ballast domain"

Reviewer #2 (Public Review):

The authors aimed to elucidate the structural rearrangements and activation mechanisms of P2X7 upon ATP application by voltage clamp fluorometry (VCF) using fluorescent unnatural amino acid (fUAA) and other fluorophores. They improved the fUAA methodology and detected ATP binding evoked changes in the ATP binding region and other regions. They also observed facilitation of fluorescence (F) changes by repeated application of ATP associated with gating. The F change in the cytoplasmic ballast region was minor, and with their experimental data, they discussed this region is involved in activation by other cytoplasmic factors, such as Ca2+.

The strengths of the study are as follows.

(1) fUAA methodology was improved to enable experiments by one time injection to oocytes (Figs. 1 and Suppl).

(2) They performed intensive mutagenesis study of as many as 61 mutants (Figs. 3, 4, 5).

(3) A careful evaluation of the successful Anap incorporation and formation of full length proteins was performed by western blot analysis (Fig. 2).

(4) By three wave lengths F recording, they obtained better information, i.e. they classified the interpretation of F changes to, quenching, dequenching, increase in polarity and decrease in polarity (Fig. 3E).

(5) They detected F changes upon ATP application in various regions of P2X7, but not many in the ballast region, showing that the ballast region is not well involved in the ATP evoked gating.

(6) They analyzed the kinetics of F and current and their changes upon repeated ATP application to approach the known facilitation mechanisms. The data are very interesting. They concluded that it is intrinsic to the P2X7 molecule and that it is associated not with the ATP binding but with the gating process (Figs. 3F, 4D, 6A).

(7) They performed interesting analysis to clarify the mechanisms of activation by cytoplasmic factors, especially Ca2+ entered via P2X7 (Fig. 6).

The weaknesses of the study are as follows.

(1) As both structures of P2X in the open and closed states are already solved, and the ATP binding evoked structural rearrangements from the ATP binding site to the gate are already known in detail. The structural rearrangements detected in the extracellular region (Fig. 3) and TM region (Fig. 4) upon ATP application are just as expected. The impact and scientific merits of this part are rather limited.

We generally agree that the cryo-EM structures clarified basic principles of receptor function. However, considering the specific features of the P2X7 receptor and its likely regulation/modulation by membrane components and environment and the fact that the actual states of the receptor structures (e.g. facilitated or not?) is not known, we think that VCF analysis of its dynamics in a more native cellular environment is still required to confirm the predicted motions and also has the potential to identify details of "P2X7 fine tuning".

(2) The facilitation mechanism is of high interest. The authors showed it is intrinsic to P2X2 and associated with the gating rather than ATP binding. However, this reviewer cannot have better understanding about the actual mechanism. (a) What is the mechanistic trigger of facilitation? Possibilities are discussed, but it appears there is no clear answer with experimental evidences yet. (b) How is the memory of the 1st ATP application stored in the molecule, i.e. how does the P2X7 structure just before the 1st application differ from that just before the 2nd application of ATP?

These are indeed fundamental questions but based on the available information we do not see a rational approach to address this issue any further. Additional extensive "screening" for ideal fluorophore positions would probably be required and is beyond our possibilities in the present study.

(3) The structural rearrangement of the CaM-M13 region (Fig. 6B, C) attached at the C-terminus by Ca2+ influx through P2X7 upon ATP application is natural due course and not very surprising. Also, it is not accepted as an evidence proving that Ca2+ is the mediator of facilitation.

We apologize, this is a misunderstanding. We only provided protocols for parallel recordings of ANAP with other fluorophores for further analysis of downstream signaling pathways but we did not show or propose any functional consequences of the Ca2+ influx (see also point 7 above).

(4) As to the ballast region, data showed its limited involvement in the ATP-induced structural rearrangements. The function of the ballast region is not clear yet. A possible involvement in GDP binding and/ or metabolism is discussed, but there is no clear experimental evidence.

We are aware of these limitations. In the absence of a clear fluorescence change around the GTP/GDP-binding site or information about its role, it is difficult to investigate its molecular function by VCF. The fact, that (un-)binding of the guanosine nucleotide does not seem to be related to channel opening (McCarthy et al., 2019) further limits our options to study its function and currently it is not even known whether GDP/GTP has just a structural role. However, we identified A564* as a potential reporter for yet undefined processes that might affect GTP/GDP binding and/or metabolism.

