Multilingual individuals no longer feel bound by traditional language structures and may freely switch between languages to express themselves more authentically. The result is a dynamic evolution of language, where new hybrid forms emerge, and old norms of linguistic purity become increasingly blurred.
I think they should still adhere to traditional structures in a professional setting, but outside of that is fine.
Integrating translanguaging into educational practices can enhance students' linguistic flexibility and prepare them for a multilingual, interconnected world.
This is more the case in communities that see a lot of code switching on a daily basis, such as in the Southwest parts of America
For technology, developers are encouraged to design platforms that accommodate diverse linguistic needs, promoting inclusivity and ensuring equitable access for users from various linguistic backgrounds, ultimately contributing to a more accessible and interconnected digital world.
How would they do this without accidentally ostracizing the English language?
To ensure the inclusivity of multilingual digital environments, educational systems and digital platforms must foster policies that promote multilingualism and ensure equal representation of minority languages.
What kind of policies would they use?
This process challenges the traditional boundaries of written or spoken language by enabling users to express themselves through a combination of linguistic and semiotic resources, thus promoting a more inclusive and flexible understanding of communication.
Translanguaging is not just constrained to words, it also uses non verbal elements as well.
For example, memes are a prominent form of digital communication where users frequently combine different languages or dialects, often mixing English with regional languages or even incorporating slang and emojis. This practice reflects the fluidity of linguistic boundaries in digital spaces and highlights how translanguaging can function as a creative tool for expressing complex ideas or social commentary
I had never thought of memes in this way before.
Utilizing a mixed-methods approach, the research analyzed the communication habits of 120 multilingual participants on platforms such as Twitter, Instagram, and WhatsApp. The findings highlight distinct patterns of code-switching and translanguaging influenced by factors like audience demographics, contextual demands, and the technological features of each platform.
The author went through a lot to get this information, so this article is very accurate in terms of statistics
For instance, bilingual individuals may switch from one language to another to signal a shift in topic or to evoke a certain emotion or cultural reference.
This happens quite a bit in New Mexico, because of the Hispanic population
Marisa Senngam; Suphannika Pornwattanakavee; Nattawut Leelakanok; Teerarat Todsarot; Gabrielle Angele Tatta Guinto; Ratchanon Takun; Assadawut Sumativit
The correct order is: Suphannika Pornwattanakavee; Nattawut Leelakanok; Teerarat Todsarot; Gabrielle Angele Tatta Guinto; Ratchanon Takun; Assadawut Sumativit; Marisa Senngam. This will be changed in the next revision and the final manuscript.
Marisa Senngam; Suphannika Pornwattanakavee; Nattawut Leelakanok; Teerarat Todsarot; Gabrielle Angele Tatta Guinto; Ratchanon Takun; Assadawut Sumativit
The correct order is: Suphannika Pornwattanakavee; Nattawut Leelakanok; Teerarat Todsarot; Gabrielle Angele Tatta Guinto; Ratchanon Takun; Assadawut Sumativit; Marisa Senngam. This will be changed in the next revision and the final manuscript.
Marisa Senngam; Suphannika Pornwattanakavee; Nattawut Leelakanok; Teerarat Todsarot; Gabrielle Angele Tatta Guinto; Ratchanon Takun; Assadawut Sumativit
The correct order is: Suphannika Pornwattanakavee; Nattawut Leelakanok; Teerarat Todsarot; Gabrielle Angele Tatta Guinto; Ratchanon Takun; Assadawut Sumativit; Marisa Senngam. This will be changed in the next revision and the final manuscript.
'CBS Evening News' co-anchor John Dickerson will leave the network this year<br /> by [[Stephen Battaglio]] - Los Angeles Times<br /> accessed on 2025-10-31T23:37:40
Download the complete Review Process [PDF] including:
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A study group for this class
This would be ideal with 3-5 people, five is even pushing it, but with this, it's good because the group could talk and communicate but also have a goal without being distracted. if there's more than five it's too many and people could start having side conversations about the things that aren't associated with the class
Groups develop a shared identity based on their task or purpose, previous accomplishments, future goals, and an identity that sets their members apart from other groups.
This reminds me of my philosophy class's small group, we mostly discuss the class and each of our respective opinions on the discussions we have in class, and it's so much fun honestly, and also, I love to have each discussion with people
Participating in groups can also increase our exposure to diversity and broaden our perspectives. Although groups vary in the diversity of their members, we can strategically choose groups that expand our diversity, or we can unintentionally end up in a diverse group. When we participate in small groups, we expand our social networks, which increase the possibility to interact with people who have different cultural identities than ourselves.
This reminds me of how my groups at homeschool co-ops were. My experience was small classes, which increased discussion, and also helped with the intellectual diversity of the class.
In such groups, like a committee or study group, interactions and decisions are primarily evaluated based on the quality of the final product or output. The three main types of tasks are production, discussion, and problem-solving tasks (Ellis & Fisher, 1994).
Most study groups hit all three main types of tasks, production, of productivity, discussion, such as discussion of class content, and working together to find and solve the problems for class
Size and structure also affect communication within a group (Ellis & Fisher, 1994). In terms of size, the more people in a group, the more issues with scheduling and coordination of communication.
This is why the circle structure is easier to deal with in this situation. One person makes a set time for people to figure out their respective schedules versus a decentralized group that has to deal with everyone's specific and respective schedules.
LooksRare clone script helps startups and entrepreneurs
Discover the potential of launching your own NFT marketplace with the LooksRare Clone Script. This ready-to-deploy solution is equipped with various features, enabling businesses to enter the NFT ecosystem quickly and efficiently. With complete customization and high-end security, it provides a cost-effective way to build a feature-rich marketplace. Read the full blog to explore how this solution can help you start your NFT business: LooksRare Clone Script – White Label LooksRare Clone software
as she does in the bus segregation case –then she engages in direct civil disobedience. When she breachesa law other than the one she opposes – suppose she engages in trespassto protest the bus segregation laws – then she engages in indirect civildisobedience. 2
a civil disobedient transgressing the law itself they wish to oppose is direct civil disobedience
indirect civil disobedience is protest by transgression of other laws
Commands mkdocs new [dir-name] - Create a new project. mkdocs serve - Start the live-reloading docs server. mkdocs build - Build the documentation site. mkdocs -h - Print help message and exit.
TORQUE TABLES
What it is: Raw torque lookups keyed by RPM with an accompanying “compression/negative-Nm” channel from the file. Structure in XML:
Row0 starts a table (b0, comp, tq), then multiple rows as row-i (int RPM) or row-f (float RPM). Optional endvar tail exists. Sanity constraints: rpm 0–25000, comp −300…300, tq −4000…10000. Tuning cue: This is the ground truth for engine output. Any rescale you do should respect the defined ranges to avoid invalid parses.
BOOST TABLES
What it is: Turbo/boost lookup by RPM with throttle columns. Structure in XML:
Row0: b0, then throttle columns t0/t25/t50/t75/t100 (bar).
Row-i: adds rpm + the same five throttle columns. Sanity constraints: each throttle cell 0.5–3.0 bar. Tuning cue: Shape the five throttle traces per RPM to control response; keep within bounds to remain parsable.
LIMITS & RPM CONTROL
RevLimitRange Two encodings (float/float/byte or int/int/byte). Defines limit_min, limit_max, steps (unit rpm). Use for hard/soft limiter windows in the map.
RevLimitSetting Single byte selector for the active limiter slot/index. Selects which limit from the defined range to use.
RevLimitLogic Float logic scalar used by the limiter behavior. Acts as a tuning knob for how the limiter applies.
LifetimeEngineRPM Float or int variant: avg, max (rpm). Book-keeping fields embedded in the file; not a control.
FUEL & ENGINE MAPPING
FuelConsumption | FuelEstimate Single float each. Consumption/estimation scalars carried with the engine.
EngineFuelMapRange Three bytes: min, max, step. Index range for fuel map selection.
EngineFuelMapSetting Byte map_index. Chooses current fuel map within the above range.
EngineBrakingMapRange float min, float max, byte steps. Defines decel/engine-brake map scale.
EngineBrakingMapSetting Byte map_index. Selects active engine-brake map.
EngineInertia Float (unit kg·m², bounded in XML). Rotational inertia used by the solver.
Unknown_EngineFreeRevs Float placeholder. Keep as-is unless you’ve correlated it.
IdleRPMLogic Two variants: floats or ints for rpm_low, rpm_high. Idle window/reference used by the map.
LaunchEfficiency | LaunchRPMLogic Float efficiency and two-value launch RPM logic. Affect launch behavior in the engine context.
THERMAL MODEL
OptimumOilTemp Float °C. Target/nominal oil temp.
CombustionHeat | EngineSpeedHeat Floats contributing to heat generation terms.
OilMinimumCooling | WaterMinimumCooling Floats: baseline cooling capacities.
OilWaterHeatTransfer | RadiatorCooling Pair of floats each: coupling and radiator cooling terms.
LifetimeOilTemp float avg, float max °C. Book-keeping values; not controls.
EMISSIONS & MISC FLOATS
EngineEmission Three floats e1/e2/e3. Generic emissions scalars recorded in the file.
LifetimeAvg | LifetimeVar Float each. Statistical placeholders carried in the data.
Unknown_Float_2265DD60 | Unknown_Float_229217E0 Floats with unknown semantics; retain original values.
STARTER & AUX BYTES
OnboardStarter Byte presence flag.
EDF_UNKN_005 Byte unnamed control; do not alter without evidence.
StarterTiming Three floats t1/t2/t3. Timing scalars used by start sequence.
AirRestrictorRange float min, float max, byte steps. Defines restrictor band and discretization.
Unknown_Byte_2B3ED340 Byte placeholder.
BoostRange | BoostSetting Range: byte min, float max (bar), byte steps; Setting: byte. Caps and selects boost within allowed envelope.
ENGINE LAYOUT TAGS
What it is: Byte sequences near the file tail that identify cylinder/rotor layout (e.g., Straight 4, V8, Flat 6, V12, etc.). Usage: Read-only hints stored in the binary; they do not change maps but help classify engines in tooling
emale are the distinctions of nature, good and bad the distinctions of Heaven; but how arace of men came into the world so exalted above the rest, and distinguished like some newspecies, is worth inquiring into, and whether they are the means of happiness or of misery tomankind
This part asks why some people are seen as better than others, like a different kind of human. It says that being a king or ruler is not natural and might cause problems instead of helping people.
But there is another and great distinction for which no truly natural or religious reason canbe assigned, and that is the distinction of men into KINGS and SUBJECTS.
Paine argue that dividing people into kings and subjects is unnatural and unfair. He says God never created some people to rule and others to obey. This challenges the idea of monarchy and supports equality among all men.
Perhaps the sentiments contained in the following pages, are not YET sufficientlyfashionable to procure them general favor; a long habit of not thinking a thing WRONG,gives it a superficial appearance of being RIGHT,
Paine is writing in 1776 to Americans in the colonies. He wants them to question British rule, even though most are used to it. He is trying to open their eyes and make them think for themselves instead of just following tradition. Paine believes that with time and reason, more people will see that independence is the right choice.
Thomas Paine Calls for Americanindependence, 1776
Paine's words are emotional and persuasive. He inspire readers to value freedom, equality, and independence, showing that ordinary people have the power to create change. His writing gave many colonists hope and courage to stand up to Britain. Paine's ideas helped spread the belief that a new, fair government could be built by the people themselves.
In the early ages of the world, according to the scripture chronology there were no kings; theconsequence of which was, there were no wars;
Paine uses historical and biblical examples to argue that monarchy causes conflict, contrasting peaceful early societies with violent monarchies.
Every spot of the old world is overrun with oppression. Freedom hath been huntedround the globe. Asia, and Africa, have long expelled her. — Europe regards her like astranger, and England hath given her warning to depart. O! receive the fugitive, and preparein time an asylum for mankind
Paine shows the revolution has global significance and urges America to protect liberty, emphasizing the importance of action.
O ye that love mankind! Ye that dare oppose, not only the tyranny, but the tyrant, standforth!
How does Paine's call to action compare to petitions, letters, or other revolutionary writings we've read in the course?
Society in every state is a blessing, but Government, even in its best state, is but a necessaryevil
Why does Paine separate society and government? How does this connect to Enlightenment ideas about natural rights or the social contract?
The cause of America is in a great measure the cause of all mankind.
Paine believes America's fight for independence is part of a larger global struggle for freedom, not just a local issue. This helps convince readers that their actions matter worldwide.
As a long and violent abuse of power, is generally the Means of calling the right of it inquestion (and in Matters too which might never have been thought of, had not the Sufferersbeen aggravated into the inquiry) and as the King of England hath undertaken in his OWNRIGHT, to support the Parliament in what he calls THEIRS, and as the good people of thiscountry are grievously oppressed by the combination, they have an undoubted privilege toinquire into the pretensions of both, and equally to reject the usurpation of either
Paine argues that when leaders abuse power, people have the right to question and reject them. This shows why colonists could challenge British rules.
But the tumult soon subsides. Time makes more converts than reason
Even if people resist new ideas at first, over time most will agree. Paine predicts that support for independence will grow naturally.
On a more practical level, such qualitiescan improve human lives wholesale through the exchange ofideas, advancement of thought, and facilitation of trade.
I can agree to this; it helps me be able to think a little more on wording and if I can use a better sentence structure.
Very recently, though, some scholars have resurrectedarguments rooted in the 1970‟s. These arguments questionthe established practice of supporting Standard LanguageIdeologies, and perhaps the most direct and visibleopposition to embracing Standard English of late hasemerged from Dr. Asao B. Inoue, the past President of theConference on College Communication and Composition(CCCC) and keynote speaker at the 2019 CCCC
A speech from Dr. Inoue's 'How Do We Language So People Stop Killing Each Other, or "What Do We Do About White Language Supremacy?" doesnt specify
That Subject A examoperated until the early twenty-first century when thenomenclature changed to the Analytical Writing PlacementExam (AWPE)
when did they start implementing the change of name to AWPE?
The traditionalresponse is remarkably pragmatic: Students with such skillsare far more likely to excel in courses that use Standard5 One need look no further than Ferdinand de Saussure‟s famous textCourse in General Linguistics and the poignant concept of sign and signifier.The greater the linguistic difference between sign and signifier leads to thegreater the possibility of slippage between sign and signifier.English textbooks, rely on lectures in English, and routinelyassign papers to be submitted in English.
That could be said to dialectical people as well as long as they use their language in these questions, and their responses.
In the“Report of The National Commission on Writing forAmerica‟s Families, Schools, and Colleges” generated byCollege Board in September of 2004, writing was recognizedas a “‟threshold skill‟ for both employment and promotion,particularly salaried employees.” (3) [14]. The surveyconsidered 120 major American corporations thatrepresented almost eight million workers.
that's a good study to conduct research on.
Smith is one of the more recentscholars to forward such a particularly lucid example of theantagonism towards Standard English, but Smith‟s argumentis notable and potent because the authors suggest changes tothe mechanism of testing.4
check for more info on Smith
Melinda J. McBee Orzulak, for example,supports the idea that Deficit Language Ideologies – the ideathat Englishes that diverge from Standard English –“marginalize nondominant groups and promote dominantgroups‟ interests” (180), presumably both within the confinesof education and in society at large [12]. Embracing apedagogical approach that places Standard English in aseminal position in the composition classroom, according toOrzulak, wouldFurther advocate for linguistic separatism by ignoringthe realities of code-meshing. One aspect of deficitlanguage ideology is the belief that if something is not“standard” English, it is not grammatical or that sloppypeople use sloppy grammar. (180)
some good arguments to be read over
Displacinginstructional time to accommodate code-meshing andcode-switching has the unfortunate byproduct of limitinginstruction in Standard English and introducing linguisticconcepts that actually interfere with Standard Englishacquisition. As a result, students may be placed at adisadvantage in acquiring the linguistic skills necessary tojoin in the Burkean parlor.
Why can't we just have two separate required classes?
“it seems useful to ask them [students] to not onlymesh codes, but to consider the politically-charged origins ofthe „codes‟ they employ, and to think about ways in whichthey might interrogate – and even construct – these codes interms of their specific personal, cultural, and rhetoricalsituations” (283).
John Vance
In other words,Standard English provides a platform for inclusivity that isarguably absent in multiple Englishes.
I wish we could see these arguments that the author is talking about.
The potential pitfall of code-meshing and the subtledisplacement of Standard English rests in a slippery slope asit can apply to instruction in the composition classroom;
I don't understand this how it says apply to instruction in the comp classrooms?
If Cochran-Smith et al. are correct,then instructors might be inclined to traverse the slipperyslope of validating code-meshing, an approach thatinvalidates the concept of Standard English, and, arguably,generates confusion about the role of Standard English andexpectations around Standard English.
read up on this to see if their outcries to implication of code meshing worked
current criticism in Composition/Rhetoricthat addresses the linguistic construct of multiple Englishesand their relationship to Standard English, most especially asthe relationship is conceived in American higher education
about the topic
The Lord came down to see the city and the tower thatthe men had built. Then the Lord said: ”If now, whilethey are one people, all speaking the same language,they have started to do this, nothing will later stop themfrom doing whatever they presume to do. Let us then godown and there confuse their language, so that one willManuscript received September 9, 2019; revised January 21, 2020.Paul A. J. Beehler is with the University of California Riverside, UnitedStates (e-mail: paulb@ucr.edu).not understand what another says.”
not really related, but I would have thought The Lord would want everyone in union out of all races.
This response considers an excerpt of Dr.Inoue’s speech and then ultimately refutes the argument thatStandard English should be abandoned
What was speech segment taken from? and why does it prove the argument wrong that Standard English should be abandoned?
bstract—Scholarly debates about Standard English in the1970’s were, in part, instigated by the 1969 Task Force onRacism and Bias in the Teaching of English.
What does the Task Force on racism and bias in teaching of English do? are they still around?
If it were a war for the purpose of making democracy safe for the world, we would say that democracy must first be safe for America before it can be safe for the world.
She talking :o
and, this is why it is tempting for us, scientists, to think that this is the right and perhaps only way to tackle challenging problems. first, figure out precise mechanism by which phenomena behind these challenging problems happen, and then based on the uncovered mechanism, come up with solutions to these problems. surely once we know why and who problems manifest themselves, we can fix the problems.
Very very important argument. This is similar to the intuition of Ilya about Deep Learning. In the past, the reason why researchers prefered classical ML is because they can theoretically prove understand and prove their mechanism. But Ilya think Deep Learning is perfect because we cannot model them.
i lliscà
The information you share online can last a long time and may be seen by thousands of people all around the world.
I asked myself: What posts or comments from my past might still exist online that I would not make now, and could they affect my personal or professional life? Our digital footprints are persistent. What seems innocuous now can be seen later by employers, peers, or public audiences and may influence perceptions of us. Being proactive about past content helps manage our online identity and reputation.
When downloading an app, stop and consider: Have you read the app’s terms of use? Do you know what you’re giving the app permission to access? (e.g., your camera, microphone, location information, contacts, etc.) Can you change the permissions you’ve given the app without affecting its functionality? Who gets access to the data collected through your use of the app, and how will it be used? What kind of privacy options does the app offer?
I realize I rarely read full terms of use for apps. So my action: For two apps I use frequently and haven’t reviewed recently, I will open the permissions settings, list what the app can access, and decide whether to revoke any permissions (especially microphone, camera, location) that seem unnecessary. Many apps collect more data than we consciously realize; this collection can invade our privacy, expose identity-information, or enable profiling/tracking. By auditing permissions we reduce our exposure and increase control of our digital identity.
Child pornography and non-consensual distribution of intimate images.
This is a concept we need to explain more clearly to everyone—especially minors. Many don’t realize that even if you're underage yourself, sharing or possessing certain types of photos involving others can still be illegal and harmful. It’s not just about personal choices; it’s about understanding consent, privacy, and the law.
It’s good practice to ask permission before sharing a picture of someone else. In a Kaspersky Lab survey, 58% of people reported feeling upset or uncomfortable when a photo of them was shared that they didn’t want made public.
I agree this is an important practice to teach our students. Photos and videos taken at school are a big part of their daily lives, but they don’t always consider who might be in the background or whether that person would want to be publicly shared. Helping students think critically about consent and privacy is essential.
[my request]
https://www.mrtvarevolucia.net/
I just want to share my annotations of the website with my friend.
I think the manifesto on the website is valuable and worth discussing. Hypothesis or a similar tool could provide great means for doing that, as I wish such a tool would provide support for many discussions around the web. I wish Hypothesis got off the ground. I don't really believe an open proxy might "create substantial friction". Integration in a popular web browser seems like the only way forward and it may already be too late for that, given the spread of smartphones. I hope I am just lacking imagination.
Being able to share the annotations as a file would probably be a useful partial solution in my situation.
that would create substantial friction
Are you sure it would? Did it create substantial friction while the open proxy was running?
Over time we will implement strategies to make extending the list of allowed sites more seamless.
Is the implementation of these strategies still on the horizon?
allow list
Which domains are on the allow list (either initial or current)?
Less than a thousand domains represent the substantial corpus where most annotations happen.
Are the most popular domains the most important? Are they the most valuable objects of discussion?
k. I felt that suc-cumbing to the shortened "u"for "you" and writing in frag-mented, seemingly unintelli-gible sentences was contributing to what mybrother humorously calls the "downfall of the En-glish language
She starts off worrying that texting is ruining proper writing. I get that. A lot of teachers probably felt that way at first.
I myself have devel-oped the ability to code-switch effortlessly betweenthe text speak I use online and the Standard EnglishI use in my academic life
I like that she practices what she teaches. It makes her point stronger because she lives it too.
ely. Ifteachers and parents can ac-knowledge that text speak isindeed appropriate in the digital world that stu-dents navigate daily, then perhaps we can see its usein school as a difference, rather than a d
This sums it up perfectly—text speak isn’t a problem, it’s just a different language style. What matters is knowing when to flip the switch.
viting students to look critically at their writing, Ibrought the issues of vernacular and standard cor-rectness to the forefron
She lets students figure out their own writing habits instead of just marking errors. That teaches awareness instead of fear of being wrong.
This type of activity introduces the idea tostudents that language varies by con
Exactly, it’s not about what’s “right” or “wrong.” It’s about understanding when to be casual and when to be formal. That’s a skill, not a rule.
To begin the Flip the Switch lesson, teachersask students to identify settings in which they com-municat
This lesson is smart, it helps students realize they already switch how they talk depending on the situation. She’s just giving that a name.
t. The authors suggest that teach-ing students to navigate between home and schooldiscourses, a task they call code-switching, privi-leges both language
This is her main point: students can use both styles, but they need to understand which one fits where. That’s what real communication skills are about.
Because digital language represents such a largepart of the primary discourse of today's adolescents(Prensky), it is not surprising that the style of elec-tronic communication is "seeping into their school-work
She’s explaining why texting shows up in schoolwork, it’s what students do all day. It’s not bad writing, just habit, and that’s an important difference.
8.4.2 Bayesian Inference
One of the main differences between frequentist and Bayesian statistics is Bayesians assume the parameters themselves has a distribution and that previous information can help make predictions. Frequentists treat parameters as fixed and use repeated sampling/long running frequencies to make predictions
he regularization term is sometimes called the penalty term
Regularization is a technique that penalizes covariates when we have a lot of features. It either shrinks covariates or zeros out covariates completely. For smoother function we want a larger penalty term to shrink coefficients/
Web 2.0 discourse demonstrates what is called “digitalexceptionalism,” the idea that the internet is different from otherforms of communication and therefore not bound by the same legaland market forces.
I like how this line challenges the idea that the internet is completely separate from the real world. Even though in reality, websites and social media are shaped by money, politics, and people in power, just like any other industry. The internet was never truly equal. It has always reflected the same power structures as the real world, where a small number of companies or individuals hold most of the control.
Web2.0 ideology is the child of both Silicon Valley entrepreneurialcapitalism and activist subcultures like independent publishing, anti-globalization activism, Burning Man, cyberdelic rave culture, andFOSS.
I think this line really captures why Silicon Valley is so confusing. There's this mix of idealism and business that doesn't quite fit together. As someone who's grown up here and become more interested in business, I've seen this firsthand. People talk about changing the world and making things better for everyone, but at the same tmie they're constantly chasing investors and profits. They want to seem different from traditional corporations, but in the end, they still just want money. It's visible everywhere today. AI companies claim to be ethical while competing to dominate the market. Startups talk about helping people but really just want funding and profit. Marwick's point about Web 2.0 being born out of activism and capitalism explains that contradiction so well. It's what makes Silicon Valley interesting but also kind of fake. It's built on a constant clash between wanting to do good and wanting to make money.
. The new web is a very differentthing. It’s a tool for bringing together the small contributions ofmillions of people and making them matter.
I found it interesting how this line really captures the optimism that surrounded social media in its early days. Everyone genuinely believed it could change the world, especially the way that we communicate. It's wild to think about how sincere that excitement was compared to now. Even though platforms like TikTok and Instagram still connect to millions of people and let them share their lives, the focus has shifted. What used to feel like a happy place for community has turned into something more performative and sometimes even toxic. People's attitudes toward social media have gone from excitement about connection to serious concerns about mental health, misinformation, and negativity. It's often seen now as one of the biggest causes of anxiety, depression, and even suicide, especially among teens. I think the way that Marwick includes this quote really sets the tone for her critique. It reminds readers of what social media was supposed to be before showing what it's actually become.
The idea that the adoption of technology causesbehavioral changes and social effects, regardless of context, is false.
Marwick fights against the idea that technology is the only thing that changes civilization. She says that politics, society, and economics all affect how tools are used. This relates to everything we've read about the history of media. Just like the printing press didn't start the Reformation, social media didn't start democracy. Her criticism reminds us that we should be careful of Silicon Valley's assertion that apps and platforms automatically make the world "better."
The
This is minor but should this be step #5? Then the list of 9.1-9.5 nicely aligns with the numbered steps here
First, the script counts the number of adults and children in each household to retain household composition information. Next, it re-labels the number of vehicles to maintain consistency. The key transformation then assigns each adult as their own household by modifying the SERIALNO and SPORDER identifiers, effectively creating a new household for each adult while dropping children from the dataset. This ensures that person-level targets are accurately represented, with each adult treated as a separate entity for weighting purposes. Finally, the script verifies that the total weights remain consistent after this restructuring, ensuring that the overall population estimates are preserved.