Reviewer #3 (Public Review):

This research contributes to optimizing the amber stop-codon suppression protocol for voltage-clamp fluorometry (VCF) experiments using Xenopus oocyte heterologous expression system. By in vitro RNA synthesizing the tRNA and tRNA synthetases, combined with the dominant-negative release factor initially developed by Jason Chin's lab, L-Anap can be site-specifically labeled to proteins by a single microinjection of a mixture of molecular components into the cytoplasm of oocytes. Although it avoids nuclear microinjection to oocytes, it adds more RNA synthesis steps. This strategy of using eRF dominant negative variant (eRF1-E55D), was previously applied to the Anap incorporation system using mammalian cell lines and model proteins (Gordon et al, eLife, 2018). In this previous 2018 paper, with eRF1-E55D, the percentage of full-length protein expression increased substantially. Using oocytes in this paper, this percentage apparently did not increase significantly as shown in Fig. 1D, different from the previous paper. Nevertheless, the overall expression level increased successfully by this method, which could facilitate macroscopic fluorescence measurements, especially considering that L-Anap is relatively dim as a fluorophore.

Anap fluorescence change was measured mostly using its environmental sensitivity, which has limited information in interpreting structural changes. The structural mechanisms proposed could be potentially strengthened and the conclusions could be further validated by combining FRET or other distance ruler experiments with the VCF method. The engineered CaM-M13 FRET experiments mostly report the calcium entry, not measuring the rearrangements of P2X7 directly.

We tried FRET analyses with ANAP-labeled P2X7 and mNeonGreen-labeled CaM but unfortunately, results were inconclusive.

In addition, results of ATP dose-response relationship for channel activation correlated with ATP dose-dependent Anap fluorescence change, especially for sites showing a large percentage of ATP-induced change in fluorescence, would provide more insights regarding the allosteric mechanism of the channel.

We agree, but unfortunately, bleaching of ANAP and the variation of background fluorescence in individual oocytes prevented such analyses .

Author Response

Reviewer #1 (Public Review):

Liau and colleagues have previously reported an approach that uses PAM-saturating CRISPR screens to identify mechanisms of resistance to active site enzyme inhibitors, allosteric inhibitors, and molecular glue degraders. Here, Ngan et al report a PAM-saturating CRISPR screen for resistance to the hypomethylating agent, decitabine, and focus on putatively allosteric regulatory sites. Integrating multiple computational approaches, they validate known - and discover new - mechanisms that increase DNMT1 activity. The work described is of the typical high quality expected from this outstanding group of scientists, but I find several claims to be slightly overreaching.

Major points:

The paper is presented as a new method - activity-based CRISPR scanning - to identify allosteric regulatory sites using DNMT1 as a proof-of-concept. Methodologically, the key differentiating feature from past work is that the inhibitor being used is an activity-based substrate analog inhibitor that forms a covalent adduct with the enzyme. I find the argument that this represents a new method for identifying allosteric sites to be relatively unconvincing and I would have preferred more follow-up of the compelling screening hits instead. The basic biology of DNMT1 and the translational relevance of decitabine resistance are undoubtedly of interest to researchers in diverse fields. In contrast, I am unconvinced that there is any qualitative or quantitative difference in the insights that can be derived from "activity-based CRISPR scanning" (using an activity-based inhibitor) compared to their standard "CRISPR suppressor scanning" (not using an activity-based inhibitor). Key to their argument, which is expanded upon at length in the manuscript, is that decitabine - being an activity-based inhibitor that only differs from the substrate by 2 atoms - will enrich for mutations in allosteric sites versus orthosteric sites because it will be more difficult to find mutations that selectively impact analog binding than it is for other active-site inhibitors. However, other work from this group clearly shows that non-activity-based allosteric and orthosteric inhibitors can just as easily identify resistance mutations in allosteric sites distal from the active site of an enzyme (https://www.biorxiv.org/content/10.1101/2022.04.04.486977v1). If the authors had compared their decitabine screen to a reversible DNMT1 inhibitor, such as GSK3685032, and found that decitabine was uniquely able to identify resistance mutations in allosteric sites, then I would be convinced. But with the data currently available, I see no reason to conclude that "activity-based CRISPR scanning" biases for different functional outcomes compared to the "CRISPR suppressor scanning" approach.