Consider reformatting this into a bulleted list
Group quarters residents
"Residents of group quarters," since the examples cite group quarters, not residents
households so that estimates align with the population
I suggest rephrasing, something like: "data so that estimates align with the targets (i.e.,population)."
Second, I pointed at the productive nature of small disagreements.
I love showing the students how this works with the students when they have different answers to problems or differences in opinions. We discuss the different ideas and dissect or unpack the ideas together and see if one answer has a better reason than the other. This helps the students understand how everyone thinks differently and comes to their own conclusions differently. Some of the responses from the students are mind blowing and fantastic rationals due to personal experiences.
Legend
I feel like this section is very important for the article, but there is no citation supporting these claims.
The Golden Apple of Discord by Jacob Jordaens
None of the images have citations, we don't know where these pictures came from (for example, a museum colection). Also, are these images folowing the Wikipedia's copyright guidlines?
In later ages playwrights, historians, and other intellectuals would create works inspired by the Trojan War. The three great tragedians of Athens: Aeschylus, Sophocles and Euripides, wrote a number of dramas that portray episodes from the Trojan War. Among Roman writers the most important is the first century BC poet Virgil; in Book 2 of his Aeneid, Aeneas narrates the sack of Troy.
There is no citation in this section. If you are planning on editing the article, maybe it would be a good idea to find sources to support these claims.
The Trojan War was a legendary conflict in Greek mythology that took place around the twelfth or thirteenth century BC. The war was waged by the Achaeans (Greeks) against the city of Troy after Paris of Troy took Helen from her husband Menelaus, king of Sparta. The war is one of the most important events in Greek mythology, and it has been narrated through many works of Greek literature, most notably Homer's Iliad. The core of the Iliad (Books II – XXIII) describes a period of four days and two nights in the tenth year of the decade-long siege of Troy; the Odyssey describes the journey home of Odysseus, one of the war's heroes. Other parts of the war are described in a cycle of epic poems, which have survived through fragments. Episodes from the war provided material for Greek tragedy and other works of Greek literature, and for Roman poets including Virgil and Ovid. The ancient Greeks believed that Troy was located near the Dardanelles and that the Trojan War was a historical event of the twelfth or thirteenth century BC. By the mid-nineteenth century AD, both the war and the city were widely seen as non-historical, but in 1868, the German archaeologist Heinrich Schliemann met Frank Calvert, who convinced Schliemann that Troy was at what is now Hisarlık in modern-day Turkey.[1] On the basis of excavations conducted by Schliemann and others, this claim is now accepted by most scholars.[2][3]
Just like we talk in class, there are many claims with no citation in this section.
Gray
This may be splitting hairs. Outside the study region is listed as "gray" here but in the figure & legend, it appears more purple. Maybe because it's the rainbow colors which make me assume the last color is purple (or violet), and also because there are other regions in the map that are gray, albeit a lighter shade of gray
(a)
Can this be displayed within the paragraph and not in this separate text box? It's odd to have to scroll horizontally to finish reading this paragraph
code-meshing in-volves the intentional incorporation of more thanone language within writing to “exploit and blendthose differences” (Young et al., 2014, p. 43) in a waythat frees students to exercise identity and agencywithin their language use.
Definition.
Researchers in bilingual education and bilitera-cy have understood code-switching as the oral useof two or more languages either within or acrosssentences (intrasententially or intersententially)
Definition.
of culture is acquired unconsciously by happenstance—that is, nobody planned to teach it, and no one made an effort to consciously try to learn it.
This has always been a question of him, how have we developed different cultures, and how does everyone not follow the same one?
Because it requires deliberate effort and people are not constantly doing it, winking can acquire special meaning in social interactions.
It is interesting how they are telling us how winking requires more effort; which is completely true because it does need more effort and sometimes people cannot do it.
Archaeologists use material artifacts as keys to understanding the technologies, social practices, and ideas of ancient peoples.
This field of anthropology is the most interesting to me because I find it interesting how we can learn how ancient people have lived.
Some live in tents made of wooden beams and covered with animal skins or cloth, in caves hollowed out of sandstone or volcanic rock, or in wooden structures built on stilts or in trees to avoid floods and predators.
Since most of Americans have house holds that are held together buy animals skins and sandstone. Its kind of interesting how some are still not as advanced as us.
The room for cooking (the kitchen) used to be separated from the room where people socialized (the living room or great room), as it was assumed that one person (the wife) would cook in the kitchen while another person (the husband) relaxed alone or with company in the living room.
It still amazes me that this was the original standard. So much has changed over time.
What should be discarded is the idea, prevalent in the mainstream media worldwide and even at the UN, that charging for the social cost of carbon and its “offsetting” can replace public planning for socio-technological transition.
I think the argument being made here, is that just having a price by itself it's a gross oversimplification about what is needed.
And just because a price has been useful in places where you have seen a transition away from fossil fuel use, it doesn't mean you should start with a price.
The price is the thing that comes in once you have a clear alternative to the fossil-powered default, to make it less attractive.
the scale required for carbon market offset projects—through environmental conservation, forest regeneration, or reforestation—to ensure net-zero emissions is absolutely unfeasible. Offsetting cannot function as an alternative to decarbonizing energy, production, and infrastructure systems simply because there is not enough land to replant forests.
I think this is quite helpful as the simplistic version of the argument about why a carbon market by itself is not good enough. You can also make a similar argument for technical carbon removal. it's just too expensive to rely on.
The strategy for transitioning to renewable energy should not, therefore, begin with raising carbon prices and waiting for the market to offer an alternative solution in the short term. Instead, it must involve government planning for the supply of new green technologies and substitute infrastructure based on sustainable energy. Only after these technologies and infrastructure are accessible can raising carbon prices induce rapid substitution. Until then, consumers and user companies will be hostage to dirty options
I think the argument here is that, you need to build the alternative before you can choke off the fossil incumbent.
A precondition of being able to raise prices is having something people can migrate TO, that meets their needs. Otherwise, people reject the premise of a lot of the time,
In practice, energy’s inelastic demand means that even if prices rise, users of dirty technologies and fossil fuels will continue to pay, even if they become poorer, because they cannot find feasible substitutes
This seem to be arguing that carbon pricing is really ineffective in the face of inelastic demand. You can point the gillet jaune in France and their protests about the price of diesel was a good example of this. The demand inelasticity thing though is extremely important to get your head around, because while there are definitely clear examples that we have seen there are also many four cases where it's much less clear cut and things may indeed be more elastic than we thought.
The global carbon market approved in Baku will not overcome this problem. International competition to reduce costs to attract private investment is a systemic feature of the world economy. It creates a coordination problem, making it impossible to establish a single international price for carbon. International divergence in carbon prices undermines an effective system for reducing global emissions
So a key thing about the global covered market discussion at cop, is that there is no single Price. So just like our countries compete to have low tax resumes you have the same thing for carbon here. Oh dear.
Uruguay’s carbon tax came closest to the IPCC figure, pricing the emission of one metric ton of greenhouse gases at $167.17. At the other end of the spectrum, however, Indonesia’s carbon market charged only $0.61 for the same ton.
Wow, Uruguay actually has a high carbon tax?
but how a race of men came into the world so exalted above the rest, and distinguished like some new species, is worth inquiring into, and whether they are the means of happiness or of misery to mankind.
Paine wanted everyone equal. His idea of America withough Britain's control over them was something a lot of people may not have been ready for.
In the following sheets, the author hath studiously avoided every thing which is personal among ourselves.
Paine addressed "Common Sense" to ordinary colonists his emotional style helped turn revolutionary ideas into later starting the Declaration of Independence.
O! receive the fugitive, and prepare in time an asylum for mankind.
Looking at this now in our world, do you think Paine had a different idea for freedom?
MANKIND being originally equals in the order of creation, the equality could only be destroyed by some subsequent circumstance: the distinctions of rich and poor may in a great measure be accounted for, and that without having recourse to the harsh ill-sounding names of oppression and avarice.
How does his definition of equality differ from in the Declaration of Independence? Paine's hope was for women and men of all races and ethnicities to be equal. This was not the case.
Paine’s pamphlet offered a very different portrayal of the British government. His criticisms swept across the North American continent and generated widespread support for American independence.
Paine wrote the pamphlet called, "Common Sense" to persuade other Americans to break away from Britain.
It was the most prosperous invention the Devil ever set on foot for the promotion of idolatry.
Thomas Paine used religious language to intrigue the colonists. In 1776, religious authority was very respected in the political world. Him saying the monarchy was from the Devil was a smart move for him since he was for independence and everyone valued religion at the time.
necessary evil
Necessary evil is used to describe the government as something important for the people to have so their rights aren't harmed.
monarchy
A monarchy is a form of government where a single person governs the head of state.
The deciding factor in their choice has been that all were written by Fisher.
why did Blake emphasize that not all articles are about standard English?
eLife Assessment
This study provides useful insights into the ways in which germinal center B cell metabolism, particularly lipid metabolism, affects cellular responses. The authors use sophisticated mouse models to demonstrate that ether lipids are relevant for B cell homeostasis and efficient humoral responses. Although the data were collected from in vitro and in vivo experiments and analyzed using solid and validated methodology, more careful experiments and extensive revision of the manuscript will be required to strengthen the authors' conclusions.
Reviewer #1 (Public review):
In this manuscript, Hoon Cho et al. presents a novel investigation into the role of PexRAP, an intermediary in ether lipid biosynthesis, in B cell function, particularly during the Germinal Center (GC) reaction. The authors profile lipid composition in activated B cells both in vitro and in vivo, revealing the significance of PexRAP. Using a combination of animal models and imaging mass spectrometry, they demonstrate that PexRAP is specifically required in B cells. They further establish that its activity is critical upon antigen encounter, shaping B cell survival during the GC reaction.
Mechanistically, they show that ether lipid synthesis is necessary to modulate reactive oxygen species (ROS) levels and prevent membrane peroxidation.
Highlights of the Manuscript:
The authors perform exhaustive imaging mass spectrometry (IMS) analyses of B cells, including GC B cells, to explore ether lipid metabolism during the humoral response. This approach is particularly noteworthy given the challenge of limited cell availability in GC reactions, which often hampers metabolomic studies. IMS proves to be a valuable tool in overcoming this limitation, allowing detailed exploration of GC metabolism.
The data presented is highly relevant, especially in light of recent studies suggesting a pivotal role for lipid metabolism in GC B cells. While these studies primarily focus on mitochondrial function, this manuscript uniquely investigates peroxisomes, which are linked to mitochondria and contribute to fatty acid oxidation (FAO). By extending the study of lipid metabolism beyond mitochondria to include peroxisomes, the authors add a critical dimension to our understanding of B cell biology.
Additionally, the metabolic plasticity of B cells poses challenges for studying metabolism, as genetic deletions from the beginning of B cell development often result in compensatory adaptations. To address this, the authors employ an acute loss-of-function approach using two conditional, cell-type-specific gene inactivation mouse models: one targeting B cells after the establishment of a pre-immune B cell population (Dhrs7b^f/f, huCD20-CreERT2) and the other during the GC reaction (Dhrs7b^f/f; S1pr2-CreERT2). This strategy is elegant and well-suited to studying the role of metabolism in B cell activation.
Overall, this manuscript is a significant contribution to the field, providing robust evidence for the fundamental role of lipid metabolism during the GC reaction and unveiling a novel function for peroxisomes in B cells. However, several major points need to be addressed:
Major Comments:
Figures 1 and 2
The authors conclude, based on the results from these two figures, that PexRAP promotes the homeostatic maintenance and proliferation of B cells. In this section, the authors first use a tamoxifen-inducible full Dhrs7b knockout (KO) and afterwards Dhrs7bΔ/Δ-B model to specifically characterize the role of this molecule in B cells. They characterize the B and T cell compartments using flow cytometry (FACS) and examine the establishment of the GC reaction using FACS and immunofluorescence. They conclude that B cell numbers are reduced, and the GC reaction is defective upon stimulation, showing a reduction in the total percentage of GC cells, particularly in the light zone (LZ).
The analysis of the steady-state B cell compartment should also be improved. This includes a more detailed characterization of MZ and B1 populations, given the role of lipid metabolism and lipid peroxidation in these subtypes.
Suggestions for Improvement:
- B Cell compartment characterization: A deeper characterization of the B cell compartment in non-immunized mice is needed, including analysis of Marginal Zone (MZ) maturation and a more detailed examination of the B1 compartment. This is especially important given the role of specific lipid metabolism in these cell types. The phenotyping of the B cell compartment should also include an analysis of immunoglobulin levels on the membrane, considering the impact of lipids on membrane composition.
- GC Response Analysis Upon Immunization: The GC response characterization should include additional data on the T cell compartment, specifically the presence and function of Tfh cells. In Fig. 1H, the distribution of the LZ appears strikingly different. However, the authors have not addressed this in the text. A more thorough characterization of centroblasts and centrocytes using CXCR4 and CD86 markers is needed.<br /> The gating strategy used to characterize GC cells (GL7+CD95+ in IgD− cells) is suboptimal. A more robust analysis of GC cells should be performed in total B220+CD138− cells.
- The authors claim that Dhrs7b supports the homeostatic maintenance of quiescent B cells in vivo and promotes effective proliferation. This conclusion is primarily based on experiments where CTV-labeled PexRAP-deficient B cells were adoptively transferred into μMT mice (Fig. 2D-F). However, we recommend reviewing the flow plots of CTV in Fig. 2E, as they appear out of scale. More importantly, the low recovery of PexRAP-deficient B cells post-adoptive transfer weakens the robustness of the results and is insufficient to conclusively support the role of PexRAP in B cell proliferation in vivo.
- In vitro stimulation experiments: These experiments need improvement. The authors have used anti-CD40 and BAFF for B cell stimulation; however, it would be beneficial to also include anti-IgM in the stimulation cocktail. In Fig. 2G, CTV plots do not show clear defects in proliferation, yet the authors quantify the percentage of cells with more than three divisions. These plots should clearly display the gating strategy. Additionally, details about histogram normalization and potential defects in cell numbers are missing. A more in-depth analysis of apoptosis is also required to determine whether the observed defects are due to impaired proliferation or reduced survival.
Reviewer #2 (Public review):
Summary:
In this study, Cho et al. investigate the role of ether lipid biosynthesis in B cell biology, particularly focusing on GC B cell, by inducible deletion of PexRAP, an enzyme responsible for the synthesis of ether lipids.
Strengths:
Overall, the data are well-presented, the paper is well-written and provides valuable mechanistic insights into the importance of PexRAP enzyme in GC B cell proliferation.
Weaknesses:
More detailed mechanisms of the impaired GC B cell proliferation by PexRAP deficiency remain to be further investigated. In the minor part, there are issues with the interpretation of the data which might cause confusion for the readers.
Author response:
eLife Assessment
This study provides useful insights into the ways in which germinal center B cell metabolism, particularly lipid metabolism, affects cellular responses. The authors use sophisticated mouse models to demonstrate that ether lipids are relevant for B cell homeostasis and efficient humoral responses. Although the data were collected from in vitro and in vivo experiments and analyzed using solid and validated methodology, more careful experiments and extensive revision of the manuscript will be required to strengthen the authors' conclusions.
In addition to praise for the eLife system and transparency (public posting of the reviews; along with an opportunity to address them), we are grateful for the decision of the Editors to select this submission for in-depth peer review and to the referees for the thoughtful and constructive comments.
In overview, we mostly agree with the specific comments and evaluation of strengths of what the work adds as well as with indications of limitations and caveats that apply to the breadth of conclusions. One can view these as a combination of weaknesses, of instances of reading more into the work than what it says, and of important future directions opened up by the findings we report. Regarding the positives, we appreciate the reviewers' appraisal that our work unveils a novel mechanism in which the peroxisomal enzyme PexRAP mediates B cell intrinsic ether lipid synthesis and promotes a humoral immune response. We are gratified by a recognition that a main contribution of the work is to show that a spatial lipidomic analysis can set the stage for discovery of new molecular processes in biology that are supported by using 2-dimensional imaging mass spectrometry techniques and cell type specific conditional knockout mouse models.
By and large, the technical issues are items we will strive to improve. Ultimately, an over-arching issue in research publications in this epoch are the questions "when is enough enough?" and "what, or how much, advance will be broadly important in moving biological and biomedical research forward?" It appears that one limitation troubling the reviews centers on whether the mechanism of increased ROS and multi-modal death - supported most by the in vitro evidence - applies to germinal center B cells in situ, versus either a mechanism for decreased GC that mostly applies to the pre-GC clonal amplification (or recruitment into GC). Overall, we agree that this leap could benefit from additional evidence - but as resources ended we instead leave that question for the future other than the findings with S1pr2-CreERT2-driven deletion leading to less GC B cells. While we strove to be very careful in framing such a connection as an inference in the posted manuscript, we will revisit the matter via rechecking the wording when revising the text after trying to get some specific evidence.
In the more granular part of this provisional response (below), we will outline our plan prompted by the reviewers but also comment on a few points of disagreement or refinement (longer and more detailed explanation). The plan includes more detailed analysis of B cell compartments, surface level of immunoglobulin, Tfh cell population, a refinement of GC B cell markers, and the ex vivo GC B cell analysis for ROS, proliferation, and cell death. We will also edit the text to provide more detailed information and clarify our interpretation to prevent the confusion of our results. At a practical level, some evidence likely is technologically impractical, and an unfortunate determinant is the lack of further sponsored funding for further work. The detailed point-by-point response to the reviewer’s comments is below.
Public Reviews:
Reviewer #1 (Public review):
In this manuscript, Sung Hoon Cho et al. presents a novel investigation into the role of PexRAP, an intermediary in ether lipid biosynthesis, in B cell function, particularly during the Germinal Center (GC) reaction. The authors profile lipid composition in activated B cells both in vitro and in vivo, revealing the significance of PexRAP. Using a combination of animal models and imaging mass spectrometry, they demonstrate that PexRAP is specifically required in B cells. They further establish that its activity is critical upon antigen encounter, shaping B cell survival during the GC reaction.
Mechanistically, they show that ether lipid synthesis is necessary to modulate reactive oxygen species (ROS) levels and prevent membrane peroxidation.
Highlights of the Manuscript:
The authors perform exhaustive imaging mass spectrometry (IMS) analyses of B cells, including GC B cells, to explore ether lipid metabolism during the humoral response. This approach is particularly noteworthy given the challenge of limited cell availability in GC reactions, which often hampers metabolomic studies. IMS proves to be a valuable tool in overcoming this limitation, allowing detailed exploration of GC metabolism.
The data presented is highly relevant, especially in light of recent studies suggesting a pivotal role for lipid metabolism in GC B cells. While these studies primarily focus on mitochondrial function, this manuscript uniquely investigates peroxisomes, which are linked to mitochondria and contribute to fatty acid oxidation (FAO). By extending the study of lipid metabolism beyond mitochondria to include peroxisomes, the authors add a critical dimension to our understanding of B cell biology.
Additionally, the metabolic plasticity of B cells poses challenges for studying metabolism, as genetic deletions from the beginning of B cell development often result in compensatory adaptations. To address this, the authors employ an acute loss-of-function approach using two conditional, cell-type-specific gene inactivation mouse models: one targeting B cells after the establishment of a pre-immune B cell population (Dhrs7b^f/f, huCD20-CreERT2) and the other during the GC reaction (Dhrs7b^f/f; S1pr2-CreERT2). This strategy is elegant and well-suited to studying the role of metabolism in B cell activation.
Overall, this manuscript is a significant contribution to the field, providing robust evidence for the fundamental role of lipid metabolism during the GC reaction and unveiling a novel function for peroxisomes in B cells.
We appreciate these positive reactions and response, and agree with the overview and summary of the paper's approaches and strengths.
However, several major points need to be addressed:
Major Comments:
Figures 1 and 2
The authors conclude, based on the results from these two figures, that PexRAP promotes the homeostatic maintenance and proliferation of B cells. In this section, the authors first use a tamoxifen-inducible full Dhrs7b knockout (KO) and afterwards Dhrs7bΔ/Δ-B model to specifically characterize the role of this molecule in B cells. They characterize the B and T cell compartments using flow cytometry (FACS) and examine the establishment of the GC reaction using FACS and immunofluorescence. They conclude that B cell numbers are reduced, and the GC reaction is defective upon stimulation, showing a reduction in the total percentage of GC cells, particularly in the light zone (LZ).
The analysis of the steady-state B cell compartment should also be improved. This includes a more detailed characterization of MZ and B1 populations, given the role of lipid metabolism and lipid peroxidation in these subtypes.
Suggestions for Improvement:
B Cell compartment characterization: A deeper characterization of the B cell compartment in non-immunized mice is needed, including analysis of Marginal Zone (MZ) maturation and a more detailed examination of the B1 compartment. This is especially important given the role of specific lipid metabolism in these cell types. The phenotyping of the B cell compartment should also include an analysis of immunoglobulin levels on the membrane, considering the impact of lipids on membrane composition.
Although the manuscript is focused on post-ontogenic B cell regulation in Ab responses, we believe we will be able to polish a revised manuscript through addition of results of analyses suggested by this point in the review: measurement of surface IgM on and phenotyping of various B cell subsets, including MZB and B1 B cells, to extend the data in Supplemental Fig 1H and I. Depending on the level of support, new immunization experiments to score Tfh and analyze a few of their functional molecules as part of a B cell paper may be feasible.
- GC Response Analysis Upon Immunization: The GC response characterization should include additional data on the T cell compartment, specifically the presence and function of Tfh cells. In Fig. 1H, the distribution of the LZ appears strikingly different. However, the authors have not addressed this in the text. A more thorough characterization of centroblasts and centrocytes using CXCR4 and CD86 markers is needed.
The gating strategy used to characterize GC cells (GL7+CD95+ in IgD− cells) is suboptimal. A more robust analysis of GC cells should be performed in total B220+CD138− cells.
We first want to apologize the mislabeling of LZ and DZ in Fig 1H. The greenish-yellow colored region (GL7<sup>+</sup> CD35<sup>+</sup>) indicate the DZ and the cyan-colored region (GL7<sup>+</sup> CD35<sup>+</sup>) indicates the LZ.
As a technical note, we experienced high background noise with GL7 staining uniquely with PexRAP deficient (Dhrs7b<sup>f/f</sup>; Rosa26-CreER<sup>T2</sup>) mice (i.e., not WT control mice). The high background noise of GL7 staining was not observed in B cell specific KO of PexRAP (Dhrs7b<sup>f/f</sup>; huCD20-CreER<sup>T2</sup>). Two formal possibilities to account for this staining issue would be if either the expression of the GL7 epitope were repressed by PexRAP or the proper positioning of GL7<sup>+</sup> cells in germinal center region were defective in PexRAP-deficient mice (e.g., due to an effect on positioning cues from cell types other than B cells). In a revised manuscript, we will fix the labeling error and further discuss the GL7 issue, while taking care not to be thought to conclude that there is a positioning problem or derepression of GL7 (an activation antigen on T cells as well as B cells).
While the gating strategy for an overall population of GC B cells is fairly standard even in the current literature, the question about using CD138 staining to exclude early plasmablasts (i.e., analyze B220<sup>+</sup> CD138<sup>neg</sup> vs B220<sup>+</sup> CD138<sup>+</sup>) is interesting. In addition, some papers like to use GL7<sup>+</sup> CD38<sup>neg</sup> for GC B cells instead of GL7<sup>+</sup> Fas (CD95)<sup>+</sup>, and we thank the reviewer for suggesting the analysis of centroblasts and centrocytes. For the revision, we will try to secure resources to revisit the immunizations and analyze them for these other facets of GC B cells (including CXCR4/CD86) and for their GL7<sup>+</sup> CD38<sup>neg</sup>. B220<sup>+</sup> CD138<sup>-</sup> and B220<sup>+</sup> CD138<sup>+</sup> cell populations.
We agree that comparison of the Rosa26-CreERT2 results to those with B cell-specific loss-of-function raise a tantalizing possibility that Tfh cells also are influenced by PexRAP. Although the manuscript is focused on post-ontogenic B cell regulation in Ab responses, we hope to add a new immunization experiments that scores Tfh and analyzes a few of their functional molecules could be added to this B cell paper, depending on the ability to wheedle enough support / fiscal resources.
- The authors claim that Dhrs7b supports the homeostatic maintenance of quiescent B cells in vivo and promotes effective proliferation. This conclusion is primarily based on experiments where CTV-labeled PexRAP-deficient B cells were adoptively transferred into μMT mice (Fig. 2D-F). However, we recommend reviewing the flow plots of CTV in Fig. 2E, as they appear out of scale. More importantly, the low recovery of PexRAP-deficient B cells post-adoptive transfer weakens the robustness of the results and is insufficient to conclusively support the role of PexRAP in B cell proliferation in vivo.
In the revision, we will edit the text and try to adjust the digitized cytometry data to allow more dynamic range to the right side of the upper panels in Fig. 2E, and otherwise to improve the presentation of the in vivo CTV result. However, we feel impelled to push back respectfully on some of the concern raised here. First, it seems to gloss over the presentation of multiple facets of evidence. The conclusion about maintenance derives primarily from Fig. 2C, which shows a rapid, statistically significant decrease in B cell numbers (extending the finding of Fig. 1D, a more substantial decrease after a bit longer a period). As noted in the text, the rate of de novo B cell production does not suffice to explain the magnitude of the decrease.
In terms of proliferation, we will improve presentation of the Methods but the bottom line is that the recovery efficiency is not bad (comparing to prior published work) inasmuch as transferred B cells do not uniformly home to spleen. In a setting where BAFF is in ample supply in vivo, we transferred equal numbers of cells that were equally labeled with CTV and counted B cells. The CTV result might be affected by lower recovered B cell with PexRAP deficiency, generally, the frequencies of CTV<sup>low</sup> divided population are not changed very much. However, it is precisely because of the pitfalls of in vivo analyses that we included complementary data with survival and proliferation in vitro. The proliferation was attenuated in PexRAP-deficient B cells in vitro; this evidence supports the conclusion that proliferation of PexRAP knockout B cells is reduced. It is likely that PexRAP deficient B cells also have defect in viability in vivo as we observed the reduced B cell number in PexRAP-deficient mice. As the reviewer noticed, the presence of a defect in cycling does, in the transfer experiments, limit the ability to interpret a lower yield of B cell population after adoptive transfer into µMT recipient mice as evidence pertaining to death rates. We will edit the text of the revision with these points in mind.