We appreciate the reviewer’s comments and thank them for their constructive feedback. We agree with the reviewer that our claims regarding the utility of activity-based CRISPR scanning would be more strongly supported with a head-to-head comparison against a non-covalent, reversible inhibitor. To address this point, we conducted a CRISPR scanning experiment on DNMT1 and UHRF1 using GSK3484862 (GSKi), which is shown in Fig. 1e–h. We observed that the top enriched sgRNA under GSKi treatment targets H1507, which directly interacts with the drug and contributes to compound binding. (Fig. 1e,h, Supplementary Fig. 1e). Our results are consistent with previous structural and biochemical studies of these inhibitors (reported in Pappalardi, M.B. et al., Nat. Cancer 2021), in which they demonstrate that the H1507Y mutation reduces GSK3685032 (a derivative of GSK3484862) inhibition of DNMT1 by >350-fold compared to wild-type DNMT1. By contrast, the top enriched sgRNA under decitabine (DAC) treatment targets D702 in the autoinhibitory linker region (Fig. 1c). Furthermore, comparison of sgRNA resistance scores across DAC and GSKi treatment conditions reveals highly distinct sgRNA enrichment profiles (Fig. 1g). Taken together, our data suggest that these two mechanistic classes of inhibitors may exert differential selective pressures that lead to unique enrichment profiles.

While we consider these data to strengthen our claim that activity-based CRISPR scanning can preferentially enrich for mutations in allosteric sites versus orthosteric sites, we also recognize that allosteric site mutations can be identified without the use of activity-based inhibitors, as the reviewer points out. To address this point, we have modified the text to suggest that the use of activity-based inhibitors may exert a greater bias for the enrichment of allosteric site mutations but clarifying that the enrichment of such mutations are not exclusive to the use of activity-based inhibitors.

How can LOF mutations from cluster 2 be leading to drug resistance? It is speculated in the paper that a change in gene dosage decreases the DNA crosslinks that cause toxicity. However, the immediate question then would be why do the resistance mutations cluster around the catalytic site? If it's just gene dosage from LOF editing outcomes, would you not expect the effect to occur more or less equally across the entire CDS?

This is an excellent point. As outlined previously above, we recognize that our gene dosage hypothesis regarding the mechanism of cluster 2 sgRNAs may lack sufficient explanation to convey our reasoning clearly, and we have added more text and data to clarify and support our claim.

Mutations that are highly likely to lead to a nonfunctional protein product (i.e., frameshift, nonsense, splice site disrupting) are annotated as “loss-of-function” (LOF) in the text, with all other protein coding mutations designated as “in-frame.” The key insight underlying our gene dosage hypothesis is that sgRNAs targeting essential protein regions and functional domains generate greater proportions of null (i.e., knockout) mutations and undergo stronger negative selection compared to sgRNAs targeting non-essential protein regions (see Shi, J. et al., Nat. Biotechnol. 2015). This is because in-frame coding mutations in protein regions that are functionally important (e.g., DNMT1 catalytic domain) are more likely to disrupt protein function than those in non-essential protein regions. As a result, sgRNAs targeting functional protein regions are more likely to generate in-frame mutations resulting in a null allele and are thus “effectively LOF.” Importantly, the observation that sgRNAs targeting specific protein regions are more likely to lead to null mutations also implies that 1. not all CDS-targeting sgRNAs are equivalent at inducing LOF effects and 2. sgRNAs that are more effective at generating null mutations may exhibit preferential clustering within functionally important protein regions.