- In vitro stimulation experiments: These experiments need improvement. The authors have used anti-CD40 and BAFF for B cell stimulation; however, it would be beneficial to also include anti-IgM in the stimulation cocktail. In Fig. 2G, CTV plots do not show clear defects in proliferation, yet the authors quantify the percentage of cells with more than three divisions. These plots should clearly display the gating strategy. Additionally, details about histogram normalization and potential defects in cell numbers are missing. A more in-depth analysis of apoptosis is also required to determine whether the observed defects are due to impaired proliferation or reduced survival.
As suggested by reviewer, testing additional forms of B cell activation can help explore the generality (or lack thereof) of findings. We plan to test anti-IgM stimulation together with anti-CD40 + BAFF as well as anti-IgM + TLR7/8, and add the data to a revised and final manuscript.
With regards to Fig. 2G (and 2H), in the revised manuscript we will refine the presentation (add a demonstration of the gating, and explicate histogram normalization of FlowJo).
It is an interesting issue in bioscience, but in our presentation 'representative data' really are pretty representative, so a senior author is reminded of a comment Tak Mak made about a reduction (of proliferation, if memory serves) to 0.7 x control. [His point in a comment to referees at a symposium related that to a salary reduction by 30% :) A mathematical alternative is to point out that across four rounds of division for WT cells, a reduction to 0.7x efficiency at each cycle means about 1/4 as many progeny.]
We will try to edit the revision (Methods, Legends, Results, Discussion] to address better the points of the last two sentences of the comment, and improve the details that could assist in replication or comparisons (e.g., if someone develops a PexRAP inhibitor as potential therapeutic).
For the present, please note that the cell numbers at the end of the cultures are currently shown in Fig 2, panel I. Analogous culture results are shown in Fig 8, panels I, J, albeit with harvesting at day 5 instead of day 4. So, a difference of ≥ 3x needs to be explained. As noted above, a division efficiency reduced to 0.7x normal might account for such a decrease, but in practice the data of Fig. 2I show that the number of PexRAP-deficient B cells at day 4 is similar to the number plated before activation, and yet there has been a reasonable amount of divisions. So cell numbers in the culture of mutant B cells are constant because cycling is active but decreased and insufficient to allow increased numbers ("proliferation" in the true sense) as programmed death is increased. In line with this evidence, Fig 8G-H document higher death rates [i.e., frequencies of cleaved caspase3<sup>+</sup> cell and Annexin V<sup>+</sup> cells] of PexRAP-deficient B cells compared to controls. Thus, the in vitro data lead to the conclusion that both decreased division rates and increased death operate after this form of stimulation.
An inference is that this is the case in vivo as well - note that recoveries differed by ~3x (Fig. 2D), and the decrease in divisions (presentation of which will be improved) was meaningful but of lesser magnitude (Fig. 2E, F).
Reviewer #2 (Public review):
Summary:
In this study, Cho et al. investigate the role of ether lipid biosynthesis in B cell biology, particularly focusing on GC B cell, by inducible deletion of PexRAP, an enzyme responsible for the synthesis of ether lipids.
Strengths:
Overall, the data are well-presented, the paper is well-written and provides valuable mechanistic insights into the importance of PexRAP enzyme in GC B cell proliferation.
We appreciate this positive response and agree with the overview and summary of the paper's approaches and strengths.
Weaknesses:
More detailed mechanisms of the impaired GC B cell proliferation by PexRAP deficiency remain to be further investigated. In the minor part, there are issues with the interpretation of the data which might cause confusion for the readers.
Issues about contributions of cell cycling and divisions on the one hand, and susceptibility to death on the other, were discussed above, amplifying on the current manuscript text. The aggregate data support a model in which both processes are impacted for mature B cells in general, and mechanistically the evidence and work focus on the increased ROS and modes of death. Although the data in Fig. 7 do provide evidence that GC B cells themselves are affected, we agree that resource limitations had militated against developing further evidence about cycling specifically for GC B cells. We will hope to be able to obtain sufficient data from some specific analysis of proliferation in vivo (e.g., Ki67 or BrdU) as well as ROS and death ex vivo when harvesting new samples from mice immunized to analyze GC B cells for CXCR4/CD86, CD38, CD138 as indicated by Reviewer 1. As suggested by Reviewer 2, we will further discuss the possible mechanism(s) by which proliferation of PexRAP-deficient B cells is impaired. We also will edit the text of a revision where to enhance clarity of data interpretation - at a minimum, to be very clear that caution is warranted in assuming that GC B cells will exhibit the same mechanisms as cultures in vitro-stimulated B cells.
The Investigative Function Presentation,” “What is Civic Engagement?,” “Using Technology for Civic Engagement in Criminal Justice,” “Course Skills & Motivation,” and “Rights Talk”
The case studies we will be focusing on.
I understood the problems plaguing poor communities of color, including issues associated with crime and rising incarceration rates, to be a function of poverty and lack of access to quality education- the continuing legacy of slavery and Jim Crow.
During the event of Jim Crow Laws in 1877 to 1950s, the problems have occurred that crime was functional to be the high associations for a lack of access to quality within increased incarceration levels.
In the era of colorblindness, it is no longer socially permissible to use race, explicitly, as a justification for discrimination, exclusion, and social contempt.
I rely on its exhibition in the era of colorblindness, it was justified for discrimination as in result for insolence must be punished. We shouldn't harm each other.
Jarvious Cotton's great-grandfather could not vote as a slave. His great-grandfather was beaten to death by the Ku Klux Klan for attempting to vote. His grandfather was prevented from voting by Klan intimidation. His father was barred from voting by poll taxes and literacy tests.
Cotton's fate of his grandfather's death at the hands of Klan was a tragedy, but he believes his father was trying to prevent form voting by poll taxes and literal expectations.
Europeans called the Americas “The New World.” But for the millions of Native Americans they encountered, it was anything but. Human beings have lived here for over ten millennia.
The Europeans claimed this land was "new". That was not the case, Native Americans had been there for years. It was nothing "new" to them.
Columbian Exchange
The Columbian Exchange was the transfer of goods between the East and the West. This later shape the whole world
Unlike the group low in autismcharacteristics, who showed normal mu suppression when viewing videos ofactions and pictures of manipulable
for those with high autism traits, this "mental rehearsal" system doesn't work as effectively, so their brain doesn't have the same response when just watching.
“flavor” of traditionalcognitive science to reveal some of its more implicit commitments
The author uses the term "flavor" to refer to the core, often unstated, assumptions or a general approach that characterizes traditional cognitive science. This "flavor" includes key implicit commitments that define the framework within which research is conducted.
eLife Assessment
This paper presents a computational method to infer from data a key feature of affinity maturation: the relationship between the affinity of B-cell receptors and their fitness. The approach, which is based on a simple population dynamics model but inferred using AI-powered Simulation-Based Inference, is novel and valuable. It exploits recently published data on replay experiments of affinity maturation. While the method is well-argued and the validation solid, the potential impact of the study is hindered by its complex presentation, which makes it hard to assess its claims reliably.
Reviewer #1 (Public review):
Summary:
This paper aims to characterize the relationship between affinity and fitness in the process of affinity maturation. To this end, the authors develop a model of germinal center reaction and a tailored statistical approach, building on recent advances in simulation-based inference. The potential impact of this work is hindered by the poor organization of the manuscript. In crucial sections, the writing style and notations are unclear and difficult to follow.
Strengths:
The model provides a framework for linking affinity measurements and sequence evolution and does so while accounting for the stochasticity inherent to the germinal center reaction. The model's sophistication comes at the cost of numerous parameters and leads to intractable likelihood, which are the primary challenges addressed by the authors. The approach to inference is innovative and relies on training a neural network on extensive simulations of trajectories from the model.
Weaknesses:
The text is challenging to follow. The descriptions of the model and the inference procedure are fragmented and repetitive. In the introduction and the methods section, the same information is often provided multiple times, at different levels of detail. This organization sometimes requires the reader to move back and forth between subsections (there are multiple non-specific references to "above" and "below" in the text).
The choice of some parameter values in simulations appears arbitrary and would benefit from more extensive justification. It remains unclear how the "significant uncertainty" associated with these parameters affects the results of inference. In addition, the performance of the inference scheme on simulated data is difficult to evaluate, as the reported distributions of loss function values are not very informative.
Finally, the discussion of the similarities and differences with an alternative approach to this inference problem, presented in Dewitt et al. (2025), is incomplete.
Reviewer #2 (Public review):
Summary:
This paper presents a new approach for explicitly transforming B-cell receptor affinity into evolutionary fitness in the germinal center. It demonstrates the feasibility of using likelihood-free inference to study this problem and demonstrates how effective birth rates appear to vary with affinity in real-world data.
Strengths:
(1) The authors leverage the unique data they have generated for a separate project to provide novel insights into a fundamental question.
(2) The paper is clearly written, with accessible methods and a straightforward discussion of the limits of this model.
(3) Code and data are publicly available and well-documented.
Weaknesses (minor):
(1) Lines 444-446: I think that "affinity ceiling" and "fitness ceiling" should be considered independent concepts. The former, as the authors ably explain, is a physical limitation. This wouldn't necessarily correspond to a fitness ceiling, though, as Figure 7 shows. Conversely, the model developed here would allow for a fitness ceiling even if the physical limit doesn't exist.
(2) Lines 566-569: I would like to see this caveat fleshed out more and perhaps mentioned earlier in the paper. While relative affinity is far more important, it is not at all clear to me that absolute affinity can be totally ignored in modeling GC behavior.
(3) One other limitation that is worth mentioning, though beyond the scope of the current work to fully address: the evolution of the repertoire is also strongly shaped by competition from circulating antibodies. (Eg: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3600904/, http://www.sciencedirect.com/science/article/pii/S1931312820303978). This is irrelevant for the replay experiment modeled here, but still an important factor in general repertoires.
eLife Assessment
This valuable study proposes a theoretical model of clathrin coat formation based on membrane elasticity that seeks to determine whether this process occurs by increasing the area of a protein-coated patch with constant curvature, or by increasing the curvature of a protein-coated patch that forms in an initially flat conformation (so called constant curvature or constant area models). Identifying energetically favorable pathways and comparing the obtained shapes with experiments provides solid support to the constant-area pathway. This work will be of interest for biologists and biophysicists interested in membrane remodelling and endocytosis. It provides an innovative approach to tackle the question of constant curvature vs. constant area coat protein formation, although some of the model's assumption are only partially supported by experimental evidence.
Reviewer #1 (Public review):
Summary:
The authors develop a set of biophysical models to investigate whether a constant area hypothesis or a constant curvature hypothesis explains the mechanics of membrane vesiculation during clathrin-mediated endocytosis.
Strengths:
The models that the authors choose are fairly well-described in the field and the manuscript is well-written.
Weaknesses:
One thing that is unclear is what is new with this work. If the main finding is that the differences are in the early stages of endocytosis, then one wonders if that should be tested experimentally. Also, the role of clathrin assembly and adhesion are treated as mechanical equilibrium but perhaps the process should not be described as equilibria but rather a time-dependent process. Ultimately, there are so many models that address this question that without direct experimental comparison, it's hard to place value on the model prediction.
While an attempt is made to do so with prior published EM images, there is excessive uncertainty in both the data itself as is usually the case but also in the methods that are used to symmetrize the data. This reviewer wonders about any goodness of fit when such uncertainty is taken into account.
Comments on revisions:
I appreciate the authors edits, but I found that the major concerns I had still hold. Therefore, I did not alter my review.
Reviewer #2 (Public review):
Summary:
In this manuscript, the authors employ theoretical analysis of an elastic membrane model to explore membrane vesiculation pathways in clathrin-mediated endocytosis. A complete understanding of clathrin-mediated endocytosis requires detailed insight into the process of membrane remodeling, as the underlying mechanisms of membrane shape transformation remain controversial, particularly regarding membrane curvature generation. The authors compare constant area and constant membrane curvature as key scenarios by which clathrins induce membrane wrapping around the cargo to accomplish endocytosis. First, they characterize the geometrical aspects of the two scenarios and highlight their differences by imposing coating area and membrane spontaneous curvature. They then examine the energetics of the process to understand the driving mechanisms behind membrane shape transformations in each model. In the latter part, they introduce two energy terms: clathrin assembly or binding energy, and curvature generation energy, with two distinct approaches for the latter. Finally, they identify the energetically favorable pathway in the combined scenario and compare their results with experiments, showing that the constant-area pathway better fits the experimental data.
Strengths:
The manuscript is well-written, well-organized, and presents the details of the theoretical analysis with sufficient clarity.<br /> The calculations are valid, and the elastic membrane model is an appropriate choice for addressing the differences between the constant curvature and constant area models.<br /> The authors' approach of distinguishing two distinct free energy terms-clathrin assembly and curvature generation-and then combining them to identify the favorable pathway is both innovative and effective in addressing the problem.<br /> Notably, their identification of the energetically favorable pathways, and how these pathways either lead to full endocytosis or fail to proceed due to insufficient energetic drives, is particularly insightful.
Comments on revisions:
The authors have carefully addressed all my comments, and the revised manuscript is now clear, rigorous, and satisfactory.
Author response:
The following is the authors’ response to the original reviews
Reviewer #1:
Summary
The authors develop a set of biophysical models to investigate whether a constant area hypothesis or a constant curvature hypothesis explains the mechanics of membrane vesiculation during clathrin-mediated endocytosis.
Strengths
The models that the authors choose are fairly well-described in the field and the manuscript is wellwritten.
Thank you for your positive comments on our work.
Weaknesses
One thing that is unclear is what is new with this work. If the main finding is that the differences are in the early stages of endocytosis, then one wonders if that should be tested experimentally. Also, the role of clathrin assembly and adhesion are treated as mechanical equilibrium but perhaps the process should not be described as equilibria but rather a time-dependent process. Ultimately, there are so many models that address this question that without direct experimental comparison, it's hard to place value on the model prediction.
Thank you for your insightful questions. We fully agree that distinguishing between the two models should ultimately be guided by experimental tests. This is precisely the motivation for including Fig. 5 in our manuscript, where we compare our theoretical predictions with experimental data. In the middle panel of Fig. 5, we observe that the predicted tip radius as a function of 𝜓<sub>𝑚𝑎𝑥</sub> from the constant curvature model (magenta curve) deviates significantly from both the experimental data points and the rolling median, highlighting the inconsistency of this model with the data.
Regarding our treatment of clathrin assembly and membrane adhesion as mechanical equilibrium processes, our reasoning is based on a timescale separation argument. Clathrin assembly typically occurs over approximately 1 minute. In contrast, the characteristic relaxation time for a lipid membrane to reach mechanical equilibrium is given by 
, where 𝜇∼5 × 10<sup>-9</sup> 𝑁𝑠𝑚<sup>-1</sup> is the membrane viscosity, 𝑅<sub>0</sub> =50𝑛𝑚 is the vesicle size, 𝜅=20 𝑘<sub>𝐵</sub>𝑇 is the bending rigidity. This yields a relaxation time of 𝜏≈1.5 × 10<sup>−4</sup>𝑠, which is several orders of magnitude shorter than the timescale of clathrin assembly. Therefore, it is reasonable to treat the membrane shape as being in mechanical equilibrium throughout the assembly process.
We believe the value of our model lies in the following key novelties:
(1) Model novelty: We introduce an energy term associated with curvature generation, a contribution that is typically neglected in previous models.
(2) Methodological novelty: We perform a quantitative comparison between theoretical predictions and experimental data, whereas most earlier studies rely on qualitative comparisons.
(3) Results novelty: Our quantitative analysis enables us to unambiguously exclude the constant curvature hypothesis based on time-independent electron microscopy data.
In the revised manuscript (line 141), we have added a statement about why we treat the clathrin assembly as in mechanical equilibrium.
While an attempt is made to do so with prior published EM images, there is excessive uncertainty in both the data itself as is usually the case but also in the methods that are used to symmetrize the data. This reviewer wonders about any goodness of fit when such uncertainty is taken into account.
Author response: We thank the reviewer for raising this important point. We agree that there is uncertainty in the experimental data. Our decision to symmetrize the data is based on the following considerations:
(1) The experimental data provide a one-dimensional membrane profile corresponding to a cross-sectional view. To reconstruct the full two-dimensional membrane surface, we must assume rotational symmetry.
(2)In addition to symmetrization, we also average membrane profiles within a certain range of 𝜓<sub>𝑚𝑎𝑥</sub> values (see Fig. 5d). This averaging helps reduce the uncertainty (due to biological and experimental variability) inherent to individual measurements.
(3)To further address the noise in the experimental data, we compare our theoretical predictions not only with individual data points but also with a rolling median, which provides a smoothed representation of the experimental trends.
These steps are taken to ensure a more robust and meaningful comparison between theory and experiments.
In the revised manuscript (line 338), we have explained why we have to symmetrize the data:
“To facilitate comparison between the axisymmetric membrane shapes predicted by the model and the non-axisymmetric profiles obtained from electron microscopy, we apply a symmetrization procedure to the experimental data, which consist of one-dimensional membrane profiles extracted from cross-sectional views, as detailed in Appendix 3 (see also Appendix 3--Fig. 1).”
Reviewer #2:
Summary
In this manuscript, the authors employ theoretical analysis of an elastic membrane model to explore membrane vesiculation pathways in clathrin-mediated endocytosis. A complete understanding of clathrin-mediated endocytosis requires detailed insight into the process of membrane remodeling, as the underlying mechanisms of membrane shape transformation remain controversial, particularly regarding membrane curvature generation. The authors compare constant area and constant membrane curvature as key scenarios by which clathrins induce membrane wrapping around the cargo to accomplish endocytosis. First, they characterize the geometrical aspects of the two scenarios and highlight their differences by imposing coating area and membrane spontaneous curvature. They then examine the energetics of the process to understand the driving mechanisms behind membrane shape transformations in each model. In the latter part, they introduce two energy terms: clathrin assembly or binding energy, and curvature generation energy, with two distinct approaches for the latter. Finally, they identify the energetically favorable pathway in the combined scenario and compare their results with experiments, showing that the constant-area pathway better fits the experimental data.
Thank you for your clear and comprehensive summary of our work.
Strengths
The manuscript is well-written, well-organized, and presents the details of the theoretical analysis with sufficient clarity. The calculations are valid, and the elastic membrane model is an appropriate choice for addressing the differences between the constant curvature and constant area models.
The authors' approach of distinguishing two distinct free energy terms-clathrin assembly and curvature generation-and then combining them to identify the favorable pathway is both innovative and effective in addressing the problem.
Notably, their identification of the energetically favorable pathways, and how these pathways either lead to full endocytosis or fail to proceed due to insufficient energetic drives, is particularly insightful.
Thank you for your positive remarks regarding the innovative aspects of our work.
Weaknesses and Recommendations
Weakness: Membrane remodeling in cellular processes is typically studied in either a constant area or constant tension ensemble. While total membrane area is preserved in the constant area ensemble, membrane area varies in the constant tension ensemble. In this manuscript, the authors use the constant tension ensemble with a fixed membrane tension, σe. However, they also use a constant area scenario, where 'area' refers to the surface area of the clathrin-coated membrane segment. This distinction between the constant membrane area ensemble and the constant area of the coated membrane segment may cause confusion.
Recommendation: I suggest the authors clarify this by clearly distinguishing between the two concepts by discussing the constant tension ensemble employed in their theoretical analysis.
Thank you for raising this question.
In the revised manuscript (line 136), we have added a sentence, emphasizing the implication of the term “constant area model”:
“We emphasize that the constant area model refers to the assumption that the clathrin-coated area 𝑎<sub>0</sub> remains fixed. Meanwhile, the membrane tension 𝜎<sub>𝑒</sub> at the base is held constant, allowing the total membrane area 𝐴𝐴 to vary in response to deformations induced by the clathrin coat.”
Weakness: As mentioned earlier, the theoretical analysis is performed in the constant membrane tension ensemble at a fixed membrane tension. The total free energy E_tot of the system consists of membrane bending energy E_b and tensile energy E_t, which depends on membrane tension, σe. Although the authors mention the importance of both E_b and E_t, they do not present their individual contributions to the total energy changes. Comparing these contributions would enable readers to cross-check the results with existing literature, which primarily focuses on the role of membrane bending rigidity and membrane tension.
Recommendation: While a detailed discussion of how membrane tension affects their results may fall outside the scope of this manuscript, I suggest the authors at least discuss the total membrane area variation and the contribution of tensile energy E_t for the singular value of membrane tension used in their analysis.
Thank you for the insightful suggestion. In the revised manuscript (line 916), we have added Appendix 6 and a supplementary figure to compare the bending energy 𝐸<sub>𝑏</sub> and the tension energy 𝐸<sub>𝑡</sub>. Our analysis shows that both energy components exhibit an energy barrier between the flat and vesiculated membrane states, with the tension energy contributing more significantly than the bending energy.
In the revised manuscript (line 151), we have also added one paragraph explaining why we set the dimensionless tension 
. This choice is motivated by our use of the characteristic length 
as the length scale, and 
as the energy scale. In this way, the dimensionless tension energy is written as
Where 
is the dimensionless area.
Weakness: The authors introduce two different models, (1,1) and (1,2), for generating membrane curvature. Model 1 assumes a constant curvature growth, corresponding to linear curvature growth, while Model 2 relates curvature growth to its current value, resembling exponential curvature growth. Although both models make physical sense in general, I am concerned that Model 2 may lead to artificial membrane bending at high curvatures. Normally, for intermediate bending, ψ > 90, the bending process is energetically downhill and thus proceeds rapidly. The bending process is energetically downhill and thus proceeds rapidly. However, Model 2's assumption would accelerate curvature growth even further. This is reflected in the endocytic pathways represented by the green curves in the two rightmost panels of Fig. 4a, where the energy steeply increases at large ψ. I believe a more realistic version of Model 2 would require a saturation mechanism to limit curvature growth at high curvatures.
Recommendation 1: I suggest the authors discuss this point and highlight the pros and cons of Model 2. Specifically, addressing the potential issue of artificial membrane bending at high curvatures and considering the need for a saturation mechanism to limit excessive curvature growth. A discussion on how Model 2 compares to Model 1 in terms of physical relevance, especially in the context of high curvature scenarios, would provide valuable insights for the reader.
Thank you for raising the question of excessive curvature growth in our models and the constructive suggestion of introducing a saturation mechanism. In the revised manuscript (line 405), following your recommendation, we have added a subsection “Saturation effect at high membrane curvatures” in the discussion to clarify the excessive curvature issue and a possible way to introduce a saturation mechanism:
“Note that our model involves two distinct concepts of curvature growth. The first is the growth of imposed curvature — referred to here as intrinsic curvature and denoted by the parameter 𝑐<sub>0</sub> — which is driven by the reorganization of bonds between clathrin molecules within the coat. The second is the growth of the actual membrane curvature, reflected by the increasing value of 𝜓<sub>𝑚𝑎𝑥</sub>.
The latter process is driven by the former.
Models (1,1) and (1,2) incorporate energy terms (Equation 6) that promote the increase of intrinsic curvature 𝑐<sub>0</sub>, which in turn drives the membrane to adopt a more curved shape (increasing 𝜓<sub>𝑚𝑎𝑥</sub>). In the absence of these energy contributions, the system faces an energy barrier separating a weakly curved membrane state (low 𝜓<sub>𝑚𝑎𝑥</sub>) from a highly curved state (high 𝜓<sub>𝑚𝑎𝑥</sub>). This barrier can be observed, for example, in the red curves of Figure 3(a–c) and in Appendix 6—Figure 1. As a result, membrane bending cannot proceed spontaneously and requires additional energy input from clathrin assembly.
The energy terms described in Equation 6 serve to eliminate this energy barrier by lowering the energy difference between the uphill and downhill regions of the energy landscape. However, these same terms also steepen the downhill slope, which may lead to overly aggressive curvature growth.
To mitigate this effect, one could introduce a saturation-like energy term of the form:
where 𝑐<sub>𝑠</sub> represents a saturation curvature. Importantly, adding such a term would not alter the conclusions of our study, since the energy landscape already favors high membrane curvature (i.e., it is downward sloping) even without the additional energy terms. “
Recommendation 2: Referring to the previous point, the green curves in the two rightmost panels of Fig. 4a seem to reflect a comparison between slow and fast bending regimes. The initial slow vesiculation (with small curvature growth) in the left half of the green curves is followed by much more rapid curvature growth beyond a certain threshold. A similar behavior is observed in Model 1, as shown by the green curves in the two rightmost panels of Fig. 4b. I believe this transition between slow and fast bending warrants a brief discussion in the manuscript, as it could provide further insight into the dynamic nature of vesiculation.
Thank you for your constructive suggestion regarding the transition between slow and fast membrane bending. As you pointed out, in both Fig. 4a (model (1,2)) and Fig. 4b (model (1,1)), the green curves tend to extend vertically at the late stage. This suggests a significant increase in 𝑐<sub>0</sub> on the free energy landscape. However, we remain cautious about directly interpreting this vertical trend as indicative of fast endocytic dynamics, since our model is purely energetic and does not explicitly incorporate kinetic details. Meanwhile, we agree with your observation that the steep decrease in free energy along the green curve could correspond to an acceleration in dynamics. To address this point, we have added a paragraph in the revised manuscript (in Subsection “Cooperativity in the curvature generation process”) discussing this potential transition and its consistency with experimental observations (line 395):
“Furthermore, although our model is purely energetic and does not explicitly incorporate dynamics, we observe in Figure 3(a) that along the green curve—representing the trajectory predicted by model (1,2)—the total free energy (𝐸<sub>𝑡𝑜𝑡</sub>) exhibits a much sharper decrease at the late stage (near the vesiculation line) compared to the early stage (near the origin). This suggests a transition from slow to fast dynamics during endocytosis. Such a transition is consistent with experimental observations, where significantly fewer number of images with large 𝜓<sub>𝑚𝑎𝑥</sub> are captured compared to those with small 𝜓<sub>𝑚𝑎𝑥</sub> (Mund et al., 2023).”
The geometrical properties of both the constant-area and constant-curvature scenarios, as well depicted in Fig. 1, are somewhat straightforward. I wonder what additional value is presented in Fig. 2. Specifically, the authors solve differential shape equations to show how Rt and Rcoat vary with the angle ψ, but this behavior seems predictable from the simple schematics in Fig. 1. Using a more complex model for an intuitively understandable process may introduce counter-intuitive results and unnecessary complications, as seen with the constant-curvature model where Rt varies (the tip radius is not constant, as noted in the text) despite being assumed constant. One could easily assume a constant-curvature model and plot Rt versus ψ. I wonder What is the added value of solving shape equations to measure geometrical properties, compared to a simpler schematic approach (without solving shape equations) similar to what they do in App. 5 for the ratio of the Rt at ψ=30 and 150.
Thank you for raising this important question. While simple and intuitive theoretical models are indeed convenient to use, their validity must be carefully assessed. The approximate model becomes inaccurate when the clathrin shell significantly deviates from its intrinsic shape, namely a spherical cap characterized by intrinsic curvature 𝑐<sub>0</sub>. As shown in the insets of Fig. 2b and 2c (red line and black points), our comparison between the simplified model and the full model demonstrates that the simple model provides a good approximation under the constant-area constraint. However, it performs poorly under the constant-curvature constraint, and the deviation between the full model and the simplified model becomes more pronounced as 𝑐<sub>0</sub> increases.
In the revised manuscript, we have added a sentence emphasizing the discrepancy between the exact calculation with the idealized picture for the constant curvature model (line 181):
“For the constant-curvature model, the ratio remains close to 1 only at small values of 𝑐<sub>0</sub>, as expected from the schematic representation of the model in Figure 1. However, as 𝑐<sub>0</sub> increases, the deviation from this idealized picture becomes increasingly pronounced.”
Recommendation: The clathrin-mediated endocytosis aims at wrapping cellular cargos such as viruses which are typically spherical objects which perfectly match the constant-curvature scenario. In this context, wrapping nanoparticles by vesicles resembles constant-curvature membrane bending in endocytosis. In particular analogous shape transitions and energy barriers have been reported (similar to Fig.3 of the manuscript) using similar theoretical frameworks by varying membrane particle binding energy acting against membrane bending:
DOI: 10.1021/la063522m
DOI: 10.1039/C5SM01793A
I think a short comparison to particle wrapping by vesicles is warranted.
Thank you for your constructive suggestion to compare our model with particle wrapping. In the revised manuscript (line 475), we have added a subsection “Comparison with particle wrapping” in the discussion:
“The purpose of the clathrin-mediated endocytosis studied in our work is the recycling of membrane and membrane-protein, and the cellular uptake of small molecules from the environment — molecules that are sufficiently small to bind to the membrane or be encapsulated within a vesicle. In contrast, the uptake of larger particles typically involves membrane wrapping driven by adhesion between the membrane and the particle, a process that has also been studied previously (Góźdź, 2007; Bahrami et al., 2016). In our model, membrane bending is driven by clathrin assembly, which induces curvature. In particle wrapping, by comparison, the driving force is the adhesion between the membrane and a rigid particle. In the absence of adhesion, wrapping increases both bending and tension energies, creating an energy barrier that separates the flat membrane state from the fully wrapped state. This barrier can hinder complete wrapping, resulting in partial or no engulfment of the particle. Only when the adhesion energy is sufficiently strong can the process proceed to full wrapping. In this context, adhesion plays a role analogous to curvature generation in our model, as both serve to overcome the energy barrier. If the particle is spherical, it imposes a constant-curvature pathway during wrapping. However, the role of clathrin molecules in this process remains unclear and will be the subject of future investigation.”
Minor points:
Line 20, abstract, "....a continuum spectrum ..." reads better.
Line 46 "...clathrin results in the formation of pentagons ...." seems Ito be grammatically correct.
Line 106, proper citation of the relevant literature is warranted here.
Line 111, the authors compare features (plural) between experiments and calculations. I would write "....compare geometric features calculated by theory with those ....".
Line 124, "Here, we choose a ..." (with comma after Here).
Line 134, "The membrane tension \sigma_e and bending rigidity \kappa define a ...."
Line 295, "....tip radius, and invagination ...." (with comma before and).
Line 337, "abortive tips, and ..." (with comma before and).
We thank you for your thorough review of our manuscript and have corrected all the issues raised.
Some writers explicitly identify and summarize a view they are responding to at the outset of their text and then return to it frequently as their text unfolds. Some refer only obliquely to a view that is motivating them, assuming that readers will be able to reconstruct that view on their own.
In such terms I expected, summary and identifications can respond to our text, and some case, readers can be able to review a different topic on their own statement.
Without such deep, attentive listening, any critique you make will be superficial and decidedly uncritical. It will be a critique that says more about you than about the writer or idea you're supposedly responding to.
Critique was pronounced that is official, but it is unclear that you will be said to respond to your writer without listening deeply.
Sometimes it is difficult to figure out the views that writers are responding to, not because these writers do not identify those views, but because their language and the concepts they are dealing with are particularly challenging.
These views can be important, but difficult, however, to those who responds the language, the concepts of the text adheres their consideration.
Another challenge in reading for the conversation is that writers sometimes build their arguments by responding to a lack of discussion.
With the lack of discussion, readers cannot understand to build arguments that can never read through conversation.
If you read the passage this way, however, you would be mistaken. Draut is not questioning whether a college degree has become the "ticket to middle-class security", but whether most Americans can obtain that ticket, whether college is within the financial reach of most American families.
In one big mistake to read the passage in the wrong way, it will be denied. The author is questioning about whether most Americans obtain the ticket rather than ticket to middle-class security in a word.
Readers need to be alert for any changes in voice that a writer might make, since instead of using explicit road-mapping phrases like "although many believe", authors may summarize the view that they want to engage with and indicate only subtly that it is not their own.
According to my opinion, "although many believe" is too clear for any observations for road-mapping phrase.
In other words, imagine an ongoing, multisided conversation in which all participants are trying to persuade others to agree or at least to take their positions seriously.
I imagine the multisided statement is to persuade the agreement and make an offer for ongoing request that they're up for. It can be a serious claim what I have known.
The results were often striking. The discussions that followed tended to be far livelier and to draw in a greater number of students. We were still asking students to look for the main argument, but we were now asking them to see that argument as a response to some other argument that provoked it, gave it a reason for being, and helped all of us see why we should care about it.
It is unclear that why did the provocation of this argument responds to any students, but the results are often impacted in undisclosed measures.
For a long time we didn't worry much about these halting discussions, justifying them to ourselves as the predictable result of assigning difficult, challenging readings.
At the same time, a challenge can be resourceful and important to justify the result to our stratagem.
The discussion that resulted was often halting, as our students struggled to get a handle on the argument, but eventually, after some awkward silences, the class would come up with something we could all agree was an accurate summary of the author's main thesis.
As I assure the discussion, this thesis can be often essential for a summary, according to a author's central thesis task.
We call for a Phase VI to address colonial legacies in the field and ongoing colonialism globally. Guided by turns in other disciplines, we argue that ethnobiologists' work needs to go beyond adhering to and satisfying ethical guidelines and be actively decolonizing
we need to go beyond ethical protocols and into active decolonization
‘If we can only receive by giving, then we must begin to give’ (Phatthanaphraiwan & Greene, 2025). The ecological and social challenges we collectively, albeit differentially, confront today in the form of climate change, biodiversity decline and social injustice were created over hundreds of years of appropriation, structural violence on Indigenous, Afro-descendant and other frontline communities globally (Correia, 2024). To guide sustainability management in more ethical and equitable ways, we need to address these relations by recognising the existent inherited prejudice, power asymmetry and hierarchical status. Hence, it is necessary to imagine that rebuilding healthy relations will also take time.
colonial past implications for reciprocity practice
While the role of IP and LC is increasingly recognised in academic and international policy arenas, a lack of recognition by national governments and some academic fora persists (McElwee et al., 2020; Tormos-Aponte, 2021). Lack of awareness is fourfold: epistemological (different knowledge production and validation methods), ontological (different assumptions of reality), ethical (different moral responsibilities between human and non-human beings) and political (different positions of power to enforce perspectives in collaborative practices, Ludwig & El-Hani, 2020).
the political problem. How to go beyond academic reciprocity and into social reciprocity.
This relationship between the human and the non-human has been conceptualised as ‘kincentric ecology’ (Bird-David, 1999; Salmón, 2000). From this perspective, plants, animals and fungi are not seen as food or material sources, but rather as vital participants in a relational web that connects humans and animals to spirits, ancestors and other beings. For example, in Baka knowledge systems, individuals of each type of being (‘species’) only exist through their interactions with many other beings and their shared environment, which they all constantly change (Hoyte & Mangombe, 2024).
Similar to the concept of agential realism!
In many cultures, reciprocity is understood as an interpersonal and communal responsibility to ensure the welfare of the community and the social-ecological system as a whole, where ancestors and those yet to be born are equally considered (Fernández-Llamazares & Virtanen, 2020).
key term here: "ancestors". We need to think of reciprocity in a way that account for the years of exploitation of our ancestors.
By placing unparasitized T. varians into cold chambers we induced the production
This should be in the RESULTS!
we identified the origin of the behavior evoked by the parasitic organism.
Didn't identify HOW they evoked this behavior though
The obtained results enabled us to identify the origin of the evoked behaviors.
Seems like a very bold statement
parasitoids attacking spiders offers an ideal system for identifying the origin of such induced behavior, because the evoked behavior is simple
Would hesitate to call this change simple
Brygos was an ancient Greek potter, active in Athens between 490 and 470 BC. He is known as a producer of excellent drinking cups. About 200 of his pieces are known. The workshop of Brygos employed a red-figure vase painter who is conventionally called the Brygos Painter. The Brygos Painter is one of the most famous vase painters of his time. His work is characterised by its high quality and realistic depictions. The workshop of Brygos also employed the Briseis Painter, among others.
Cite more, This bibliography is not substantial. once again look at the novet catalogue
Dionysos and satyrs on a vase made by Brygos and painted by the Brygos Painter, ca. 480 BC (Cabinet des Médailles, Paris)
Perhaps comment on what this is, analyze it maybe
Brygos
a lot to talk about, maybe do some more research on "Brygos Painter". This is a very short article, try finding more information on the Novet catalogue
Configure AG-Grid Locale (Automatic)Set the AG-Grid locale in your Task Management configuration to match your application language (e.g., “en-US”, “ro-RO”, “de-DE”). AG-Grid will automatically translate all 300+ grid component keys including filters, menus, pagination, and data types
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"Today we're going to open up ChatGPT for developers to build real apps inside of ChatGPT...with the Apps SDK, your apps can reach hundreds of millions of ChatGPT users" — Sam Altman
API Parity with Consumer Products: - GPT-5 Pro - flagship model now available via API - Sora 2 & Sora 2 Pro - video generation models released - Distilled models: - gpt-realtime-mini (70% cheaper) - gpt-audio-mini - gpt-image-1-mini (80% cheaper)
"OpenAI adopted [MCP] so quickly, much less to now be the first to turn it into the basis of a full app store platform"
"they really treat it as an open protocol...they are not viewing it as this thing that is specific to Anthropic"
"launched agent kit today, full set of solutions to build, deploy and optimize agents"
"allowing you to use [traces] in the evals product and be able to grade it...over the entirety of what it's supposed to be doing"
"ChatKit itself is like an embeddable iframe...if you are using ChatKit and we come up with new...a new model that reasons in a different way...you don't actually need to rebuild"
"we end up storing quite a bit of state...we can actually end up doing a lot more creative stuff...when you're chatting with ChatGPT"
"how do you even evaluate a 20 minute task correctly? And it's like, it's a really hard problem"
"two years ago people were like, oh, at some point...prompting is going to be dead...And if anything, it is like become more and more entrenched"
"it is like pretty difficult for us to manage all of these different [fine-tuning] snapshots...if there is a way to...do this like zero gradient like optimization via prompts...I'm all for it"
"on their way to work, they're like kicking off like five Codex tasks because the bus takes 30 minutes...and it kind of helps you orient yourself for the day"
"We haven't had one [major outage] that bad since...We think we've got reliability in a spot where we're comfortable kind of putting this out there"
I mean the public performance required of those subject to elabo-rate and systematic forms of social subordination: the worker to the boss, thetenant or sharecropper to the landlord, the serf to the lord, the slave to themaster, the untouchable to the Brahmin, a member of a subject race to one ofthe dominant race. With rare, but significant, exceptions the public perfor-mance of the subordinate will, out of prudence, fear, and the desire to curryfavor, be shaped to appeal to the expectations of the powerful. I shall use theterm public transcript as a shorthand way of describing the open interactionbetween subordinates and those who dominate. 1 The public transcript, whereit is not positively misleading, is unlikely to tell the whole story about powerrelations. It is frequently in the interest of both parties to tacitly conspire inmisrepresentation. The oral history of a French tenant farmer, Old Tiennon,covering much of the nineteenth century is filled with accounts of a prudentand misleading deference: "When he [the landlord who had dismissed hisfather] crossed from Le Craux, going to Meillers, he would stop and speak tome and I forced myself to appear amiable, in spite of the contempt I felt forhim.
he’s drawing a line between everyday politeness and historically imposed performance. What begins as etiquette becomes survival strategy under domination. This “public transcript,” as he calls it, isn’t just a set of social cues it’s a choreography shaped by fear, prudence, and sometimes sheer exhaustion.
And Wald-Tinan’s anecdote is such a vivid example smiling at the landlord who dismissed your father, not because you respect him, but because appearing “amiable” protects you. It’s a performance built on unequal power, and it says so much about the psychic toll of domination.
George Eliot may not haveexaggerated in claiming that "there is no action possible without a littleacting."I
there is no action possible without a little acting that’s powerful. It blurs the line between resistance and complicity, showing that even politeness can be strategic.
Invocation of the Muse
The legendary king of Mycenae and leader of the Greek army in the Trojan War of Homer's Illiad. Agamemnon is a great warrior but also a selfish ruler who famously upset his invincible champion Achilles, a feud that prolonged the war and suffering of his men. https://www.worldhistory.org/Agamemnon_(Person)/
eLife Assessment
This important manuscript provides compelling evidence that BK and CaV1.3 channels can co-localize as ensembles early in the biosynthetic pathway, including in the ER and Golgi. The findings, supported by a range of imaging and proximity assays, offer insights into channel organization in both heterologous and endogenous systems. While the data broadly support the central claims, mechanistic aspects remain unresolved, particularly regarding the determinants of mRNA co-localization, the temporal dynamics of ensemble trafficking, and the physiological implications of pre-assembly for channel function at the plasma membrane.
Reviewer #1 (Public review):
Summary:
This manuscript by Pournejati et al investigates how BK (big potassium) channels and CaV1.3 (a subtype of voltage-gated calcium channels) become functionally coupled by exploring whether their ensembles form early-during synthesis and intracellular trafficking-rather than only after insertion into the plasma membrane. To this end, the authors use the PLA technique to assess the formation of ion channel associations in the different compartments (ER, Golgi or PM), single-molecule RNA in situ hybridization (RNAscope), and super-resolution microscopy.
Strengths:
The manuscript is well written and addresses an interesting question, combining a range of imaging techniques. The findings are generally well-presented and offer important insights into the spatial organization of ion channel complexes, both in heterologous and endogenous systems.
Weaknesses:
The authors have improved their manuscript after revisions, and some previous concerns have been addressed. Still, the main concern about this work is that the current experiments do not quantitatively or mechanistically link the ensembles observed intracellularly (in the endoplasmic reticulum (ER) or Golgi) to those found at the plasma membrane (PM). As a result, it is difficult to fully integrate the findings into a coherent model of trafficking. Specifically, the manuscript does not address what proportion of ensembles detected at the PM originated in the ER. Without data on the turnover or half-life of these ensembles at the PM, it remains unclear how many persist through trafficking versus forming de novo at the membrane. The authors report the percentage of PLA-positive ensembles localized to various compartments, but this only reflects the distribution of pre-formed ensembles. What remains unknown is the proportion of total BK and CaV1.3 channels (not just those in ensembles) that are engaged in these complexes within each compartment. Without this, it is difficult to determine whether ensembles form in the ER and are then trafficked to the PM, or if independent ensemble formation also occurs at the membrane. To support the model of intracellular assembly followed by coordinated trafficking, it would be important to quantify the fraction of the total channel population that exists as ensembles in each compartment. A comparable ensemble-to-total ratio across ER and PM would strengthen the argument for directed trafficking of pre-assembled channel complexes.
Reviewer #2 (Public review):
Summary:
The co-localization of large conductance calcium- and voltage activated potassium (BK) channels with voltage-gated calcium channels (CaV) at the plasma membrane is important for the functional role of these channels in controlling cell excitability and physiology in a variety of systems.
An important question in the field is where and how do BK and CaV channels assemble as 'ensembles' to allow this coordinated regulation - is this through preassembly early in the biosynthetic pathway, during trafficking to the cell surface or once channels are integrated into the plasma membrane. These questions also have broader implications for assembly of other ion channel complexes.
Using an imaging based approach, this paper addresses the spatial distribution of BK-CaV ensembles using both overexpression strategies in tsa201 and INS-1 cells and analysis of endogenous channels in INS-1 cells using proximity ligation and superesolution approaches. In addition, the authors analyse the spatial distribution of mRNAs encoding BK and Cav1.3.
The key conclusion of the paper that BK and CaV1.3 are co-localised as ensembles intracellularly in the ER and Golgi is well supported by the evidence. However, whether they are preferentially co-translated at the ER, requires further work. Moreover, whether intracellular pre-assembly of BK-CaV complexes is the major mechanism for functional complexes at the plasma membrane in these models requires more definitive evidence including both refinement of analysis of current data as well as potentially additional experiments.
Strengths & Weaknesses
(1) Using proximity ligation assays of overexpressed BK and CaV1.3 in tsa201 and INS-1 cells the authors provide strong evidence that BK and CaV can exist as ensembles (ie channels within 40 nm) at both the plasma membrane and intracellular membranes, including ER and Golgi. They also provide evidence for endogenous ensemble assembly at the Golgi in INS-1 cells and it would have been useful to determine if endogenous complexes are also observe in the ER of INS-1 cells. There are some useful controls but the specificity of ensemble formation would be better determined using other transmembrane proteins rather than peripheral proteins (eg Golgi 58K).
(2) Ensemble assembly was also analysed using super-resolution (dSTORM) imaging in INS-1 cells. In these cells only 7.5% of BK and CaV particles (endogenous?) co-localise that was only marginally above chance based on scrambled images. More detailed quantification and validation of potential 'ensembles' needs to be made for example by exploring nearest neighbour characteristics (but see point 4 below) to define proportion of ensembles versus clusters of BK or Cav1.3 channels alone etc. For example, it is mentioned that a distribution of distances between BK and Cav is seen but data are not shown.
(3) The evidence that the intracellular ensemble formation is in large part driven by co-translation, based on co-localisation of mRNAs using RNAscope, requires additional critical controls and analysis. The authors now include data of co-localised BK protein that is suggestive but does not show co-translation. Secondly, while they have improved the description of some controls mRNA co-localisation needs to be measured in both directions (eg BK - SCN9A as well as SCN9A to BK) especially if the mRNAs are expressed at very different levels. The relative expression levels need to be clearly defined in the paper. Authors also use a randomized image of BK mRNA to show specificity of co-localisation with Cav1.3 mRNA, however the mRNA distribution would not be expected to be random across the cell but constrained by ER morphology if co-translated so using ER labelling as a mask would be useful?
(4) The authors attempt to define if plasma membrane assemblies of BK and CaV occur soon after synthesis. However, because the expression of BK and CaV occur at different times after transient transfection of plasmids more definitive experiments are required. For example, using inducible constructs to allow precise and synchronised timing of transcription. This would also provide critical evidence that co-assembly occurs very early in synthesis pathways - ie detecting complexes at ER before any complexes at Golgi or plasma membrane.
(5) While the authors have improved the definition of hetero-clusters etc it is still not clear in superesolution analysis, how they separate a BK tetramer from a cluster of BK tetramers with the monoclonal antibody employed ie each BK channel will have 4 binding sites (4 subunits in tetramer) whereas Cav1.3 has one binding site per channel. Thus, how do authors discriminate between a single BK tetramer (molecular cluster) with potential 4 antibodies bound compared to a cluster of 4 independent BK channels.
(6) The post-hoc tests used for one way ANOVA and ANOVA statistics need to be defined throughout
Reviewer #3 (Public review):
Summary:
The authors present a clearly written and beautifully presented piece of work demonstrating clear evidence to support the idea that BK channels and Cav1.3 channels can co-assemble prior to their assertion in the plasma membrane.
Strengths:
The experimental records shown back up their hypotheses and the authors are to be congratulated for the large number of control experiments shown in the ms.
Author response:
The following is the authors’ response to the original reviews.
Recommendations for the Authors:
(1) Clarify Mechanistic Interpretations
(a) Provide stronger evidence or a more cautious interpretation regarding whether intracellular BK-CaV1.3 ensembles are precursors to plasma membrane complexes.
This is an important point. We adjusted the interpretation regarding intracellular BKCa<sub>V</sub>1.3 hetero-clusters as precursors to plasma membrane complexes to reflect a more cautious stance, acknowledging the limitations of available data. We added the following to the manuscript.
“Our findings suggest that BK and Ca<sub>V</sub>1.3 channels begin assembling intracellularly before reaching the plasma membrane, shaping their spatial organization and potentially facilitating functional coupling. While this suggests a coordinated process that may contribute to functional coupling, further investigation is needed to determine the extent to which these hetero-clusters persist upon membrane insertion.”
(b) Discuss the limitations of current data in establishing the proportion of intracellular complexes that persist on the cell surface.
We appreciate the suggestion. We expanded the discussion to address the limitations of current data in determining the proportion of intracellular complexes that persist on the cell surface. We added the following to the manuscript.
“Our findings highlight the intracellular assembly of BK-Ca<sub>V</sub>1.3 hetero-clusters, though limitations in resolution and organelle-specific analysis prevent precise quantification of the proportion of intracellular complexes that ultimately persist on the cell surface. While our data confirms that hetero-clusters form before reaching the plasma membrane, it remains unclear whether all intracellular hetero-clusters transition intact to the membrane or undergo rearrangement or disassembly upon insertion. Future studies utilizing live cell tracking and high resolution imaging will be valuable in elucidating the fate and stability of these complexes after membrane insertion.”
(2) Refine mRNA Co-localization Analysis
(a) Include appropriate controls using additional transmembrane mRNAs to better assess the specificity of BK and CaV1.3 mRNA co-localization.
We agree with the reviewers that these controls are essential. We explain better the controls used to address this concern. We added the following to the manuscript.
“To explore the origins of the initial association, we hypothesized that the two proteins are translated near each other, which could be detected as the colocalization of their mRNAs (Figure 5A and B). The experiment was designed to detect single mRNA molecules from INS-1 cells in culture. We performed multiplex in situ hybridization experiments using an RNAScope fluorescence detection kit to be able to image three mRNAs simultaneously in the same cell and acquired the images in a confocal microscope with high resolution. To rigorously assess the specificity of this potential mRNA-level organization, we used multiple internal controls. GAPDH mRNA, a highly expressed housekeeping gene with no known spatial coordination with channel mRNAs, served as a baseline control for nonspecific colocalization due to transcript abundance. To evaluate whether the spatial proximity between BK mRNA (KCNMA1) and Ca<sub>V</sub>1.3 mRNA (CACNA1D) was unique to functionally coupled channels, we also tested for Na<sup>V</sup>1.7 mRNA (SCN9A), a transmembrane sodium channel expressed in INS-1 cells but not functionally associated with BK. This allowed us to determine whether the observed colocalization reflected a specific biological relationship rather than shared expression context. Finally, to test whether this proximity might extend to other calcium sources relevant to BK activation, we probed the mRNA of ryanodine receptor 2 (RyR2), another Ca<sup>2+</sup> channel known to interact structurally with BK channels [32]. Together, these controls were chosen to distinguish specific mRNA colocalization patterns from random spatial proximity, shared subcellular distribution, or gene expression level artifacts.”
(b) Quantify mRNA co-localization in both directions (e.g., BK with CaV1.3 and vice versa) and account for differences in expression levels.
We thank the reviewer for this suggestion. We chose to quantify mRNA co-localization in the direction most relevant to the formation of functionally coupled hetero-clusters, namely, the proximity of BK (KCNMA1) mRNA to Ca<sub>V</sub>1.3 (CACNA1D) mRNA. Since BK channel activation depends on calcium influx provided by nearby Ca<sub>V</sub>1.3 channels, this directional analysis more directly informs the hypothesis of spatially coordinated translation and channel assembly. To address potential confounding effects of transcript abundance, we implemented a scrambled control approach in which the spatial coordinates of KCNMA1 mRNAs were randomized while preserving transcript count. This control resulted in significantly lower colocalization with CACNA1D mRNA, indicating that the observed proximity reflects a specific spatial association rather than expressiondriven overlap. We also assessed colocalization of CACNA1D with both KCNMA1, GAPDH mRNAs and SCN9 (NaV1.7); as you can see in the graph below these data support t the same conclusion but were not included in the manuscript.
Author response image 1.
(c) Consider using ER labeling as a spatial reference when analyzing mRNA localization
We thank the reviewers for this suggestion. Rather than using ER labeling as a spatial reference, we assess BK and CaV1.3 mRNA localization using fluorescence in situ hybridization (smFISH) alongside BK protein immunostaining. This approach directly identifies BK-associated translation sites, ensuring that observed mRNA localization corresponds to active BK synthesis rather than general ER association. By evaluating BK protein alongside its mRNA, we provide a more functionally relevant measure of spatial organization, allowing us to assess whether BK is synthesized in proximity to CaV1.3 mRNA within micro-translational complexes. The results added to the manuscript is as follows.
“To further investigate whether KCNMA1 and CACNA1D are localized in regions of active translation (Figure 7A), we performed RNAScope targeting KCNMA1 and CACNA1D alongside immunostaining for BK protein. This strategy enabled us to visualize transcript-protein colocalization in INS-1 cells with subcellular resolution. By directly evaluating sites of active BK translation, we aimed to determine whether newly synthesized BK protein colocalized with CACNA1D mRNA signals (Figure 7A). Confocal imaging revealed distinct micro-translational complex where KCNMA1 mRNA puncta overlapped with BK protein signals and were located adjacent to CACNA1D mRNA (Figure 7B). Quantitative analysis showed that 71 ± 3% of all KCNMA1 colocalized with BK protein signal which means that they are in active translation. Interestingly, 69 ± 3% of the KCNMA1 in active translation colocalized with CACNA1D (Figure 7C), supporting the existence of functional micro-translational complexes between BK and Ca<sub>V</sub>1.3 channels.”