In this context, we reasoned that cluster 2 sgRNAs, which target the essential catalytic domain, may be more effective at reducing DNMT1 gene dosage than other DNMT1-targeting sgRNAs because in-frame mutations generated by these sgRNAs are more likely to lead to nonfunctional DNMT1 protein. That is, cluster 2 sgRNAs may generate greater proportions of “effectively LOF” in-frame mutations that disrupt DNMT1’s essential function. Consequently, we posited that the observed clustering of these sgRNAs in the catalytic domain is likely a reflection of its functional importance. To test this idea, we transduced WT K562 cells with 6 individual sgRNAs targeting the N-terminus, RFTS domain, and catalytic domain of DNMT1, and performed genotyping on the cellular pools over 28 days (Fig. 4f). We observed that sgRNAs targeting outside of the catalytic domain exhibited increasing frequencies of in-frame mutations over time, consistent with the idea that these sgRNAs generate functional in-frame mutations that are not under strong negative selection. By contrast, catalytic-targeting sgRNAs exhibited significant depletion of inframe mutations over time, supporting the notion that in-frame mutations in essential regions are functional knockouts and thus negatively selected under normal growth conditions. Consequently, the ability of catalytic-targeting sgRNAs to generate greater proportions of null mutations would therefore make them more effective at conferring resistance through gene dosage reduction than other DNMT1-targeting sgRNAs.

Our hypothesis implies that a large proportion of in-frame mutations generated by cluster 2 sgRNAs are functionally equivalent to LOF mutations (i.e., frameshift, nonsense, splice site disruption), and therefore neither in-frame or LOF mutations should be preferentially selected for under DAC treatment, in contrast to the positive selection of gain-of-function (GOF) in-frame mutations in cluster 1 sgRNAs. Consistent with this idea, our data indicate that the relative proportions of in-frame and LOF mutations in cluster 2 sgRNAs remain comparable across vehicle and DAC treatments (Fig. 4b). Furthermore, since the selective pressure on in-frame and LOF mutations should be similar if they are functionally equivalent, the relative proportions of in-frame versus LOF mutations in cluster 2 sgRNAs should be primarily dictated by their frequencies as editing outcomes. Consistent with this idea, the observed proportions of in-frame versus LOF mutations in cluster 2 sgRNAs under DAC treatment do not deviate significantly from their expected proportions as predicted by inDelphi (Supplementary Fig. 4c). Conversely, cluster 1 sgRNAs exhibit greater ratios of in-frame versus LOF mutations under DAC treatment than their predicted ratios from inDelphi (Supplementary Fig. 4c,d). Altogether, these data are consistent with the notion that cluster 2 sgRNAs may operate through a gene dosage reduction effect.

In general, I found the screens, and integrative analyses, highly compelling. But the follow-up was rather narrow. For example, how much do these mutations shift the IC50 curves for DAC?

To address this point, we derived two clonal cell lines from the screen harboring endogenous DNMT1 mutations in either the autoinhibitory linker or the RFTS domain (Supplementary Fig. 3g). We treated these cell lines, in addition to WT K562 cells, with varying concentrations of DAC and observed a partial growth rescue in the mutant cell lines relative to WT K562 cells (Fig. 3i). We also show that these mutant cell lines exhibit DAC-mediated degradation of DNMT1, consistent with our fluorescent reporter results (Supplementary Fig. 3h). To further validate whether these endogenous DNMT1 mutations confer partial resistance to DAC, we transduced WT K562 cells with vectors encoding an shRNA targeting the 3' UTR of the endogenous DNMT1 transcript and a DNMT1 overexpression vector encoding WT and mutant DNMT1 constructs (Supplementary Fig. 3i). Upon treating these knockdown and overexpression cells with varying concentrations of DAC, we again observed a partial growth rescue in the presence of mutant versus WT DNMT1 (Fig. 3j).

What kinetic parameters have changed to increase catalytic activity?

We performed enzyme activity assays at various temperatures with recombinant DNMT1 protein for WT and mutant DNMT1 constructs, observing that mutant DNMT1 constructs exhibit varying degrees of overactivity relative to WT DNMT1 at different temperatures (Fig. 3h, Supplementary Fig. 4f). Whereas the autoinhibitory linker mutations display consistently higher levels of activity relative to WT DNMT1 at all temperatures tested, we observed that RFTS and CXXC mutants exhibited decreasing levels of overactivity with increasing temperature (Fig. 3h). Previous studies (see Berkyurek, A.C. et al., J. Biol. Chem. 2014) have observed similar behavior with RFTS mutations, suggesting that these mutations may disrupt critical hydrogen bonds at the autoinhibitory interface that reduce the activation energy required to release DNMT1 from an autoinhibited to active conformation. Our RFTS and CXXC mutations exhibit behavior that are consistent with this hypothesis, which may explain the decreasing levels of overactivity with increasing temperature.