(3) Improve Terminology and Definitions
(a) Clarify and consistently use terms like "ensemble," "cluster," and "complex," especially in quantitative analyses.
We agree with the reviewers, and we clarified terminology such as 'ensemble,' 'cluster,' and 'complex' and used them consistently throughout the manuscript, particularly in quantitative analyses, to enhance precision and avoid ambiguity.
(b) Consider adopting standard nomenclature (e.g., "hetero-clusters") to avoid ambiguity.
We agree with the reviewers, and we adapted standard nomenclature, such as 'heteroclusters,' in the manuscript to improve clarity and reduce ambiguity.
(4) Enhance Quantitative and Image Analysis
(a) Clearly describe how colocalization and clustering were measured in super-resolution data.
We thank the reviewers for this suggestion. We have modified the Methods section to provide a clearer description of how colocalization and clustering were measured in our super-resolution data. Specifically, we now detail the image processing steps, including binary conversion, channel multiplication for colocalization assessment, and density-based segmentation for clustering analysis. These updates ensure transparency in our approach and improve accessibility for readers, and we added the following to the manuscript.
“Super-resolution imaging:
Direct stochastic optical reconstruction microscopy (dSTORM) images of BK and 1.3 overexpressed in tsA-201 cells were acquired using an ONI Nanoimager microscope equipped with a 100X oil immersion objective (1.4 NA), an XYZ closed-loop piezo 736 stage, and triple emission channels split at 488, 555, and 640 nm. Samples were imaged at 35°C. For singlemolecule localization microscopy, fixed and stained cells were imaged in GLOX imaging buffer containing 10 mM β-mercaptoethylamine (MEA), 0.56 mg/ml glucose oxidase, 34 μg/ml catalase, and 10% w/v glucose in Tris-HCl buffer. Single-molecule localizations were filtered using NImOS software (v.1.18.3, ONI). Localization maps were exported as TIFF images with a pixel size of 5 nm. Maps were further processed in ImageJ (NIH) by thresholding and binarization to isolate labeled structures. To assess colocalization between the signal from two proteins, binary images were multiplied. Particles smaller than 400 nm<sup>2</sup> were excluded from the analysis to reflect the spatial resolution limit of STORM imaging (20 nm) and the average size of BK channels. To examine spatial localization preference, binary images of BK were progressively dilated to 20 nm, 40 nm, 60 nm, 80 nm, 100 nm, and 200 nm to expand their spatial representation. These modified images were then multiplied with the Ca<sub>V</sub>1.3 channel to quantify colocalization and determine BK occupancy at increasing distances from Ca<sub>V</sub>1.3. To ensure consistent comparisons across distance thresholds, data were normalized using the 200 nm measurement as the highest reference value, set to 1.”
(b) Where appropriate, quantify the proportion of total channels involved in ensembles within each compartment.
We thank the reviewers for this comment. However, our method does not allow for direct quantification of the total number of BK and Ca<sub>V</sub>1.3 channels expressed within the ER or ER exit sites, as we rely on proximity-based detection rather than absolute fluorescence intensity measurements of individual channels. Traditional methods for counting total channel populations, such as immunostaining or single-molecule tracking, are not applicable to our approach due to the hetero-clusters formation process. Instead, we focused on the relative proportion of BK and Ca<sub>V</sub>1.3 hetero-clusters within these compartments, as this provides meaningful insights into trafficking dynamics and spatial organization. By assessing where hetero-cluster preferentially localize rather than attempting to count total channel numbers, we can infer whether their assembly occurs before plasma membrane insertion. While this approach does not yield absolute quantification of ER-localized BK and Ca<sub>V</sub>1.3 channels, it remains a robust method for investigating hetero-cluster formation and intracellular trafficking pathways. To reflect this limitation, we added the following to the manuscript.
“Finally, a key limitation of this approach is that we cannot quantify the proportion of total BK or Ca<sub>V</sub>1.3 channels engaged in hetero-clusters within each compartment. The PLA method provides proximity-based detection, which reflects relative localization rather than absolute channel abundance within individual organelles”.
(5) Temper Overstated Claims
(a) Revise language that suggests the findings introduce a "new paradigm," instead emphasizing how this study extends existing models.
We agree with the reviewers, and we have revised the language to avoid implying a 'new paradigm.' The following is the significance statement.
“This work examines the proximity between BK and Ca<sub>V</sub>1.3 molecules at the level of their mRNAs and newly synthesized proteins to reveal that these channels interact early in their biogenesis. Two cell models were used: a heterologous expression system to investigate the steps of protein trafficking and a pancreatic beta cell line to study the localization of endogenous channel mRNAs. Our findings show that BK and Ca<sub>V</sub>1.3 channels begin assembling intracellularly before reaching the plasma membrane, revealing new aspects of their spatial organization. This intracellular assembly suggests a coordinated process that contributes to functional coupling.”
(b) Moderate conclusions where the supporting data are preliminary or correlative.
We agree with the reviewers, and we have moderated conclusions in instances where the supporting data are preliminary or correlative, ensuring a balanced interpretation. We added the following to the manuscript.
“This study provides novel insights into the organization of BK and Ca<sub>V</sub>1.3 channels in heteroclusters, emphasizing their assembly within the ER, at ER exit sites, and within the Golgi. Our findings suggest that BK and Ca<sub>V</sub>1.3 channels begin assembling intracellularly before reaching the plasma membrane, shaping their spatial organization, and potentially facilitating functional coupling. While this suggests a coordinated process that may contribute to functional coupling, further investigation is needed to determine the extent to which these hetero-clusters persist upon membrane insertion. While our study advances the understanding of BK and Ca<sub>V</sub>1.3 heterocluster assembly, several key questions remain unanswered. What molecular machinery drives this colocalization at the mRNA and protein level? How do disruptions to complex assembly contribute to channelopathies and related diseases? Additionally, a deeper investigation into the role of RNA binding proteins in facilitating transcript association and localized translation is warranted”.
(6) Address Additional Technical and Presentation Issues
(a) Include clearer figure annotations, especially for identifying PLA puncta localization (e.g., membrane vs. intracellular).
We agree with the reviewers, and we have updated the figures to include clearer annotations that distinguish PLA puncta localized at the membrane versus those within intracellular compartments.
(b) Reconsider the scale and arrangement of image panels to better showcase the data.
We agree with the reviewers, and we have adjusted the scale and layout of the image panels to enhance data visualization and readability. Enlarged key regions now provide better clarity of critical features.
(c) Provide precise clone/variant information for BK and CaV1.3 channels used.
We thank the reviewers for their suggestion, and we now provide precise information regarding the BK and Ca<sub>V</sub>1.3 channel constructs used in our experiments, including their Addgene plasmid numbers and relevant variant details. These have been incorporated into the Methods section to ensure reproducibility and transparency. We added the following to the manuscript.
“The Ca<sub>V</sub>1.3 α subunit construct used in our study corresponds to the rat Ca<sub>V</sub>1.3e splice variant containing exons 8a, 11, 31b, and 42a, with a deletion of exon 32. The BK channel construct used in this study corresponds to the VYR splice variant of the mouse BKα subunit (KCNMA1)”.
(d) Correct typographical errors and ensure proper figure/supplementary labeling throughout.
Typographical errors have been corrected, and figure/supplementary labeling has been reviewed for accuracy throughout the manuscript.
(7) Expand the Discussion
(a) Include a brief discussion of findings such as BK surface expression in the absence of CaV1.3.
We thank the reviewers for their suggestion. We expanded the Discussion to include a brief analysis of BK surface expression in the absence of Ca<sub>V</sub>1.3. We included the following in the manuscript.
“BK Surface Expression and Independent Trafficking Pathways
BK surface expression in the absence of Ca<sub>V</sub>1.3 indicates that its trafficking does not strictly rely on Ca<sub>V</sub>1.3-mediated interactions. Since BK channels can be activated by multiple calcium sources, their presence in intracellular compartments suggests that their surface expression is governed by intrinsic trafficking mechanisms rather than direct calcium-dependent regulation. While some BK and Ca<sub>V</sub>1.3 hetero-clusters assemble into signaling complexes intracellularly, other BK channels follow independent trafficking pathways, demonstrating that complex formation is not obligatory for all BK channels. Differences in their transport kinetics further reinforce the idea that their intracellular trafficking is regulated through distinct mechanisms. Studies have shown that BK channels can traffic independently of Ca<sub>V</sub>1.3, relying on alternative calcium sources for activation [13, 41]. Additionally, Ca<sub>V</sub>1.3 exhibits slower synthesis and trafficking kinetics than BK, emphasizing that their intracellular transport may not always be coordinated. These findings suggest that BK and Ca<sub>V</sub>1.3 exhibit both independent and coordinated trafficking behaviors, influencing their spatial organization and functional interactions”.
(b) Clarify why certain colocalization comparisons (e.g., ER vs. ER exit sites) are not directly interpretable.
We thank the reviewer for their suggestion. A clarification has been added to the result section and discussion of the manuscript explaining why colocalization comparisons, such as ER versus ER exit sites, are not directly interpretable. We included the following in the manuscript.
“Result:
ER was not simply due to the extensive spatial coverage of ER labeling, we labeled ER exit sites using Sec16-GFP and probed for hetero-clusters with PLA. This approach enabled us to test whether the hetero-clusters were preferentially localized to ER exit sites, which are specialized trafficking hubs that mediate cargo selection and direct proteins from the ER into the secretory pathway. In contrast to the more expansive ER network, which supports protein synthesis and folding, ER exit sites ensure efficient and selective export of proteins to their target destinations”.
“By quantifying the proportion of BK and Ca<sub>V</sub>1.3 hetero-clusters relative to total channel expression at ER exit sites, we found 28 ± 3% colocalization in tsA-201 cells and 11 ± 2% in INS-1 cells (Figure 3F). While the percentage of colocalization between hetero-clusters and the ER or ER exit sites alone cannot be directly compared to infer trafficking dynamics, these findings reinforce the conclusion that hetero-clusters reside within the ER and suggest that BK and Ca<sub>V</sub>1.3 channels traffic together through the ER and exit in coordination”.
“Colocalization and Trafficking Dynamics
The colocalization of BK and Ca<sub>V</sub>1.3 channels in the ER and at ER exit sites before reaching the Golgi suggests a coordinated trafficking mechanism that facilitates the formation of multi-channel complexes crucial for calcium signaling and membrane excitability [37, 38]. Given the distinct roles of these compartments, colocalization at the ER and ER exit sites may reflect transient proximity rather than stable interactions. Their presence in the Golgi further suggests that posttranslational modifications and additional assembly steps occur before plasma membrane transport, providing further insight into hetero-cluster maturation and sorting events. By examining BK-Ca<sub>V</sub>1.3 hetero-cluster distribution across these trafficking compartments, we ensure that observed colocalization patterns are considered within a broader framework of intracellular transport mechanisms [39]. Previous studies indicate that ER exit sites exhibit variability in cargo retention and sorting efficiency [40], emphasizing the need for careful evaluation of colocalization data. Accounting for these complexities allows for a robust assessment of signaling complexes formation and trafficking pathways”.
Reviewer #1 (Recommendations for the authors):
In addition to the general aspects described in the public review, I list below a few points with the hope that they will help to improve the manuscript:
(1) Page 3: "they bind calcium delimited to the point of entry at calcium channels", better use "sources"
We agree with the reviewer. The phrasing on Page 3 has been updated to use 'sources' instead of 'the point of entry at calcium channels' for clarity.
(2) Page 3 "localized supplies of intracellular calcium", I do not like this term, but maybe this is just silly.
We agree with the reviewer. The term 'localized supplies of intracellular calcium' on Page 3 has been revised to “Localized calcium sources”
(3) Regarding the definitions stated by the authors: How do you distinguish between "ensembles" corresponding to "coordinated collection of BK and Cav channels" and "assembly of BK clusters with Cav clusters"? I believe that hetero-clusters is more adequate. The nomenclature does not respond to any consensus in the protein biology field, and I find that it introduces bias more than it helps. I would stick to heteroclusters nomenclature that has been used previously in the field. Moreover, in some discussion sections, the term "ensemble" is used in ways that border on vague, especially when talking about "functional signaling complexes" or "ensembles forming early." It's still acceptable within context but could benefit from clearer language to distinguish ensemble (structural proximity) from complex (functional consequence).
We agree with the reviewer, and we recognize the importance of precise nomenclature and have adopted hetero-clusters instead of ensembles to align with established conventions in the field. This term specifically refers to the spatial organization of BK and Ca<sub>V</sub>1.3 channels, while functional complexes denote mechanistic interactions. We have revised sections where ensemble was used ambiguously to ensure clear distinction between structure and function.
The definition of "cluster" is clearly stated early but less emphasized in later quantitative analyses (e.g., particle size discussions in Figure 7). Figure 8 is equally confusing, graphs D and E referring to "BK ensembles" and "Cav ensembles", but "ensembles" should refer to combinations of both channels, whereas these seem to be "clusters". In fact, the Figure legend mentions "clusters".
We agree with the reviewer. Terminology has been revised throughout the manuscript to ensure consistency, with 'clusters' used appropriately in quantitative analyses and figure descriptions.
(4) Methods: how are clusters ("ensembles") analysed from the STORM data? What is the logarithm used for? More info about this is required. Equally, more information and discussion about how colocalization is measured and interpreted in superresolution microscopy are required.
We thank the reviewer for their suggestion, and additional details have been incorporated into the Methods section to clarify how clusters ('ensembles') are analyzed from STORM data, including the role of the logarithm in processing. Furthermore, we have expanded the discussion to provide more information on how colocalization is measured and interpreted in super resolution microscopy. We include the following in the manuscript.
“Direct stochastic optical reconstruction microscopy (dSTORM) images of BK and Ca<sub>V</sub>1.3 overexpressed in tsA-201 cells were acquired using an ONI Nanoimager microscope equipped with a 100X oil immersion objective (1.4 NA), an XYZ closed-loop piezo 736 stage, and triple emission channels split at 488, 555, and 640 nm. Samples were imaged at 35°C. For singlemolecule localization microscopy, fixed and stained cells were imaged in GLOX imaging buffer containing 10 mM β-mercaptoethylamine (MEA), 0.56 mg/ml glucose oxidase, 34 μg/ml catalase, and 10% w/v glucose in Tris-HCl buffer. Single-molecule localizations were filtered using NImOS software (v.1.18.3, ONI). Localization maps were exported as TIFF images with a pixel size of 5 nm. Maps were further processed in ImageJ (NIH) by thresholding and binarization to isolate labeled structures. To assess colocalization between the signal from two proteins, binary images were multiplied. Particles smaller than 400 nm<sup>2</sup> were excluded from the analysis to reflect the spatial resolution limit of STORM imaging (20 nm) and the average size of BK channels. To examine spatial localization preference, binary images of BK were progressively dilated to 20 nm, 40 nm, 60 nm, 80 nm, 100 nm, and 200 nm to expand their spatial representation. These modified images were then multiplied with the Ca<sub>V</sub>1.3 channel to quantify colocalization and determine BK occupancy at increasing distances from Ca<sub>V</sub>1.3. To ensure consistent comparisons across distance thresholds, data were normalized using the 200 nm measurement as the highest reference value, set to 1”.
(5) Related to Figure 2:
(a) Why use an antibody to label GFP when PH-PLCdelta should be a membrane marker? Where is the GFP in PH-PKC-delta (intracellular, extracellular? Images in Figure 2E are confusing, there is a green intracellular signal.
We thank the reviewer for their feedback. To clarify, GFP is fused to the N-terminus of PH-PLCδ and primarily localizes to the inner plasma membrane via PIP2 binding. Residual intracellular GFP signal may reflect non-membrane-bound fractions or background from anti-GFP immunostaining. We added a paragraph explaining the use of the antibody anti GFP in the Methods section Proximity ligation assay subsection.
(b) The images in Figure 2 do not help to understand how the authors select the PLA puncta located at the plasma membrane. How do the authors do this? A useful solution would be to indicate in Figure 2 an example of the PLA signals that are considered "membrane signals" compared to another example with "intracellular signals". Perhaps this was intended with the current Figure, but it is not clear.
We agree with the reviewer. We have added a sentence to explain how the number of PLA puncta at the plasma membrane was calculated.
“We visualized the plasma membrane with a biological sensor tagged with GFP (PHPLCδ-GFP) and then probed it with an antibody against GFP (Figure 2E). By analyzing the GFP signal, we created a mask that represented the plasma membrane. The mask served to distinguish between the PLA puncta located inside the cell and those at the plasma membrane, allowing us to calculate the number of PLA puncta at the plasma membrane”.
(c) Figure 2C: What is the negative control? Apologies if it is described somewhere, but I seem not to find it in the manuscript.
We thank the reviewer for their suggestion. For the negative control in Figure 2C, BK was probed using the primary antibody without co-staining for Ca<sub>V</sub>1.3 or other proteins, ensuring specificity and ruling out non-specific antibody binding or background fluorescence. A sentence clarifying the negative control for Figure 2C has been added to the Results section, specifying that BK was probed using the primary antibody without costaining for Ca<sub>V</sub>1.3 or other proteins to ensure specificity.
“To confirm specificity, a negative control was performed by probing only for BK using the primary antibody, ensuring that detected signals were not due to non-specific binding or background fluorescence”.
(d) What is the resolution in z of the images shown in Figure 2? This is relevant for the interpretation of signal localization.
The z-resolution of the images shown in Figure 2 was approximately 270–300 nm, based on the Zeiss Airyscan system’s axial resolution capabilities. Imaging was performed with a step size of 300 nm, ensuring adequate sampling for signal localization while maintaining optimal axial resolution.
“In a different experiment, we analyzed the puncta density for each focal plane of the cell (step size of 300 nm) and compared the puncta at the plasma membrane to the rest of the cell”.
(e) % of total puncta in PM vs inside cell are shown for transfected cells, what is this proportion in INS-1 cells?
This quantification was performed for transfected cells; however, we have not conducted the same analysis in INS-1 cells. Future experiments could address this to determine potential differences in puncta distribution between endogenous and overexpressed conditions.
(6) Related to Figure 3:
(a) Figure 3B: is this antibody labelling or GFP fluorescence? Why do they use GFP antibody labelling, if the marker already has its own fluorescence? This should at least be commented on in the manuscript.
We thank the reviewer for their concern. In Figure 3B, GFP was labeled using an antibody rather than relying on its intrinsic fluorescence. This approach was necessary because GFP fluorescence does not withstand the PLA protocol, resulting in significant fading. Antibody labeling provided stronger signal intensity and improved resolution, ensuring optimal signal-to-noise ratio for accurate analysis.
A clarification regarding the use of GFP antibody labeling in Figure 3B has been added to the Methods section, explaining that intrinsic GFP fluorescence does not endure the PLA protocol, necessitating antibody-based detection for improved signal and resolution.We added the following to the manuscript.
“For PLA combined with immunostaining, PLA was followed by a secondary antibody incubation with Alexa Fluor-488 at 2 μg/ml for 1 hour at 21˚C. Since GFP fluorescence fades significantly during the PLA protocol, resulting in reduced signal intensity and poor image resolution, GFP was labeled using an antibody rather than relying on its intrinsic fluorescence”.
(b) Why is it relevant to study the ER exit sites? Some explanation should be included in the main text (page 11) for clarification to non-specialized readers. Again, the quantification should be performed on the proportion of clusters/ensembles out of the total number of channels expressed at the ER (or ER exit sites).
We thank the reviewer for their feedback. We have modified this section to include a more detailed explanation of the relevance of ER exit sites to protein trafficking. ER exit sites serve as specialized sorting hubs that regulate the transition of proteins from the ER to the secretory pathway, distinguishing them from the broader ER network, which primarily facilitates protein synthesis and folding. This additional context clarifies why studying ER exit sites provides valuable insights into ensemble trafficking dynamics.
Regarding quantification, our method does not allow for direct measurement of the total number of BK and Ca<sub>V</sub>1.3 channels expressed at the ER or ER exit sites. Instead, we focused on the proportion of hetero-clusters localized within these compartments, which provides insight into trafficking pathways despite the limitation in absolute channel quantification. We included the following in the manuscript in the Results section.
“To determine whether the observed colocalization between BK–Ca<sub>V</sub>1.3 hetero-clusters and the ER was not simply due to the extensive spatial coverage of ER labeling, we labeled ER exit sites using Sec16-GFP and probed for hetero-clusters with PLA. This approach enabled us to test whether the hetero-clusters were preferentially localized to ER exit sites, which are specialized trafficking hubs that mediate cargo selection and direct proteins from the ER into the secretory pathway. In contrast to the more expansive ER network, which supports protein synthesis and folding, ER exit sites ensure efficient and selective export of proteins to their target destinations”.
“By quantifying the proportion of BK and Ca<sub>V</sub>1.3 hetero-clusters relative to total channel expression at ER exit sites, we found 28 ± 3% colocalization in tsA-201 cells and 11 ± 2% in INS-1 cells (Figure 3F). While the percentage of colocalization between hetero-clusters and the ER or ER exit sites alone cannot be directly compared to infer trafficking dynamics, these findings reinforce the conclusion that hetero-clusters reside within the ER and suggest that BK and Ca<sub>V</sub>1.3 channels traffic together through the ER and exit in coordination”.
(7) Related to Figure 4:
A control is included to confirm that the formation of BK-Cav1.3 ensembles is not unspecific. Association with a protein from the Golgi (58K) is tested. Why is this control only done for Golgi? No similar experiment has been performed in the ER. This aspect should be commented on.
We thank the reviewer for their suggestion. We selected the Golgi as a control because it represents the final stage of protein trafficking before proteins reach their functional destinations. If BK and Ca<sub>V</sub>1.3 hetero-cluster formation is specific at the Golgi, this suggests that their interaction is maintained throughout earlier trafficking steps, including within the ER. While we did not perform an equivalent control experiment in the ER, the Golgi serves as an effective checkpoint for evaluating specificity within the broader protein transport pathway. We included the following in the manuscript.
“We selected the Golgi as a control because it represents the final stage of protein trafficking, ensuring that hetero-cluster interactions observed at this point reflect specificity maintained throughout earlier trafficking steps, including within the ER”.
(8) How is colocalization measured, eg, in Figure 6? Are the images shown in Figure 6 representative? This aspect would benefit from a clearer description.
We thank the reviewer for their suggestion. A section clarifying colocalization measurement and the representativeness of Figure 6 images has been added to the Methods under Data Analysis. We included the following in the manuscript.
For PLA and RNAscope experiments, we used custom-made macros written in ImageJ. Processing of PLA data included background subtraction. To assess colocalization, fluorescent signals were converted into binary images, and channels were multiplied to identify spatial overlap.
(9) The text should be revised for typographical errors, for example:
(a) Summary "evidence of" (CHECK THIS ONE)
We agree with the reviewer, and we corrected the typographical errors
(b) Table 1, row 3: "enriches" should be "enrich"
We agree with the reviewer. The term 'enriches' in Table 1, row 3 has been corrected to 'enrich'.
(c) Figure 2B "priximity"
We agree with the reviewer. The typographical errors in Figure 2B has been corrected from 'priximity' to 'proximity'.
(d) Legend of Figure 7 (C) "size of BK and Cav1.3 channels". Does this correspond to individual channels or clusters?
We agree with the reviewer. The legend of Figure 7C has been clarified to indicate that 'size of BK and Cav1.3 channels' refers to clusters rather than individual channels.
(e) Methods: In the RNASCOPE section, "Fig.4-supp1" should be "Fig. 5-supp1"
(f) Page 15, Figure 5B is cited, should be Figure 6B
We agree with the reviewer. The reference in the RNASCOPE section has been updated from 'Fig.4-supp1' to 'Fig. 5-supp1,' and the citation on Page 15 has been corrected from Figure 5B to Figure 6B.
Reviewer #2 (Recommendations for the authors):
(1) The abstract could be more accessible for a wider readership with improved flow.
We thank the reviewer for their suggestion. We modified the summary as follows to provide a more coherent flow for a wider readership.
“Calcium binding to BK channels lowers BK activation threshold, substantiating functional coupling with calcium-permeable channels. This coupling requires close proximity between different channel types, and the formation of BK–Ca<sub>V</sub>1.3 hetero-clusters at nanometer distances exemplifies this unique organization. To investigate the structural basis of this interaction, we tested the hypothesis that BK and Ca<sub>V</sub>1.3 channels assemble before their insertion into the plasma membrane. Our approach incorporated four strategies: (1) detecting interactions between BK and Ca<sub>V</sub>1.3 proteins inside the cell, (2) identifying membrane compartments where intracellular hetero-clusters reside, (3) measuring the proximity of their mRNAs, and (4) assessing protein interactions at the plasma membrane during early translation. These analyses revealed that a subset of BK and Ca<sub>V</sub>1.3 transcripts are spatially close in micro-translational complexes, and their newly synthesized proteins associate within the endoplasmic reticulum (ER) and Golgi. Comparisons with other proteins, transcripts, and randomized localization models support the conclusion that BK and Ca<sub>V</sub>1.3 hetero-clusters form before their insertion at the plasma membrane”.
(2) Figure 2B - spelling of proximity.
We agree with the reviewer. The typographical errors in Figure 2B has been corrected from 'priximity' to 'proximity'.
Reviewer #3 (Recommendations for the authors):
Minor issues to improve the manuscript:
(1) For completeness, the authors should include a few sentences and appropriate references in the Introduction to mention that BK channels are regulated by auxiliary subunits.
We agree with the reviewer. We have revised the Introduction to include a brief discussion of how BK channel function is modulated by auxiliary subunits and provided appropriate references to ensure completeness. These additions highlight the broader regulatory mechanisms governing BK channel activity, complementing the focus of our study. We included the following in the manuscript.
“Additionally, BK channels are modulated by auxiliary subunits, which fine-tune BK channel gating properties to adapt to different physiological conditions. β and γ subunits regulate BK channel kinetics, altering voltage sensitivity and calcium responsiveness [18]. These interactions ensure precise control over channel activity, allowing BK channels to integrate voltage and calcium signals dynamically in various cell types. Here, we focus on the selective assembly of BK channels with Ca<sub>V</sub>1.3 and do not evaluate the contributions of auxiliary subunits to BK channel organization.”
(2) Insert a space between 'homeostasis' and the square bracket at the end of the Introduction's second paragraph.
We agree with the reviewer. A space has been inserted between 'homeostasis' and the square bracket in the second paragraph of the Introduction for clarity.
(3) The images presented in Figures 2-5 should be increased in size (if permitted by the Journal) to allow the reader to clearly see the puncta in the fluorescent images. This would necessitate reconfiguring the figures into perhaps a full A4 page per figure, but I think the quality of the images presented really do deserve to "be seen". For example, Panels A & B could be at the top of Figure 2, with C & D presented below them. However, I'll leave it up to the authors to decide on the most aesthetically pleasing way to show these.
We agree with the reviewer. We have increased the size of Figures 2–8 to enhance the visibility of fluorescent puncta, as suggested. To accommodate this, we reorganized the panel layout for each figure—for example, in Figure 2, Panels A and B are now placed above Panels C and D to support a more intuitive and aesthetically coherent presentation. We believe this revised configuration highlights the image quality and improves readability while conforming to journal layout constraints.
(4) I think that some of the sentences could be "toned down"
(a) eg, in the first paragraph below Figure 2, the authors state "that 46(plus minus)3% of the puncta were localised on intracellular membranes" when, at that stage, no data had been presented to confirm this. I think changing it to "that 46(plus minus)3% of the puncta were localised intracellularly" would be more precise.
(b) Similarly, please consider replacing the wording of "get together at membranes inside the cell" to "co-localise intracellularly".
(c) In the paragraph just before Figure 5, the authors mention that "the abundance of KCNMA1 correlated more with the abundance of CACNA1D than ... with GAPDH." Although this is technically correct, the R2 value was 0.22, which is exceptionally poor. I don't think that the paper is strengthened by sentences such as this, and perhaps the authors might tone this down to reflect this.
(d) The authors clearly demonstrate in Figure 8 that a significant number of BK channels can traffic to the membrane in the absence of Cav1.3. Irrespective of the differences in transcription/trafficking time between the two channel types, the authors should insert a few lines into their discussion to take this finding into account.
We appreciate the reviewer’s feedback regarding the clarity and precision of our phrasing.
Our responses for each point are below.
(a) We have modified the statement in the first paragraph below Figure 2, changing '46 ± 3% of the puncta were localized on intracellular membranes' to '46 ± 3% of the puncta were localized ‘intracellularly’ to ensure accuracy in the absence of explicit data confirming membrane association.
(b) Similarly, we have replaced 'get together at membranes inside the cell' with 'colocalize intracellularly' to maintain clarity and avoid unintended implications.
(c) Regarding the correlation between KCNMA1 and CACNA1D abundance, we recognize that the R² value of 0.22 is relatively low. To reflect this appropriately, we have revised the phrasing to indicate that while a correlation exists, it is modest. We added the following to the manuscript.
“Interestingly, the abundance of KCNMA1 transcripts correlated more with the abundance of CACNA1D transcripts than with the abundance of GAPDH, a standard housekeeping gene, though with a modest R² value.”
(d) To incorporate the findings from Figure 8, we have added discussion acknowledging that a substantial number of BK channels traffic to the membrane independently of Ca<sub>V</sub>1.3. This addition provides context for potential trafficking mechanisms that operate separately from ensemble formation.
(5) For clarity, please insert the word "total" in the paragraph after Figure 3 "..."63{plus minus}3% versus 50%{plus minus}6% of total PLA puncta were localised at the ER". I know this is explicitly stated later in the manuscript, but I think it needs to be clarified earlier.
We agree with the reviewer. The word 'total' has been inserted in the paragraph following Figure 3 to clarify the percentage of PLA puncta localized at the ER earlier in the manuscript
(6) In the discussion, I think an additional (short) paragraph needs to be included to clarify to the reader why the % "colocalization between ensembles and the ER or the ER exit sites can't be compared or used to understand the dynamics of the ensembles". This may permit the authors to remove the last sentence of the paragraph just before the results section, "BK and Cav1.3 ensembles go through the Golgi."
We thank the reviewer for their suggestion. We have added a short paragraph in the discussion to clarify why colocalization percentages between ensembles and the ER or ER exit sites cannot be compared to infer ensemble dynamics. This allowed us to remove the final sentence of the paragraph preceding the results section ('BK and Cav1.3 ensembles go through the Golgi).
(7) In the paragraph after Figure 6, Figure 5B is inadvertently referred to. Please correct this to Figure 6B.
We agree with the reviewer. The reference to Figure 5B in the paragraph after Figure 6 has been corrected to Figure 6B.
(8) In the discussion under "mRNA co-localisation and Protein Trafficking", please insert a relevant reference illustrating that "disruption in mRNA localization... can lead to ion channel mislocalization".
We agree with the reviewer. We have inserted a relevant reference under 'mRNA Colocalization and Protein Trafficking' to illustrate that disruption in mRNA localization can lead to ion channel mislocalization.
(9) The supplementary Figures appear to be incorrectly numbered. Please correct and also ensure that they are correctly referred to in the text.
We agree with the reviewer. The numbering of the supplementary figures has been corrected, and all references to them in the text have been updated accordingly.
(10) The final panels of the currently labelled Figure 5-Supplementary 2 need to have labels A-F included on the image.
We agree with the reviewer. Labels A-F have been added to the final panels of Figure 5-Supplementary 2.
References
(1) Shah, K.R., X. Guan, and J. Yan, Structural and Functional Coupling of Calcium-Activated BK Channels and Calcium-Permeable Channels Within Nanodomain Signaling Complexes. Frontiers in Physiology, 2022. Volume 12 - 2021.
(2) Chen, A.L., et al., Calcium-Activated Big-Conductance (BK) Potassium Channels Traffic through Nuclear Envelopes into Kinocilia in Ray Electrosensory Cells. Cells, 2023. 12(17): p. 2125.
(3) Berkefeld, H., B. Fakler, and U. Schulte, Ca2+-activated K+ channels: from protein complexes to function. Physiol Rev, 2010. 90(4): p. 1437-59.
(4) Loane, D.J., P.A. Lima, and N.V. Marrion, Co-assembly of N-type Ca2+ and BK channels underlies functional coupling in rat brain. J Cell Sci, 2007. 120(Pt 6): p. 98595.
(5) Boncompain, G. and F. Perez, The many routes of Golgi-dependent trafficking. Histochemistry and Cell Biology, 2013. 140(3): p. 251-260.
(6) Kurokawa, K. and A. Nakano, The ER exit sites are specialized ER zones for the transport of cargo proteins from the ER to the Golgi apparatus. The Journal of Biochemistry, 2019. 165(2): p. 109-114.
(7) Chen, G., et al., BK channel modulation by positively charged peptides and auxiliary γ subunits mediated by the Ca2+-bowl site. Journal of General Physiology, 2023. 155(6).
• Sleep-time compute allows models to "think" offline about contexts before queries are presented, reducing test-time compute requirements by ~5× on benchmark tasks
"by anticipating what queries users might ask and pre-computing useful quantities, we can significantly reduce the compute requirements at test-time"
• The approach works by processing context c during idle time to create an enhanced representation c', which is then used at test-time: S(c) → c', followed by Tb(q, c') → a
"In practice, this is achieved by prompting the model to generate a new context consisting of inferences about the existing context, which may be potentially useful for answering test-time queries"
• Performance improvements: Sleep-time compute reduces test-time compute needed to achieve same accuracy by ~5× on Stateful GSM-Symbolic and Stateful AIME
"Sleep-time compute produces a pareto improvement in the test-time compute vs. accuracy curve, reducing the test-time compute needed to achieve the same accuracy by ∼ 5×"
• Scaling benefits: By scaling up sleep-time compute, accuracy increases by up to 13% on Stateful GSM-Symbolic and 18% on Stateful AIME
• Cost amortization: When multiple queries share the same context, average cost per query decreases by 2.5×
"By amortizing sleep-time compute across related queries about the same context using Multi-Query GSM-Symbolic, we can decrease the average cost per query by 2.5×"
• Stateful GSM-Symbolic: Modified from GSM-Symbolic (P1: 5000 examples, P2: 2500 examples) by splitting problems into context and question
"We introduce two datasets to study applying sleep-time compute in stateful settings, Stateful GSM-Symbolic, and Stateful AIME – by splitting the existing problems in these datasets into a context and a question"
• Stateful AIME: Contains 60 questions from AIME 2024 and 2025, split into context and query components
• Multi-Query GSM-Symbolic: Extends GSM-Symbolic with multiple related queries per context (P1: 12,043 questions, 1,095 contexts; P2: 5,497 questions, 500 contexts)
• SWE-Features: Software engineering benchmark for multi-file feature implementation tasks (33 examples from Aider-AI/aider and ComfyUI repositories)
• Non-reasoning models: GPT-4o-mini and GPT-4o on GSM-Symbolic tasks
• Reasoning models: OpenAI's o1, o3-mini, Anthropic's Claude Sonnet 3.7 Extended Thinking, and DeepSeek-R1 on AIME tasks
• Test-time compute scaled both sequentially (varying verbosity/reasoning effort) and in parallel (pass@k sampling)
• Query predictability correlation: Sleep-time compute is most effective when queries are predictable from context
"sleep-time compute is more effective in settings where the query is more easily predictable from the context"
• Predictability measured using log-probability of question given context under Llama2-70B base model
• Accuracy gap between sleep-time and test-time compute widens for more predictable questions (binned analysis across 5 quantiles)
• Sleep-time compute implemented via function calling with two functions:
- rethink_memory: Takes new string input and replaces current context
- finish_rethinking: Terminates sleep-time compute process
• Models allowed up to 10 calls to rethink_memory function
• Cost modeling assumes test-time tokens are 10× more expensive than sleep-time tokens (t=10) due to latency optimization
"Since at test-time, there are strict latency constraints, and latency optimized inference can be roughly 10× more expensive, we model the total cost of inference between both sleep-time and test-time, by up-weighing the cost of test-time tokens"
• Pass@k parallel scaling: Sleep-time compute consistently outperforms pass@k at same test-time token budget
"sleep-time compute consistently outperforms pass@k parallel scaling at the same test-time token budget, demonstrating that sleep-time compute can be a more effective way to scale inference-time compute than standard parallel test-time scaling"
• Context-only baseline: Sleep-time compute significantly outperforms models that only receive context and must guess the question, demonstrating questions are not trivially predictable
• At lower test-time budgets, sleep-time compute achieves ~1.5× reduction in test-time tokens with higher F1 scores
• At higher budgets, standard test-time compute performs better, with higher precision but comparable recall
• Hypothesis: sleep-time compute explores more files, leading to editing more files and slightly lower precision
• Builds on recent test-time scaling approaches: sequential (OpenAI o1, DeepSeek-R1) and parallel (pass@k, best-of-N)
• Connection to speculative decoding (Leviathan et al., 2023): Both speculate on user queries, but sleep-time compute uses generated tokens as input regardless of actual query
• Connection to pre-computation in systems: Similar to memory caches (Smith, 1982) and data cubes for OLAP workloads (Gray et al., 1997)
• Resembles representation learning but operates in natural language space rather than parameter/activation space
• Sleep-time compute less effective when queries are unpredictable or unrelated to context
• Current approach assumes simple two-phase interaction (sleep-time and test-time), but real-world scenarios involve multiple interaction rounds
• Future work: Optimal allocation of compute between sleep-time and test-time based on query predictability
• Potential application to synthetic data generation at scale for pretraining
Kevin Lin, Charlie Snell, Yu Wang, Charles Packer, Sarah Wooders, Ion Stoica, Joseph E. Gonzalez (Letta & UC Berkeley)
Code and data: https://github.com/letta-ai/sleep-time-compute
Neither transformers nor sub-quadratic architectures are well-suited for long-context training
"the cost of processing the context is too expensive in the former, too inexpensive in the latter"
Power attention introduced as solution: A linear-cost sequence modeling architecture with independently adjustable state size > "an architectural layer for linear-cost sequence modeling whose state size can be adjusted independently of parameters"
Weight-state FLOP ratio should approach 1:1 for compute-optimal models
"for compute-optimal models, the WSFR should be somewhat close to 1:1"
Exponential attention becomes unbalanced at long contexts
At 1,000,000 context: WSFR is 1:125
"exponential attention is balanced for intermediate context lengths, but unbalanced for long context lengths, where it does far more state FLOPs than weight FLOPs"
Linear attention remains unbalanced at all context lengths
"Linear attention...is unbalanced at all context lengths in the opposite direction: far more weight FLOPs than state FLOPs"
Large state size improves ICL performance
"state scaling improves performance"
Windowed attention fails ICL beyond window size
"no in-context learning occurs beyond 100 tokens for window-32 attention"
Linear attention maintains ICL across entire sequence
"linear attention...demonstrate consistent in-context learning across the entire sequence"
Power attention formula: Uses p-th power instead of exponential
"attnᵖₚₒw(Q, K, V)ᵢ = Σⱼ₌₁ⁱ (QᵢᵀKⱼ)ᵖVⱼ"
Symmetric power expansion (SPOW) reduces state size vs tensor power (TPOW)
"SPOWₚ is a state expansion that increases the state size by a factor of (ᵈ⁺ᵖ⁻¹ₚ)/d without introducing any parameters"
Fused expand-MMA kernel: Expands tiles on-the-fly during matrix multiplication
"a matrix multiplication where the tiles of one operand are expanded on-the-fly"
Tiled symmetric power expansion (TSPOW): Interpolates between TPOW and SPOW
Optimal tile size: d-tile = 8 for p=2, d-tile = 4 for p=3
Chunked form enables practical efficiency
"The chunked form interpolates between the recurrent form and the attention form, capturing benefits of both"
"In all cases, the ICL curve becomes steeper as we scale the respective axis"
"Most sequences in OpenWebText have length less than 1k"
"autoregressive prediction of natural language is largely dominated by short-context dependencies"
Comprehensive taxonomy: "We establish a structured understanding of prompt engineering by assembling a taxonomy of prompting techniques and analyzing their applications. We present a detailed vocabulary of 33 vocabulary terms, a taxonomy of 58 LLM prompting techniques, and 40 techniques for other modalities."
Scope limitation: "We limit our study to focus on prefix prompts rather than cloze prompts, because modern LLM transformer architectures widely employ prefix prompts"
Focus on hard prompts: "Additionally, we refined our focus to hard (discrete) prompts rather than soft (continuous) prompts and leave out papers that make use of techniques using gradient-based updates (i.e. fine-tuning). Hard prompts contain only tokens (vectors) that correspond to words in the model's vocabulary"
Prompt definition: "A prompt is an input to a Generative AI model, that is used to guide its output"
Prompt template: "A prompt template is a function that contains one or more variables which will be replaced by some media (usually text) to create a prompt"
Prompting: "Prompting is the process of providing a prompt to a GenAI, which then generates a response"
Consolidated definition: "Prompt engineering is the iterative process of developing a prompt by modifying or changing the prompting technique that you are using"
Process description: "The Prompt Engineering Process consists of three repeated steps 1) performing inference on a dataset 2) evaluating performance and 3) modifying the prompt template"
Directive: "Many prompts issue a directive in the form of an instruction or question. This is the core intent of the prompt"
Examples/Exemplars: "Examples, also known as exemplars or shots, act as demonstrations that guide the GenAI to accomplish a task"
Output formatting: "It is often desirable for the GenAI to output information in certain formats, for example, CSV, Markdown, XML, or even custom formats"
Style instructions: "Style instructions are a type of output formatting used to modify the output stylistically rather than structurally"
Role/Persona: "A Role, also known as a persona, is a frequently discussed component that can improve writing and style text"
Approach: "We conducted a machine-assisted systematic review grounded in the PRISMA process to identify 58 different text-based prompting techniques"
Data sources: "Our main data sources were arXiv, Semantic Scholar, and ACL. We query these databases with a list of 44 keywords narrowly related to prompting and prompt engineering"
Pipeline: "We retrieve papers from arXiv based on a simple set of keywords and boolean rules. Then, human annotators label a sample of 1,661 articles"
Inter-rater reliability: "A set of 300 articles are reviewed independently by two annotators, with 92% agreement (Krippendorff's α = Cohen's κ = 81%)"
Final dataset: "The combined human and LLM annotations generate a final set of 1,565 papers"
Definition: "ICL refers to the ability of GenAIs to learn skills and tasks by providing them with exemplars and or relevant instructions within the prompt, without the need for weight updates/retraining"
Few-Shot Prompting: "Brown et al. (2020) is the paradigm seen in Figure 2.4, where the GenAI learns to complete a task with only a few examples (exemplars)"
Exemplar quantity: "Increasing the quantity of exemplars in the prompt generally improves model performance, particularly in larger models. However, in some cases, the benefits may diminish beyond 20 exemplars"
Exemplar ordering: "The order of exemplars affects model behavior. On some tasks, exemplar order can cause accuracy to vary from sub-50% to 90%+"
Label distribution impact: "As in traditional supervised machine learning, the distribution of exemplar labels in the prompt affects behavior"
Label quality: "Despite the general benefit of multiple exemplars, the necessity of strictly valid demonstrations is unclear. Some work suggests that the accuracy of labels is irrelevant—providing models with exemplars with incorrect labels may not negatively diminish performance"
Exemplar format: "The formatting of exemplars also affects performance. One of the most common formats is 'Q: {input}, A: {label}', but the optimal format may vary across tasks"
Exemplar similarity: "Selecting exemplars that are similar to the test sample is generally beneficial for performance. However, in some cases, selecting more diverse exemplars can improve performance"
K-Nearest Neighbor (KNN): "Liu et al. (2021) is part of a family of algorithms that selects exemplars similar to test samples to boost performance"
Vote-K: "Su et al. (2022) is another method to select similar exemplars to the test sample... Vote-K also ensures that newly added exemplars are sufficiently different than existing ones to increase diversity"
Self-Generated In-Context Learning (SG-ICL): "Kim et al. (2022) leverages a GenAI to automatically generate exemplars. While better than zero-shot scenarios when training data is unavailable, the generated samples are not as effective as actual data"
Prompt Mining: "Jiang et al. (2020) is the process of discovering optimal 'middle words' in prompts through large corpus analysis"
Role Prompting: "Wang et al. (2023j); Zheng et al. (2023d), also known as persona prompting, assigns a specific role to the GenAI in the prompt"
Style Prompting: "Lu et al. (2023a) involves specifying the desired style, tone, or genre in the prompt to shape the output"
Emotion Prompting: "Li et al. (2023a) incorporates phrases of psychological relevance to humans (e.g., 'This is important to my career') into the prompt, which may lead to improved LLM performance"
System 2 Attention (S2A): "Weston and Sukhbaatar (2023) first asks an LLM to rewrite the prompt and remove any information unrelated to the question therein"
Rephrase and Respond (RaR): "Deng et al. (2023) instructs the LLM to rephrase and expand the question before generating the final answer"
Re-reading (RE2): "Xu et al. (2023) adds the phrase 'Read the question again:' to the prompt in addition to repeating the question"
Self-Ask: "Press et al. (2022) prompts LLMs to first decide if they need to ask follow up questions for a given prompt"
Chain-of-Thought (CoT): "Wei et al. (2022b) leverages few-shot prompting to encourage the LLM to express its thought process before delivering its final answer"
Zero-Shot-CoT: "The most straightforward version of CoT contains zero exemplars. It involves appending a thought inducing phrase like 'Let's think step by step.' to the prompt"
Step-Back Prompting: "Zheng et al. (2023c) is a modification of CoT where the LLM is first asked a generic, high-level question about relevant concepts or facts before delving into reasoning"
Thread-of-Thought (ThoT): "Zhou et al. (2023) consists of an improved thought inducer for CoT reasoning. Instead of 'Let's think step by step,' it uses 'Walk me through this context in manageable parts step by step, summarizing and analyzing as we go.'"