Do the mutants with increased catalytic activity alter the abundance of methylated DNA (naively or in response to the drug)? It is speculated that several UHRF1 sgRNAs disrupt PPIs and not DNA binding, but this is never tested.

While we derived clonal cell lines containing DNMT1 mutations, as noted above, it proved too difficult to compare these drug-resistant cells to naïve cells because they were cultured in the presence of DAC for 2 months, leading to large changes in DNA methylation that may confound any conclusions about the effects of the mutations alone. Additionally, the reviewer also brings up valid limitations regarding our studies on UHRF1, which also proved very difficult to biochemically purify and beyond our expertise. After some initial studies, we chose not to pursue these additional experiments further but instead prioritized the GSKi CRISPR-suppressor scan and cluster 2 studies, as suggested by the reviewers. We acknowledge these limitations in the text.

Reviewer #2 (Public Review):

In this manuscript, Ngan and coworkers described a CRISPER-based screening approach to identify potential variants of DNMT1 and UHRF1 that can suppress the anti-proliferation role of decitabine. In theory, such an effect can be achieved by at least two types of gain-of-activity DNMT1/UHRF1 mutants by directly boosting the enzymatic activity or by indirectly abolishing the intrinsic inhibitory activity of the DNMT1-UHRF1 axis. Through systematically targeting the DNMT1-UHRF1 reading frames with a rationally designed sgRNA library, the authors identified and characterized a few potential hotspots within multiple autoinhibitory motifs. While the approach has its merits in regard to the unbiased screening of the target proteins in living cells, there are the following serious concerns in terms of how the data were interpreted and the limitation of the approach itself as detailed below.

(1) Although the authors identified multiple hotspots in the DNMT1-UHRF1 complex with their alterations associated with the resistance to decitabine, it is risky to argue these mutations increase DNMT1 activity simply because they are clustered within known auto-inhibitory regions. There are many alternative explanations for this observation. For instance, some mutants may allosterically alter how DNMT1 recognizes decitabine-containing vs native GpC motifs; others may recruit other proteins as modulators. The key gap here is to associate the decitabine-resistance phenotype to the loss of auto-inhibitory functions because multiple hotspots were in the auto-inhibitory regions.

In our original manuscript, we supported our claim that gain-of-function DNMT1 mutations enhance DNMT1 activity with experimental data using purified DNMT1 protein constructs in enzyme activity assays (Fig. 3g, Fig. 4g), so our conclusion was not solely inferred from sgRNA clustering at the autoinhibitory interface, but also experimentally validated. In our revised manuscript, we provide additional experimental biochemical characterization to further support the claim that autoinhibition is weakened in the DNMT1 mutants we identified (Fig. 3h, Supplementary Fig. 4f). Moreover, we provide cellular data using clonal cell lines harboring endogenous DNMT1 mutations in addition to knockdown/overexpression experiments, demonstrating that RFTS and autoinhibitory linker mutations confer partial growth rescue to DAC treatment (Fig. 3i,j). We agree that we cannot rule out the possibility that these mutations may exert other effects that independently contribute to the observed resistance phenotype (e.g., altered CpG recognition), and we have added a statement acknowledging this limitation.

(2) Lack of general biological relevance of the corresponding findings. Through this work, the author identified multiple DNMT1-UHRF1 variants that alter the anti-proliferation role of decitabine. However, the observation that the multiple mutants were clustered in a hotspot doesn't mean that these mutants have to act via the same mechanism. The authors seem to underestimate the complexity of how these mutants can render the same biological readouts and even haven't considered the possibility of transcriptional modulation of antagonists or agonists in the DNMT1-UHRF1. Therefore, the biological relevance of these findings remains unclear.

We agree that although the cluster 1 mutations share a common property of increased DNMT1 activity, it does not preclude alternative mechanisms. Indeed, it is likely that these mutations have complex and nuanced mechanistic differences in the biochemical alterations underlying their observed increases in DNMT1 activity. Indeed, we have included enzyme activity data suggesting that autoinhibitory linker mutations may exhibit a different biochemical basis for increased DNMT1 activity than RFTS and CXXC mutations. That said, we did not intend to make broader claims regarding biological relevance and were instead focused on conveying that this activity-based methodology can identify gain-of-function mutations, which we directly support with experimental data. To clarify these points, we have adapted the text to more precisely convey our intended claims and have acknowledged that other complex mechanisms may also be involved.

(3) Collectively for reasons (1) and (2), the mechanistic analysis seems only to associate the current findings with known regulatory pathways. Without detailed in vitro and in-cell characterization of the DNMT1-UHRF1 mutants, the novel regulatory mechanisms, which may exist, could be largely missed.

We have added some additional characterization of these mutations in the revised manuscript, which have been detailed above, and we would like to note that we identified new sites in DNMT1 and UHRF1 that may be functional based off our allele analysis. However, since this manuscript is intended more as a methodology, we believe that extensively exploring novel regulatory mechanisms and their mechanism is beyond the scope of this report.

(4) The current CRISPER-based screening approach has the technical limitation of mainly screen deletion with some exceptions for point mutations. As a result, the majority of loss/gain-of-function point mutations will be missed by the CRISPER-based screening method.

We acknowledge that a technical limitation of this Cas nuclease-based mutational scan is that it is biased toward insertion/deletion mutations versus point mutations. However, we disagree with the reviewer’s claim that this means that the majority of the loss-/gain-of-function mutations will be missed, since insertion/deletions are often larger perturbations than point mutations and thus have stronger effect sizes in many cases. In principle, the selection modalities (e.g., activity-based inhibitors) used here — which are the primary focus of the study — can also be combined with alternative genomic editing approaches to assess distinct mutational perturbations, such as base editing for point mutations (see Lue, N.Z. et al., Nat. Chem. Biol. 2022). To acknowledge the reviewer’s concern, however, we have added additional text explicitly stating that the screen is biased against point mutations and that future integration with base editing and other mutational modalities may be useful to complement our nuclease-based approach.

(5) The current CRISPER-based screening approach can work only in the context of living cells. As a result, robust cellular readouts are needed. The DNMT1-UHRF1 in combination with decitabine is among few suitable targets for such application.

While running CRISPR-based screens requires robust cellular assays, the main advantage of CRISPRbased mutational scanning is the ability to mutagenize the endogenous protein target in situ and assess the effect of the perturbation in the native cellular and genomic context. Resistance to activity-based probes — and small molecules more broadly — provides a robust phenotypic readout that has been extensively used by our group and many others. Alternatively, other types of phenotypic readouts that do not rely on cell viability can also be employed with these screens, including those used to assess DNA methylation (see Lue, N.Z. et al., Nat. Chem. Biol. 2022). Given the increasingly large body of literature applying CRISPR-based screens towards a multitude of biological pathways and diverse targets, we disagree with the reviewer’s claim that only a few targets can be evaluated in such a manner.

(6) Although the authors claim that their mutants are "gain-of-function" for DNMT1/UHRF1, they were indeed due to the loss of inhibitory regulation. It is a little disappointing because the screening outcomes still fall into the conventional expectation of the loss-of-function variants.

We agree that the mutations are not truly neomorphic, but instead likely hypermorphic due to loss of an autoinhibitory mechanism, resulting in gain-of-function increase in catalytic activity. While discovering neomorphic mutations would be extraordinary, we do not believe that our results are disappointing since the identification of autoinhibitory mechanisms is nevertheless impactful.

Collectively, the current status of the manuscript is short of merits in terms of the impacts of technology and biological findings.

We respectfully disagree with the reviewer’s comment as we believe that the experimental and computational methodology may be broadly useful for the field. Indeed, we have already implemented many of the tools developed here in our current ongoing work.

excellent sequence of links

Sleep tactic for solving programming challenges

Ha! Cf. Lawson,