Tabular Chain-of-Thought (Tab-CoT): "Jin and Lu (2023) consists of a Zero-Shot CoT prompt that makes the LLM output reasoning as a markdown table"
Contrastive CoT: "Chia et al. (2023) adds both exemplars with incorrect and correct explanations to the CoT prompt in order to show the LLM how not to reason"
Complexity-based Prompting: "Fu et al. (2023b) involves two major modifications to CoT. First, it selects complex examples for annotation and inclusion in the prompt... Second, during inference, it samples multiple reasoning chains"
Active Prompting: "Diao et al. (2023) starts with some training questions/exemplars, asks the LLM to solve them, then calculates uncertainty (disagreement in this case) and asks human annotators to rewrite the exemplars with highest uncertainty"
Memory-of-Thought: "Li and Qiu (2023b) leverage unlabeled training exemplars to build Few-Shot CoT prompts at test time"
Automatic Chain-of-Thought (Auto-CoT): "Zhang et al. (2022b) uses Wei et al. (2022b)'s Zero-Shot prompt to automatically generate chains of thought. These are then used to build a Few-Shot CoT prompt"
Least-to-Most Prompting: "Zhou et al. (2022a) starts by prompting a LLM to break a given problem into sub-problems without solving them. Then, it solves them sequentially, appending model responses to the prompt each time"
Decomposed Prompting (DECOMP): "Khot et al. (2022) Few-Shot prompts a LLM to show it how to use certain functions. These might include things like string splitting or internet searching"
Plan-and-Solve Prompting: "Wang et al. (2023f) consists of an improved Zero-Shot CoT prompt, 'Let's first understand the problem and devise a plan to solve it. Then, let's carry out the plan and solve the problem step by step'"
Tree-of-Thought (ToT): "Yao et al. (2023b), also known as Tree of Thoughts, creates a tree-like search problem by starting with an initial problem then generating multiple possible steps in the form of thoughts"
Program-of-Thoughts: "Chen et al. (2023d) uses LLMs like Codex to generate programming code as reasoning steps. A code interpreter executes these steps to obtain the final answer"
Skeleton-of-Thought: "Ning et al. (2023) focuses on accelerating answer speed through parallelization. Given a problem, it prompts an LLM to create a skeleton of the answer"
Demonstration Ensembling (DENSE): "Khalifa et al. (2023) creates multiple few-shot prompts, each containing a distinct subset of exemplars from the training set. Next, it aggregates over their outputs"
Self-Consistency: "Wang et al. (2022) is based on the intuition that multiple different reasoning paths can lead to the same answer. This method first prompts the LLM multiple times to perform CoT, crucially with a non-zero temperature"
Universal Self-Consistency: "Chen et al. (2023e) is similar to Self-Consistency except that rather than selecting the majority response by programmatically counting how often it occurs, it inserts all outputs into a prompt template"
DiVeRSe: "Li et al. (2023i) creates multiple prompts for a given problem then performs Self-Consistency for each, generating multiple reasoning paths"
Prompt Paraphrasing: "Jiang et al. (2020) transforms an original prompt by changing some of the wording, while still maintaining the overall meaning"
Self-Calibration: "Kadavath et al. (2022) first prompts an LLM to answer a question. Then, it builds a new prompt that includes the question, the LLM's answer, and an additional instruction asking whether the answer is correct"
Self-Refine: "Madaan et al. (2023) is an iterative framework where, given an initial answer from the LLM, it prompts the same LLM to provide feedback on the answer, and then prompts the LLM to improve the answer based on the feedback"
Self-Verification: "Weng et al. (2022) generates multiple candidate solutions with Chain-of-Thought (CoT). It then scores each solution by masking certain parts of the original question"
Chain-of-Verification (COVE): "Dhuliawala et al. (2023) first uses an LLM to generate an answer to a given question. Then, it creates a list of related questions that would help verify the correctness of the answer"
AutoPrompt: "Shin et al. (2020b) uses a frozen LLM as well as a prompt template that includes some 'trigger tokens', whose values are updated via backpropagation at training time"
Automatic Prompt Engineer (APE): "Zhou et al. (2022b) uses a set of exemplars to generate a Zero-Shot instruction prompt. It generates multiple possible prompts, scores them, then creates variations of the best ones"
Gradientfree Instructional Prompt Search (GrIPS): "Prasad et al. (2023) is similar to APE, but uses a more complex set of operations including deletion, addition, swapping, and paraphrasing"
RLPrompt: "Deng et al. (2022) uses a frozen LLM with an unfrozen module added. It uses this LLM to generate prompt templates, scores the templates on a dataset, and updates the unfrozen module using Soft Q-Learning"
Answer Shape: "The shape of an answer is its physical format. For example, it could be a token, span of tokens, or even an image or video"
Answer Space: "The space of an answer is the domain of values that its structure may contain. This may simply be the space of all tokens, or in a binary labeling task, could just be two possible tokens"
Answer Extractor: "In cases where it is impossible to entirely control the answer space... a rule can be defined to extract the final answer. This rule is often a simple function (e.g. a regular expression)"
Verbalizer: "Often used in labeling tasks, a verbalizer maps a token, span, or other type of output to a label and vice-versa (injective)"
Regex: "Regexes are often used to extract answers. They are usually used to search for the first instance of a label"
Separate LLM: "Sometimes outputs are so complicated that regexes won't work consistently. In this case, it can be useful to have a separate LLM evaluate the output and extract an answer"
Translate First Prompting: "Shi et al. (2022) is perhaps the simplest strategy and first translates non-English input examples into English"
Cross-Lingual Thought (XLT): "Huang et al. (2023a) utilizes a prompt template composed of six separate instructions, including role assignment, cross-lingual thinking, and CoT"
Cross-Lingual Self Consistent Prompting (CLSP): "Qin et al. (2023a) introduces an ensemble technique that constructs reasoning paths in different languages to answer the same question"
English advantage: "Constructing the prompt template in English is often more effective than in the task language for multilingual tasks. This is likely due to the predominance of English data during LLM pre-training"
Native language rationale: "In contrast, many multilingual prompting benchmarks such as BUFFET or LongBench use task language prompts for language-specific use cases"
Multi-Aspect Prompting and Selection (MAPS): "He et al. (2023b) mimics the human translation process, which involves multiple preparatory steps to ensure high-quality output"
Chain-of-Dictionary (CoD): "Lu et al. (2023b) first extracts words from the source phrase, then makes a list of their meanings in multiple languages, automatically via retrieval from a dictionary"
Interactive-Chain-Prompting (ICP): "Pilault et al. (2023) deals with potential ambiguities in translation by first asking the GenAI to generate sub-questions about any ambiguities in the phrase to be translated"
Prompt Modifiers: "are simply words appended to a prompt to change the resultant image. Components such as Medium (e.g. 'on canvas') or Lighting (e.g. 'a well lit scene') are often used"
Negative Prompting: "allows users to numerically weight certain terms in the prompt so that the model considers them more/less heavily than others"
Paired-Image Prompting: "shows the model two images: one before and one after some transformation. Then, present the model with a new image for which it will perform the demonstrated conversion"
Image-as-Text Prompting: "Hakimov and Schlangen (2023) generates a textual description of an image. This allows for the easy inclusion of the image (or multiple images) in a text-based prompt"
Duty Distinct Chain-of-Thought (DDCoT): "Zheng et al. (2023b) extends Least-to-Most prompting to the multimodal setting, creating subquestions, then solving them and combining the answers"
Chain-of-Images (CoI): "Meng et al. (2023) is a multimodal extension of Chain-of-Thought prompting, that generates images as part of its thought process"
Audio: "Experiments with audio ICL have generated mixed results, with some open source audio models failing to perform ICL. However, other results do show an ICL ability in audio models"
Video: "Prompting has also been extended to the video modality, for use in text-to-video generation, video editing, and video-to-text generation"
3D: "Prompting can also be used in 3D modalities, for example in 3D object synthesis, 3D surface texturing, and 4D scene generation"
Modular Reasoning, Knowledge, and Language (MRKL) System: "Karpas et al. (2022) is one of the simplest formulations of an agent. It contains a LLM router providing access to multiple tools"
Self-Correcting with Tool-Interactive Critiquing (CRITIC): "Gou et al. (2024a) first generates a response to the prompt, with no external calls. Then, the same LLM criticizes this response for possible errors"
Program-aided Language Model (PAL): "Gao et al. (2023b) translates a problem directly into code, which is sent to a Python interpreter to generate an answer"
Tool-Integrated Reasoning Agent (ToRA): "Gou et al. (2024b) is similar to PAL, but instead of a single code generation step, it interleaves code and reasoning steps for as long as necessary"
Reasoning and Acting (ReAct): "Yao et al. (2022) generates a thought, takes an action, and receives an observation (and repeats this process) when given a problem to solve"
Reflexion: "Shinn et al. (2023) builds on ReAct, adding a layer of introspection. It obtains a trajectory of actions and observations, then is given an evaluation of success/failure"
Voyager: "Wang et al. (2023a) is composed of three parts. First, it proposes tasks for itself to complete in order to learn more about the world. Second, it generates code to execute these actions. Finally, it saves these actions to be retrieved later"
Ghost in the Minecraft (GITM): "Zhu et al. (2023) starts with an arbitrary goal, breaks it down into subgoals recursively, then iteratively plans and executes actions by producing structured text"
Core concept: "RAG is a paradigm in which information is retrieved from an external source and inserted into the prompt. This can enhance performance in knowledge intensive tasks"
Verify-and-Edit: "Zhao et al. (2023a) improves on self-consistency by generating multiple chains-of-thought, then selecting some to be edited. They do this by retrieving relevant (external) information"
Interleaved Retrieval guided by Chain-of-Thought (IRCoT): "Trivedi et al. (2023) is a technique for multi-hop question answering that interleaves CoT and retrieval"
In-Context Learning: "is frequently used in evaluation prompts, much in the same way it is used in other applications"
Role-based Evaluation: "is a useful technique for improving and diversifying evaluations. By creating prompts with the same instructions for evaluation, but different roles, it is possible to effectively generate diverse evaluations"
Chain-of-Thought: "prompting can further improve evaluation performance"
Model-Generated Guidelines: "Liu et al. (2023d, h) prompt an LLM to generate guidelines for evaluation. This reduces the insufficient prompting problem arising from ill-defined scoring guidelines"
Styling: "Formatting the LLM's response using XML or JSON styling has also been shown to improve the accuracy of the judgment generated by the evaluator"
Linear Scale: "A very simple output format is a linear scale (e.g. 1-5). Many works use ratings of 1-10, 1-5, or even 0-1"
Binary Score: "Prompting the model to generate binary responses like Yes or No and True or False is another frequently used output format"
Likert Scale: "Prompting the GenAI to make use of a Likert Scale can give it a better understanding of the meaning of the scale"
LLM-EVAL: "Lin and Chen (2023) is one of the simplest evaluation frameworks. It uses a single prompt that contains a schema of variables to evaluate"
G-EVAL: "Liu et al. (2023d) is similar to LLM-EVAL, but includes an AutoCoT steps in the prompt itself"
ChatEval: "Chan et al. (2024) uses a multi-agent debate framework with each agent having a separate role"
Batch Prompting: "For improving compute and cost efficiency, some works employ batch prompting for evaluation where multiple instances are evaluated at once"
Pairwise Evaluation: "Chen et al. (2023g) find that directly comparing the quality of two texts may lead to suboptimal results and that explicitly asking LLM to generate a score for individual summaries is the most effective"
Definition: "Prompt hacking refers to a class of attacks which manipulate the prompt in order to attack a GenAI"
Prompt Injection: "is the process of overriding original developer instructions in the prompt with user input"
Jailbreaking: "is the process of getting a GenAI model to do or say unintended things through prompting"
Training Data Reconstruction: "refers to the practice of extracting training data from GenAIs. A straightforward example of this is Nasr et al. (2023), who found that by prompting ChatGPT to repeat the word 'company' forever, it began to regurgitate training data"
Prompt Leaking: "refers to the process of extracting the prompt template from an application. Developers often spend significant time creating prompt templates, and consider them to be IP worth protecting"
Package Hallucination: "occurs when LLM-generated code attempts to import packages that do not exist. After discovering what package names are frequently hallucinated by LLMs, hackers could create those packages, but with malicious code"
Prompt-based Defenses: "Multiple prompt-based defenses have been proposed, in which instructions are included in the prompt to avoid prompt injection. However, Schulhoff et al. (2023) ran a study with hundreds of thousands of malicious prompts and found that no prompt-based defense is fully secure"
Detectors: "are tools designed to detect malicious inputs and prevent prompt hacking. Many companies have built such detectors, which are often built using fine-tuned models trained on malicious prompts"
Guardrails: "are rules and frameworks for guiding GenAI outputs. Guardrails often make use of detectors, but not always. Guardrails are more concerned with the general dialogue flow in an application"
Small changes impact: "Several works show that LLMs are highly sensitive to the input prompt, i.e., even subtle changes to a prompt such as exemplar order can result in vastly different outputs"
Task format variation: "describes different ways to prompt an LLM to execute the same task... Zhao et al. (2021b) show that these minor changes can alter the accuracy of GPT-3 by up to 30%"
Prompt Drift: "Chen et al. (2023b) occurs when the model behind an API changes over time, so the same prompt may produce different results on the updated model"
Overconfidence: "LLMs are often overconfident in their answers, especially when prompted to express their own confidence in words, which may lead to user overreliance on model outputs"
Sycophancy: "refers to the concept that LLMs will often express agreement with the user, even when that view contradicts the model's own initial output"
Vanilla Prompting: "Si et al. (2023b) simply consists of an instruction in the prompt that tells the LLM to be unbiased. This technique has also been referred to as moral self-correction"
Cultural Awareness: "Yao et al. (2023a) can be injected into prompts to help LLMs with cultural adaptation"
AttrPrompt: "Yu et al. (2023) is a prompting technique designed to avoid producing text biased towards certain attributes when generating synthetic data"
Ambiguous Demonstrations: "Gao et al. (2023a) are examples that have an ambiguous label set. Including them in a prompt can increase ICL performance"
Question Clarification: "Rao and Daumé III (2019) allows the LLM to identify ambiguous questions and generate clarifying questions to pose to the user"
Performance trends: "Performance generally improved as techniques grew more complex. However, Zero-Shot-CoT dropped precipitously from Zero-Shot. Although it had a wide spread, for all variants, Zero-Shot performed better"
Best performer: "Few-Shot CoT performs the best, and unexplained performance drops from certain techniques need further research"
Self-Consistency impact: "Both cases of Self-Consistency, naturally had lower spread since they repeated a single technique, but it only improved accuracy for Zero-Shot prompts"
Problem domain: "Our illustrative problem involves detection of signal that is predictive of crisis-level suicide risk in text written by a potentially suicidal individual"
Target construct: "We focus here on the most important predictive factor in Suicide Crisis Syndrome assessments, referred to in the literature as either frantic hopelessness or entrapment"
Dataset: "Two coders trained on the recognition of the factors in Suicide Crisis Syndrome coded a set of 221 posts for presence or absence of entrapment, achieving solid inter-coder reliability (Krippendorff's alpha = 0.72)"
Development effort: "The exercise proceeded through 47 recorded development steps, cumulatively about 20 hours of work. From a cold start with 0% performance, performance was boosted to an F1 of 0.53"
Best manual approach: "10-Shot AutoDiCoT prompt includes 15 exemplars (without CoT reasoning) and one bootstrapped reasoning demonstration"
DSPy comparison: "The best resulting prompt... achieves 0.548 F1 (and 0.385 / 0.952 precision / recall) on the test set, without making any use of the professor's email nor the incorrect instruction about the explicitness of entrapment"
Sensitivity to details: "prompt engineering is fundamentally different from other ways of getting a computer to behave the way you want it to: these systems are being cajoled, not programmed, and... can be incredibly sensitive to specific details in prompts without there being any obvious reason those details should matter"
Domain expertise crucial: "the third and most important take-away is that prompt engineering should involve engagement between the prompt engineer, who has expertise in how to coax LLMs to behave in desired ways, and domain experts, who understand what those desired ways are and why"
Automation value: "Ultimately we found that there was significant promise in an automated method for exploring the prompting space, but also that combining that automation with human prompt engineering/revision was the most successful approach"
Start simple: "To those just beginning in prompt engineering, our recommendations resemble what one would recommend in any machine learning setting: understand the problem you are trying to solve (rather than just focusing on input/output and benchmark scores)"
Stay skeptical: "It is better to start with simpler approaches first, and to remain skeptical of claims about method performance"
Final corpus: "The dataset contains 1,565 research papers in PDF format. Any duplicate papers were removed automatically, though some could exist"
Time frame: "The dataset was curated the duration of the research paper, primarily in February of 2024"
Source distribution: "We wrote scripts to automatically query the APIs of Arxiv and Semantic Scholar"
Human validation: "After collecting data from different sources, we removed duplicate papers and did a manual and semi-automated review of papers to ensure they were all relevant"
LLM-assisted review: "We develop a prompt using gpt-4-1106-preview to classify the remaining articles. We validate the prompt against 100 ground-truth annotations, achieving 89% precision and 75% recall (for an F1 of 81%)"
Black art acknowledgment: "This can be interpreted both optimistically and pessimistically. Optimistically, it demonstrates how improvements can arise through exploration and fortuitous discovery. On the pessimistic side, the value of duplicating the email in the prompt highlights the extent to which prompting remains a difficult to explain black art"
Emergent vs discovered: "Many of the techniques described here have been called 'emergent', but it is perhaps more appropriate to say that they were discovered—the result of thorough experimentation, analogies from human reasoning, or pure serendipity"
Lack of standardization: "The field is new, and evaluation is variable and unstandardized—even the most meticulous experimentation may suffer from unanticipated shortcomings, and model outputs themselves are sensitive to meaning-preserving changes in inputs"
Transfer uncertainty: "As a result, we encourage the reader to avoid taking any claims at face value and to recognize that techniques may not transfer to other models, problems, or datasets"
Focus restrictions: "To keep the work approachable to less technical readers and maintain a manageable scope... we only study task-agnostic techniques"
Exclusions: "These decisions keep the work approachable to less technical readers and maintain a manageable scope"
Variable replacement: "A prompt template is a function that contains one or more variables which will be replaced by some media (usually text) to create a prompt"
Context preservation: "It is often necessary to include additional information in the prompt... Additional Information is sometimes called 'context', though we discourage the use of this term as it is overloaded with other meanings in the prompting space"
Verbalizer design: "For example, if we wish for a model to predict whether a Tweet is positive or negative, we could prompt it to output either '+' or '-' and a verbalizer would map these token sequences to the appropriate labels"
Regex patterns: "Regexes are often used to extract answers. They are usually used to search for the first instance of a label. However, depending on the output format and whether CoTs are generated, it may be better to search for the last instance"
Cascading approaches: "Sometimes outputs are so complicated that regexes won't work consistently. In this case, it can be useful to have a separate LLM evaluate the output and extract an answer"
Guardrails interference: "A take-away from this initial phase is that the 'guard rails' associated with some large language models may interfere with the ability to make progress on a prompting task, and this could influence the choice of model for reasons other than the LLM's potential quality"
Temperature settings: "For the two Self-Consistency results, we set temperature to 0.5, following Wang et al. (2022)'s guidelines. For all other prompts, a temperature of 0 was used"
In-Context Learning ambiguity: "Note that the word 'learn' is misleading. ICL can simply be task specification–the skills are not necessarily new, and can have already been included in the training data"
Brown et al. definitions: "Brown et al. (2020) seemingly offer two different definitions for ICL... However, they explicitly state that ICL does not necessarily involve learning new tasks"
Prompt vs Prompt Template: "Brown et al. (2020) consider the word 'llama' to be the prompt, while 'Translate English to French:' is the 'task description'. More recent papers, including this one, refer to the entire string passed to the LLM as the prompt"
Hard (discrete): "These prompts only contain tokens that directly correspond to words in the LLM vocabulary"
Soft (continuous): "These prompts contain tokens that may not correspond to any word in the vocabulary... Soft prompts can be used when fine-tuning is desired, but modifying the weights of the full model is prohibitively expensive"
Prefix prompts: "In Prefix prompts, the token to be predicted is at the end of the prompt. This is usually the case with modern GPT-style models"
Cloze prompts: "In Cloze prompts, the token(s) to be predicted are presented as 'slots to fill', usually somewhere in the middle of the prompt. This is usually the case for earlier transformer models such as BERT"
Algorithm description: "We call the algorithm in Figure 6.12 Automatic Directed CoT (AutoDiCoT), since it automatically directs the CoT process to reason in a particular way"
Process: "For each pair (qi, ai) in training data: Label qi as entrapment or not using the model. If correct, prompt with 'Why?' to generate reasoning. If incorrect, prompt 'It is actually [is/is not] entrapment, please explain why.'"
Generalizability: "This technique can be generalized to any labeling task. It combines the automatic generation of CoTs with showing the LLM examples of bad reasoning, as in the case of Contrastive CoT"
Six critical factors: "We highlight six separate design decisions, including the selection and order of exemplars that critically influence the output quality"
Tradeoffs: "Although effective, employing KNN during prompt generation may be time and resource intensive"
FLARE approach: "Forward-Looking Active REtrieval augmented generation (FLARE) and Imitate, Retrieve, Paraphrase (IRP) perform retrieval multiple times during long-form generation"
Three-step process: "1) generating a temporary sentence to serve as a content plan; 2) retrieving external knowledge using the temporary sentence as a query; 3) injecting the retrieved knowledge into the temporary sentence"
Query quality: "These temporary sentences have been shown to be better search queries compared to the document titles provided in long-form generation tasks"
Most cited techniques: "The prevalence of citations for Few-Shot and Chain-of-Thought prompting is unsurprising and helps to establish a baseline for understanding the prevalence of other techniques"
Model usage: Citation analysis shows GPT family dominates research, followed by PaLM and open-source alternatives
Dataset popularity: MMLU, GSM8K, and arithmetic reasoning benchmarks most frequently used
Paper growth: 1,565 relevant papers identified from broader corpus of 4,247 unique records
Quality metrics: Inter-annotator agreement of 92% (Krippendorff's α = Cohen's κ = 81%) for relevance labeling
LLM assistance: "We validate the prompt against 100 ground-truth annotations, achieving 89% precision and 75% recall (for an F1 of 81%)" for automated paper screening
Basic prompt conditioning: "p(A|T,Q) = ∏(i=1 to |A|) p_LM(ai|T,Q,a1:i-1)" where T is prompt template, Q is question, A is answer
Few-shot extension: "p(A|T(X,x)) = ∏(i=1 to |A|) p_LM(ai|T(X,x),a1:i-1)" where X is set of training exemplars
Optimization objective: "T* = argmax_T E_{xi,yi~D}[S(p_LM(A|T(xi)),yi)]" maximizing scoring function S over dataset D
Answer engineering: "A ~ p_LM(A|T(xi),yi); T* = argmax_{T,E} E_{xi,yi~D}[S(E(A),yi)]" where E is extraction function
Critical restriction: "NEVER use localStorage, sessionStorage, or ANY browser storage APIs in artifacts. These APIs are NOT supported and will cause artifacts to fail in the Claude.ai environment"
Alternatives: "Instead, you MUST: Use React state (useState, useReducer) for React components; Use JavaScript variables or objects for HTML artifacts; Store all data in memory during the session"
Context Engineering over RAG: "RAG" as a term is fundamentally flawed and confusing
"RAG. We never use the term rag. I hate the term rag... retrieval, augmented generation. Are three concepts put together into one thing? Like, that's just really confusing."
Context Engineering Definition: The job of determining optimal context window contents
"Context engineering is the job of figuring out what should be in the context window any given LLM generation step. And there's both an inner loop, which is setting up the, you know, what should be in the context window this time. And there's the outer loop, which is how do you get better over time at filling the context window with only the relevant information."
Models degrade with longer contexts: Performance is not invariant to token count
"The performance of LLMs is not invariant to how many tokens you use. As you use more and more tokens, the model can pay attention to less and then also can reason sort of less effectively."
Needle in Haystack is misleading: Lab benchmarks don't reflect real-world usage
"There was this bit of, like, this sort of implication where, like, oh, look, our model is perfect on this task, needle in a haystack. Therefore, the context window you can use for whatever you want. There was an implication there. And, well, I hope that that is true someday. That is not the case."
Claude Sonnet 4.5 performs best: Based on area under curve for context utilization
"I don't have much commentary. That is what we found for this particular task... I think it shows here if this is true, that's a big explanation for why" developers love Claude
Multiple signals for initial culling: Dense vectors, lexical search, metadata filtering
"One pattern is to use what a lot of people call first stage retrieval to do a big cull down... using signals like vector search, like full text search, like metadata filtering, metadata search, and others to go from, let's say 10,000 down to 300."
LLMs can handle more than 10 results: Unlike traditional search for humans
"You don't have to give an LLM 10 blue links. You can brute force a lot more."
LLM re-ranking is cost-effective and emerging: From 300 candidates down to 30
"Using an LLM as a re-ranker and brute forcing from 300 down to 30, I've seen now emerging a lot, like a lot of people are doing this and it actually is like way more cost effective than I think a lot of people realize I've heard of people that are running models themselves that are getting like a penny per million input tokens"
Purpose-built re-rankers will decline: Like specialized hardware, only needed at extreme scale
"I actually think that like probably purpose built re-rankers will go away. And the same way that like purpose built... if you're at extreme scale, extreme cost, yes, you'll care to optimize that... the same way that if you're running with hardware... you're just going to use a CPU or GPU. Unless you absolutely have to."
Structured ingestion matters: Extract metadata and signals at write time
"As much structured information as you can put into your write or your ingestion pipeline, you should. So all of the metadata you can extract, do it at ingestion. All of the chunk rewriting you can do, do it at ingestion."
Chunk rewriting for code: Generate natural language descriptions
"Instead of just embedding the code, you first have an LLM generate like a natural language description of like what this code is doing. And either you embed like just the natural language description or you embed that and the code"
Regex remains dominant: 85-90% of queries satisfied, but embeddings add value
"My guess is that like for code today, it's something like 90% of queries or 85% of queries can be satisfactorily run with Regex... But you maybe can get like 15% or 10% or 5% improvement by also using embeddings."
Chroma supports native regex search: With indexing for scale
"We've actually worked on now inside of Chroma, both single load and distributed, we support regex search natively. So you can do regex search inside of Chroma because we've seen that as like a very powerful tool for code search."
Fork-based indexing for versioning: Fast branch/commit-specific indexes
"Another feature we added to Chroma is the ability to do forking. So you can take an existing index and you can create a copy of that index in under a hundred milliseconds for pennies... you now can like have an index for like different each commit."
Small golden datasets are highly valuable: Few hundred examples sufficient
"The returns to a very high-quality small label data set are so high. Everybody thinks you have to have, like, a million examples or whatever. No. Actually, just, like, a couple hundred even, like, high-quality examples is extremely beneficial."
Generate synthetic QA pairs: When you have chunks but need queries
"We did a whole technical report around how do you teach an LLM to write good queries from chunks? Because, again, you want, like, chunk query pairs. And so if you have the chunks, you need the queries."
Data labeling parties work: Simple, practical approach
"Thursday night, we're all going to be in the conference room. We're ordering pizza. And we're just going to have a data labeling party for a few hours. That's all it takes to bootstrap this."
Memory is context engineering's benefit: Same problem, different framing
"Memory again is like the memory is the term that like everybody can understand... but what is memory under the hood? It's still just context engineering... the domain of how do you put the right information into the context window?"
Compaction enables offline improvement: Re-indexing and refinement
"Offline processing is helpful, and I think that is also helpful in this case... You're taking data. You're like, oh, maybe those two data points should be merged. Maybe they should be split. Maybe they should be, like, rewritten."
Stay in latent space: Avoid natural language round-trip
"Why are we going back to natural language? Why aren't we just like passing the embeddings like directly to the models who are just going to functionally like re put it into latent space."
Continuous retrieval during generation: Not just one-shot before generation
"For the longest time we've done one retrieval per generation... why are we not continually retrieving as we need to"
Current approaches are crude: Will seem primitive in 5-10 years
"I think when we look back in things, this was like, like hilariously crude, the way we do things today."
Developer experience is paramount: Zero-config, serverless approach
"In the same way that you could run pip install ChromaDB and be up and running in five seconds... That same story had to be true for the cloud... It needed to be like zero config, zero knobs to tune. It should just be always fast, always very cost-effective and always fresh without you having to do or think about anything."
Usage-based billing: True serverless pricing
"We only charge you for the minimal slice of compute that you use and like nothing more, which not all serverless databases can claim"
Slow, intentional hiring: Culture and craft over speed
"The slope of our future growth is entirely dependent on the people that are here in this office... we've just really decided to hire very slowly and be really picky."
eLife Assessment
This useful study reports a method to detect and analyze a novel post-translational modification, lysine acetoacetylation (Kacac), finding it regulates protein metabolism pathways. The study unveils epigenetic modifiers involved in placing this mark, including key histone acetyltransferases such as p300, and concomitant HDACs, which remove the mark. Proteomic and bioinformatics analysis identified many human proteins with Kacac sites, potentially suggesting broad effects on cellular processes and disease mechanisms. The data presented are solid, although some concerns persist regarding inconsistencies in molecular weight of the enzyme used. The study will be of interest to those studying protein and metabolic regulation.
Reviewer #2 (Public review):
In the manuscript by Fu et al., the authors developed a chemo-immunological method for the reliable detection of Kacac, a novel post-translational modification, and demonstrated that acetoacetate and AACS serve as key regulators of cellular Kacac levels. Furthermore, the authors identified the enzymatic addition of the Kacac mark by acyltransferases GCN5, p300, and PCAF, as well as its removal by deacetylase HDAC3. These findings indicate that AACS utilizes acetoacetate to generate acetoacetyl-CoA in the cytosol, which is subsequently transferred into the nucleus for histone Kacac modification. A comprehensive proteomic analysis has identified 139 Kacac sites on 85 human proteins. Bioinformatics analysis of Kacac substrates and RNA-seq data reveal the broad impacts of Kacac on diverse cellular processes and various pathophysiological conditions. This study provides valuable additional insights into the investigation of Kacac and would serve as a helpful resource for future physiological or pathological research.
Comments on revised version:
The authors have made efforts to revise this manuscript and address my concerns. The revisions are appropriate and have improved the quality of the manuscript.
Reviewer #3 (Public review):
Summary:
This paper presents a timely and significant contribution to the study of lysine acetoacetylation (Kacac). The authors successfully demonstrate a novel and practical chemo-immunological method using the reducing reagent NaBH4 to transform Kacac into lysine β-hydroxybutyrylation (Kbhb).
Strengths:
This innovative approach enables simultaneous investigation of Kacac and Kbhb, showcasing its potential in advancing our understanding of post-translational modifications and their roles in cellular metabolism and disease.
Weaknesses:
The experimental evidence presented in the article is insufficient to fully support the authors' conclusions. In the in vitro assays, the proteins used appear to be highly inconsistent with their expected molecular weights, as shown by Coomassie Brilliant Blue staining (Figure S3A). For example, p300, which has a theoretical molecular weight of approximately 270 kDa, appeared at around 37 kDa; GCN5/PCAF, expected to be ~70 kDa, appeared below 20 kDa. Other proteins used in the in vitro experiments also exhibited similarly large discrepancies from their predicted sizes. These inconsistencies severely compromise the reliability of the in vitro findings. Furthermore, the study lacks supporting in vivo data, such as gene knockdown experiments, to validate the proposed conclusions at the cellular level.
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
Summary
Lysine acetoacetylation (Kacac) is a recently discovered histone post-translational modification (PTM) connected to ketone body metabolism. This research outlines a chemo-immunological method for detecting Kacac, eliminating the requirement for creating new antibodies. The study demonstrates that acetoacetate acts as the precursor for Kacac, which is catalyzed by the acyltransferases GCN5, p300, and PCAF, and removed by the deacetylase HDAC3. AcetoacetylCoA synthetase (AACS) is identified as a central regulator of Kacac levels in cells. A proteomic analysis revealed 139 Kacac sites across 85 human proteins, showing the modification's extensive influence on various cellular functions. Additional bioinformatics and RNA sequencing data suggest a relationship between Kacac and other PTMs, such as lysine βhydroxybutyrylation (Kbhb), in regulating biological pathways. The findings underscore Kacac's role in histone and non-histone protein regulation, providing a foundation for future research into the roles of ketone bodies in metabolic regulation and disease processes.
Strengths
(1) The study developed an innovative method by using a novel chemo-immunological approach to the detection of lysine acetoacetylation. This provides a reliable method for the detection of specific Kacac using commercially available antibodies.
(2) The research has done a comprehensive proteome analysis to identify unique Kacac sites on 85 human proteins by using proteomic profiling. This detailed landscape of lysine acetoacetylation provides a possible role in cellular processes.
(3) The functional characterization of enzymes explores the activity of acetoacetyltransferase of key enzymes like GCN5, p300, and PCAF. This provides a deeper understanding of their function in cellular regulation and histone modifications.
(4) The impact of acetyl-CoA and acetoacetyl-CoA on histone acetylation provides the differential regulation of acylations in mammalian cells, which contributes to the understanding of metabolic-epigenetic crosstalk.
(5) The study examined acetoacetylation levels and patterns, which involve experiments using treatment with acetohydroxamic acid or lovastatin in combination with lithium acetoacetate, providing insights into the regulation of SCOT and HMGCR activities.
We thank all the reviewers for their positive, insightful comments which have helped us improve our manuscript. We have revised the manuscript as suggested by the reviewers.
Weakness
(1) There is a limitation to functional validation, related to the work on the biological relevance of identified acetoacetylation sites. Hence, the study requires certain functional validation experiments to provide robust conclusions regarding the functional implications of these modifications on cellular processes and protein function. For example, functional implications of the identified acetoacetylation sites on histone proteins would aid the interpretation of the results.
We agree with the reviewer that investigating the functional role of individual histone Kacac sites is essential for understanding the epigenetic impact of Kacac marks on gene expression, signaling pathways, and disease mechanisms. This topic is out of the scope of this paper which focuses on biochemical studies and proteomics. Functional elucidation in specific pathways will be a critical direction for future investigation, ideally with the development of site-specific anti-Kacac antibodies.
(2) The authors could have studied acetoacetylation patterns between healthy cells and disease models like cancer cells to investigate potential dysregulation of acetoacetylation in pathological conditions, which could provide insights into their PTM function in disease progression and pathogenesis.
We appreciate the reviewer’s valuable suggestion. In our study, we measured Kacac levels in several types of cancer cell lines, including HCT116 (Fig. 2B), HepG2 (Supplementary Fig. S2), and HeLa cells (data not shown in the manuscript), and found that acetoacetate-mediated Kacac is broadly present in all these cancer cell lines. Our proteomics analysis linked Kacac to critical cellular functions, e.g. DNA repair, RNA metabolism, cell cycle regulation, and apoptosis, and identified promising targets that are actively involved in cancer progression such as p53, HDAC1, HMGA2, MTA2, LDHA. These findings suggest that Kacac has significant, non-negligible effects on cancer pathogenesis. We concur that exploring the acetoacetylation patterns in cancer patient samples with comparison with normal cells represents a promising direction for next-step research. We plan to investigate these big issues in future studies.
(3) The time-course experiments could be performed following acetoacetate treatment to understand temporal dynamics, which can capture the acetoacetylation kinetic change, thereby providing a mechanistic understanding of the PTM changes and their regulatory mechanisms.
As suggested, time-course experiments were performed, and the data have been included in the revised manuscript (Supplementary Fig. S2A).
(4) Though the discussion section indeed provides critical analysis of the results in the context of existing literature, further providing insights into acetoacetylation's broader implications in histone modification. However, the study could provide a discussion on the impact of the overlap of other post-translational modifications with Kacac sites with their implications on protein functions.
We appreciate the reviewer’s helpful suggestion. We have added more discussions on the impact of the Kacac overlap with other post-translational modifications in the discussion section of the revised manuscript.
Impact
The authors successfully identified novel acetoacetylation sites on proteins, expanding the understanding of this post-translational modification. The authors conducted experiments to validate the functional significance of acetoacetylation by studying its impact on histone modifications and cellular functions.
We appreciate the reviewer’s comments.
Reviewer #2 (Public review):
In the manuscript by Fu et al., the authors developed a chemo-immunological method for the reliable detection of Kacac, a novel post-translational modification, and demonstrated that acetoacetate and AACS serve as key regulators of cellular Kacac levels. Furthermore, the authors identified the enzymatic addition of the Kacac mark by acyltransferases GCN5, p300, and PCAF, as well as its removal by deacetylase HDAC3. These findings indicate that AACS utilizes acetoacetate to generate acetoacetyl-CoA in the cytosol, which is subsequently transferred into the nucleus for histone Kacac modification. A comprehensive proteomic analysis has identified 139 Kacac sites on 85 human proteins. Bioinformatics analysis of Kacac substrates and RNA-seq data reveals the broad impacts of Kacac on diverse cellular processes and various pathophysiological conditions. This study provides valuable additional insights into the investigation of Kacac and would serve as a helpful resource for future physiological or pathological research.
The following concerns should be addressed:
(1) A detailed explanation is needed for selecting H2B (1-26) K15 sites over other acetylation sites when evaluating the feasibility of the chemo-immunological method.
The primary reason for selecting the H2B (1–26) K15acac peptide to evaluate the feasibility of our chemo-immunological method is that H2BK15acac was one of the early discovered modification sites in our preliminary proteomic screening data. The panKbhb antibody used herein is independent of peptide sequence so different modification sites on histones can all be recognized. We have added the explanation to the manuscript.
(2) In Figure 2(B), the addition of acetoacetate and NaBH4 resulted in an increase in Kbhb levels. Specifically, please investigate whether acetoacetylation is primarily mediated by acetoacetyl-CoA and whether acetoacetate can be converted into a precursor of β-hydroxybutyryl (bhb-CoA) within cells. Additional experiments should be included to support these conclusions.
We appreciate the reviewer’s valuable comments. In our paper, we had the data showing that acetoacetate treatment had very little effect on histone Kbhb levels in HEK293T cells, as observed in lanes 1–4 of Fig. 2A, demonstrating that acetoacetate minimally contributes to Kbhb generation. We drew the conclusion that histone Kacac is primarily mediated by acetoacetyl-CoA based on multiple pieces of evidence: first, we observed robust Kacac formation from acetoacetyl-CoA upon incubation with HATs and histone proteins or peptides, as confirmed by both western blotting (Figs. 3A, 3B; Supplementary Figs. S3C– S3F) and MALDI-MS analysis (Supplementary Fig. S4A). Second, treatment with hymeglusin—a specific inhibitor of hydroxymethylglutaryl-CoA synthase, which catalyzes the conversion of acetoacetyl-CoA to HMG-CoA—led to increased Kacac levels in HepG2 cells (PMID: 37382194). Third, we demonstrated that AACS whose function is to convert acetoacetate into acetoacetyl-CoA leads to marked histone Kacac upregulation (Fig. 2E). Collectively, these findings strongly support the conclusion that acetoacetate promotes Kacac formation primarily via acetoacetyl-CoA.
(3) In Figure 2(E), the amount of pan-Kbhb decreased upon acetoacetate treatment when SCOT or AACS was added, whereas this decrease was not observed with NaBH4 treatment. What could be the underlying reason for this phenomenon?
In the groups without NaBH₄ treatment (lanes 5–8, Figure 2E), the Kbhb signal decreased upon the transient overexpression of SCOT or AACS, owing to protein loading variation in these two groups (lanes 7 and 8). Both Ponceau staining and anti-H3 results showed a lower amount of histones in the AACS- or SCOT-treated samples. On the other hand, no decrease in the Kbhb signal was observed in the NaBH₄-treated groups (lanes 1–4), because NaBH₄ treatment elevated Kacac levels, thereby compensating for the reduced histone loading. The most important conclusion from this experiment is that AACS overexpression increased Kacac levels, whereas SCOT overexpression had no/little effect on histone Kacac levels in HEK293T cells.
(4) The paper demonstrates that p300, PCAF, and GCN5 exhibit significant acetoacetyltransferase activity and discusses the predicted binding modes of HATs (primarily PCAF and GCN5) with acetoacetyl-CoA. To validate the accuracy of these predicted binding models, it is recommended that the authors design experiments such as constructing and expressing protein mutants, to assess changes in enzymatic activity through western blot analysis.
We appreciate the reviewer’s valuable suggestion. Our computational modeling shows that acetoacetyl-CoA adopts a binding mode similar to that of acetyl-CoA in the tested HATs. This conclusion is supported by experimental results showing that the addition of acetyl-CoA significantly competed for the binding of acetoacetyl-CoA to HATs, leading to reduced enzymatic activity in mediating Kacac (Fig. 3C). Further structural biology studies to investigate the key amino acid residues involved in Kacac binding within the GCN5/PCAF binding pocket, in comparison to Kac binding—will be a key direction of future studies.
(5) HDAC3 shows strong de-acetoacetylation activity compared to its de-acetylation activity. Specific experiments should be added to verify the molecular docking results. The use of HPLC is recommended, in order to demonstrate that HDAC3 acts as an eraser of acetoacetylation and to support the above conclusions. If feasible, mutating critical amino acids on HDAC3 (e.g., His134, Cys145) and subsequently analyzing the HDAC3 mutants via HPLC and western blot can further substantiate the findings.
We appreciate the reviewer’s helpful suggestion. In-depth characterizations of HDAC3 and other HDACs is beyond this manuscript. We plan in the future to investigate the enzymatic activity of recombinant HDAC3, including the roles of key amino acid residues and the catalytic mechanism underlying Kacac removal, and to compare its activity with that involved in Kac removal.
(6) The resolution of the figures needs to be addressed in order to ensure clarity and readability.
Edits have been made to enhance figure resolutions in the revised manuscript.
Reviewer #3 (Public review):
Summary:
This paper presents a timely and significant contribution to the study of lysine acetoacetylation (Kacac). The authors successfully demonstrate a novel and practical chemo-immunological method using the reducing reagent NaBH4 to transform Kacac into lysine β-hydroxybutyrylation (Kbhb).
Strengths:
This innovative approach enables simultaneous investigation of Kacac and Kbhb, showcasing their potential in advancing our understanding of post-translational modifications and their roles in cellular metabolism and disease.
Weaknesses:
The paper's main weaknesses are the lack of SDS-PAGE analysis to confirm HATs purity and loading consistency, and the absence of cellular validation for the in vitro findings through knockdown experiments. These gaps weaken the evidence supporting the conclusions.
We appreciate the reviewer’s positive comments on the quality of this work and the importance to the field. The SDS-PAGE results of HAT proteins (Supplementary Fig. S3A) was added in the revised manuscript. The cellular roles of p300 and GCN5 as acetoacetyltransferases were confirmed in a recent study (PMID: 37382194). Their data are consistent with our studies herein and provide further support for our conclusion. We agree that knockdown experiments are essential to further validate the activities of these enzymes and plan to address this in future studies.
Reviewer #1 (Recommendations for the authors):
This study conducted the first comprehensive analysis of lysine acetoacetylation (Kacac) in human cells, identifying 139 acetoacetylated sites across 85 proteins in HEK293T cells. Kacac was primarily localized to the nucleus and associated with critical processes like chromatin organization, DNA repair, and gene regulation. Several previously unknown Kacac sites on histones were discovered, indicating its widespread regulatory role. Key enzymes responsible for adding and removing Kacac marks were identified: p300, GCN5, and PCAF act as acetoacetyltransferases, while HDAC3 serves as a remover. The modification depends on acetoacetate, with AACS playing a significant role in its regulation. Unlike Kbhb, Kacac showed unique cellular distribution and functional roles, particularly in gene expression pathways and metabolic regulation. Acetoacetate demonstrated distinct biological effects compared to βhydroxybutyrate, influencing lipid synthesis, metabolic pathways, and cancer cell signaling. The findings suggest that Kacac is an important post-translational modification with potential implications for disease, metabolism, and cellular regulation.
Major Concerns
(1) The authors could expand the study by including different cell lines and also provide a comparative study by using cell lines - such as normal vs disease (eg. Cancer cell like) - to compare and to increase the variability of acetoacetylation patterns across cell types. This could broaden the understanding of the regulation of PTMs in pathological conditions.
We sincerely appreciate the reviewer’s valuable suggestions. We concur that a
deeper investigation into Kacac patterns in cancer cell lines would significantly enhance understanding of Kacac in the human proteome. Nevertheless, due to constraints such as limited resource availability, we are currently unable to conduct very extensive explorations as proposed. Nonetheless, as shown in Fig. 2A, Fig. 2B, and Supplementary Fig. S2, our present data provide strong evidence for the widespread occurrence of acetoacetatemediated Kacac in both normal and cancer cell lines. Notably, our proteomic profiling identified several promising targets implicated in cancer progression, including p53, HDAC1, HMGA2, MTA2, and LDHA. We plan to conduct more comprehensive explorations of acetoacetylation patterns in cancer samples in future studies.
(2) The paper lacks inhibition studies silencing the enzyme genes or inhibiting the enzyme using available inhibitors involved in acetoacetylation or using aceto-acetate analogues to selectively modulate acetoacetylation levels. This can validate their impact on downstream cellular pathways in cellular regulation.
We appreciate the reviewer’s valuable suggestions. Our study, along with the previous research, has conducted initial investigations into the inhibition of key enzymes involved in the Kacac pathway. For example, inhibition of HMGCS, which catalyzes the conversion of acetoacetyl-CoA to HMG-CoA, was shown to enhance histone Kacac levels (PMID: 37382194). In our study, we examined the inhibitory effects of SCOT and HMGCR, both of which potentially influence cellular acetoacetyl-CoA levels. However, their respective inhibitors did not significantly affect histone Kacac levels. We also investigated the role of acetyl-CoA, which competes with acetoacetyl-CoA for binding to HAT enzymes and can function as a competitive inhibitor in histone Kacac generation. Furthermore, inhibition of HDAC activity by SAHA led to increased histone Kacac levels in HepG2 cells (PMID: 37382194), supporting our conclusion that HDAC3 functions as the eraser responsible for Kacac removal. These inhibition studies confirmed the functions of these enzymes and provided insights into their regulatory roles in modulating Kacac and its downstream pathways. Further in-depth investigations will explore the specific roles of these enzymes in regulating Kacac within cellular pathways.
(3) The authors could validate the functional impact of pathways using various markers through IHC/IFC or western blot to confirm their RNA-seq analysis, since pathways could be differentially regulated at the RNA vs protein level.
We agree that pathways can be differentially regulated at the RNA and protein levels. It is our future plan to select and fully characterize one or two gene targets to elaborate the presence and impact of Kacac marks on their functional regulation at both the gene expression and protein level.
(4) Utilize in vitro reconstitution assays to confirm the direct effect of acetoacetylation on histone modifications and nucleosome assembly, establishing a causal relationship between acetoacetylation and chromatin regulation.
We appreciate this suggestion, and this will be a very fine biophysics project for us and other researchers for the next step. We plan to do this and related work in a future paper to characterize the impact of lysine acetoacetylation on chromatin structure and gene expression. Technique of site-specific labelling will be required. Also, we hope to obtain monoclonal antibodies that directly recognize Kacac in histones to allow for ChIP-seq assays in cells.
(5) The authors could provide a site-directed mutagenesis experiment by mutating a particular site, which can validate and address concerns regarding the specificity of a particular site involved in the mechanism.
We agree that validating and characterizing the specificity of individual Kacac sites and understanding their functional implications are important for elucidating the mechanisms by which Kacac affects these substrate proteins. Such work will involve extensive biochemical and cellular studies. It is our future goal to select and fully characterize one or two gene targets in detail and in depth to elaborate the presence and impact of Kacac on their function regulation using comprehensive techniques (transfection, mutation, pulldown, and pathway analysis, etc.).
(6) If possible, the authors could use an in vivo model system, such as mice, to validate the physiological relevance of acetoacetylation in a more complex system.
We currently do not have access to resources of relevant animal models. We will conduct in vivo screening and characterization of protein acetoacetylation in animal models and clinical samples in collaboration with prospective collaborators.
Minor Concerns
(1) The authors could discuss the overlap of Kacac sites with other post-translational modifications and their implications on protein functions. They could provide comparative studies with other PTMs, which can improvise a comprehensive understanding of acetoacetylation function in epigenetic regulation.
We have expanded the discussion in the revised manuscript to address the overlap between Kacac and other post-translational modifications, along with their potential functional implications.
(2) The authors could provide detailed information on the implications of their data, which would enhance the impact of the research and its relevance to the scientific community. Specifically, they could clarify the acetoacetylation (Kacac) significance in nucleosome assembly and its correlation with RNA processing.
In the revised manuscript, we have added more elaborations on the implication and significance of Kacac in nucleosome assembly and RNA processing.
Reviewer #3 (Recommendations for the authors):
Major Comments:
(1) Figures 3A, 3B, Supplementary Figures S3A-D
I could not find the SDS-PAGE analysis results for the purified HATs used in the in vitro assay. It is imperative to display these results to confirm consistent loading amounts and sufficient purity of the HATs across experimental groups. Additionally, I did not observe any data on CBP, even though it was mentioned in the results section. If CBP-related experiments were not conducted, please remove the corresponding descriptions.
We appreciate the reviewer’s valuable suggestion. The SDS-PAGE results for the HAT proteins have been included, and the part in the results section discussing CBP has been updated according to the reviewer’s suggestion in the revised manuscript.
(2) Knockdown of Selected HATs and HDAC3 in cells
The authors should perform gene knockdown experiments in cells, targeting the identified HATs and HDAC3, followed by Western blot and mass spectrometry analysis of Kacac expression levels. This would validate whether the findings from the in vitro assays are biologically relevant in cellular contexts.
We appreciate the reviewer’s valuable suggestion. Our identified HATs, including p300 and GCN5, were reported as acetoacetyltransferases in cellular contexts by a recent study (PMID: 37382194). Their findings are precisely consistent with our biochemical results, providing additional evidence that p300 and GCN5 mediate Kacac both in vitro and in vivo. In addition, inhibition of HDAC activity by SAHA greatly increased histone Kacac levels in HepG2 cells (PMID: 37382194), supporting the role of HDAC3 as an eraser responsible for Kacac removal. We plan to further study these enzymes’ contributions to Kacac through gene knockdown experiments and investigate the specific functions of enzyme-mediated Kacac under some pathological contexts.
Minor Comments:
(1) Abstract accuracy
In the Abstract, the authors state, "However, regulatory elements, substrate proteins, and epigenetic functions of Kacac remain unknown." Please revise this statement to align with the findings in Reference 22 and describe these elements more appropriately. If similar issues exist in other parts of the manuscript, please address them as well.
The issues have been addressed in the revised manuscript based on the reviewer's comments.
(2) Terminology issue
GCN5 and PCAF are both members of the GNAT family. It is not accurate to describe "GCN5/PCAF/HAT1" as one family. Please refine the terminology to reflect the classification accurately.
The description has been refined in the revised manuscript to accurately reflect the classification, in accordance with the reviewer's suggestion.
(3) Discussion on HBO1
Reference 22 has already established HBO1 as an acetoacetyltransferase. This paper should include a discussion of HBO1 alongside the screened p300, PCAF, and GCN5 to provide a more comprehensive perspective.
More discussion on HBO1 alongside the other screened HATs has been added in the revised manuscript.
eLife Assessment
This useful study explores the role of RAP2A in asymmetric cell division (ACD) regulation in glioblastoma stem cells (GSCs), drawing parallels to Drosophila ACD mechanisms and proposing that an imbalance toward symmetric divisions drives tumor progression. While findings on RAP2A's role in GSC expansion are promising, and the reviewers found the study innovative and technically sound, the study is nevertheless still considered incomplete because of its reliance on neurosphere models without in vivo confirmation and insufficient mechanistic validation. Addressing those gaps would substantiate the study's claims.
Reviewer #1 (Public review):
Summary:
The authors validate the contribution of RAP2A to GB progression. RAp2A participates in asymetric cell division, and the localization of several cell polarity markers including cno and Numb.
Strengths:
The use of human data, Drosophila models and cell culture or neurospheres is a good scenario to validate the hypothesis using complementary systems.
Moreover, the mechanisms that determine GB progression, and in particular glioma stem cells biology, are relevant for the knowledge on glioblastoma and opens new possibilities to future clinical strategies.
Weaknesses:
While the manuscript presents a well-supported investigation into RAP2A's role in GBM, some methodological aspects could benefit from further validation. The major concern is the reliance on a single GB cell line (GB5), including multiple GBM lines, particularly primary patient-derived 3D cultures with known stem-like properties, would significantly enhance the study's robustness.
Several specific points raised in previous reviews have improved this version of the manuscript:
• The specificity of Rap2l RNAi has been further confirmed by using several different RNAi tools.
• Quantification of phenotypic penetrance and survival rates in Rap2l mutants would help determine the consistency of ACD defects. The authors have substantially increased the number of samples analyzed including three different RNAi lines (both the number of NB lineages and the number of different brains analyzed) to confirm the high penetrance of the phenotype.
• The observations on neurosphere size and Ki-67 expression require normalization (e.g., Ki-67+ cells per total cell number or per neurosphere size). This is included in the manuscript and now clarified in the text.
• The discrepancy in Figures 6A and 6B requires further discussion. The authors have included a new analysis and further explanations and they can conclude that in 2 cell-neurospheres there are more cases of asymmetric divisions in the experimental condition (RAP2A) than in the control.
• Live imaging of ACD events would provide more direct evidence. Live imaging was not done due to technical limitations. Despite being a potential contribution to the manuscript, the current conclusions of the manuscript are supported by the current data, and live experiments can be dispensable
• Clarification of terminology and statistical markers (e.g., p-values) in Figure 1A would improve clarity. This has been improved.
Comments on revisions:
The manuscript has improved the clarity in general, and I think that it is suitable for publication. However, for future experiments and projects, I would like to insist in the relevance of validating the results in vivo using xenografts with 3D-primary patient-derived cell lines or GB organoids.
Reviewer #2 (Public review):
This study investigates the role of RAP2A in regulating asymmetric cell division (ACD) in glioblastoma stem cells (GSCs), bridging insights from Drosophila ACD mechanisms to human tumor biology. They focus on RAP2A, a human homolog of Drosophila Rap2l, as a novel ACD regulator in GBM is innovative, given its underexplored role in cancer stem cells (CSCs). The hypothesis that ACD imbalance (favoring symmetric divisions) drives GSC expansion and tumor progression introduces a fresh perspective on differentiation therapy. However, the dual role of ACD in tumor heterogeneity (potentially aiding therapy resistance) requires deeper discussion to clarify the study's unique contributions against existing controversies.
Comments on revisions:
More experiments as suggested in the original assessment of the submission are needed to justify the hypothesis drawn in the manuscript.
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
Summary:
The authors validate the contribution of RAP2A to GB progression. RAp2A participates in asymmetric cell division, and the localization of several cell polarity markers, including cno and Numb.
Strengths:
The use of human data, Drosophila models, and cell culture or neurospheres is a good scenario to validate the hypothesis using complementary systems.
Moreover, the mechanisms that determine GB progression, and in particular glioma stem cells biology, are relevant for the knowledge on glioblastoma and opens new possibilities to future clinical strategies.
Weaknesses:
While the manuscript presents a well-supported investigation into RAP2A's role in GBM, several methodological aspects require further validation. The major concern is the reliance on a single GB cell line (GB5), which limits the generalizability of the findings. Including multiple GBM lines, particularly primary patient-derived 3D cultures with known stem-like properties, would significantly enhance the study's relevance.
Additionally, key mechanistic aspects remain underexplored. Further investigation into the conservation of the Rap2l-Cno/aPKC pathway in human cells through rescue experiments or protein interaction assays would be beneficial. Similarly, live imaging or lineage tracing would provide more direct evidence of ACD frequency, complementing the current indirect metrics (odd/even cell clusters, Numb asymmetry).
Several specific points require attention:
(1) The specificity of Rap2l RNAi needs further confirmation. Is Rap2l expressed in neuroblasts or intermediate neural progenitors? Can alternative validation methods be employed?
There are no available antibodies/tools to determine whether Rap2l is expressed in NB lineages, and we have not been able either to develop any. However, to further prove the specificity of the Rap2l phenotype, we have now analyzed two additional and independent RNAi lines of Rap2l along with the original RNAi line analyzed. We have validated the results observed with this line and found a similar phenotype in the two additional RNAi lines now analyzed. These results have been added to the text ("Results section", page 6, lines 142-148) and are shown in Supplementary Figure 3.
(2) Quantification of phenotypic penetrance and survival rates in Rap2l mutants would help determine the consistency of ACD defects.
In the experiment previously mentioned (repetition of the original Rap2l RNAi line analysis along with two additional Rap2l RNAi lines) we have substantially increased the number of samples analyzed (both the number of NB lineages and the number of different brains analyzed). With that, we have been able to determine that the penetrance of the phenotype was 100% or almost 100% in the 3 different RNAi lines analyzed (n>14 different brains/larvae analyzed in all cases). Details are shown in the text (page 6, lines 142-148), in Supplementary Figure 3 and in the corresponding figure legend.
(3) The observations on neurosphere size and Ki-67 expression require normalization (e.g., Ki-67+ cells per total cell number or per neurosphere size). Additionally, apoptosis should be assessed using Annexin V or TUNEL assays.
The experiment of Ki-67+ cells was done considering the % of Ki-67+ cells respect the total cell number in each neurosphere. In the "Materials and methods" section it is well indicated: "The number of Ki67+ cells with respect to the total number of nuclei labelled with DAPI within a given neurosphere were counted to calculate the Proliferative Index (PI), which was expressed as the % of Ki67+ cells over total DAPI+ cells"
Perhaps it was not clearly showed in the graph of Figure 5A. We have now changed it indicating: "% of Ki67+ cells/ neurosphere" in the "Y axis".
Unfortunately, we currently cannot carry out neurosphere cultures to address the apoptosis experiments.
(4) The discrepancy in Figures 6A and 6B requires further discussion.
We agree that those pictures can lead to confusion. In the analysis of the "% of neurospheres with even or odd number of cells", we included the neurospheres with 2 cells both in the control and in the experimental condition (RAP2A). The number of this "2 cell-neurospheres" was very similar in both conditions (27,7 % and 27 % of the total neurospheres analyzed in each condition), and they can be the result of a previous symmetric or asymmetric division, we cannot distinguish that (only when they are stained with Numb, for example, as shown in Figure 6B). As a consequence, in both the control and in the experimental condition, these 2-cell neurospheres included in the group of "even" (Figure 6A) can represent symmetric or asymmetric divisions. However, in the experiment shown in Figure 6B, it is shown that in these 2 cellneurospheres there are more cases of asymmetric divisions in the experimental condition (RAP2A) than in the control.
Nevertheless, to make more accurate and clearer the conclusions, we have reanalyzed the data taking into account only the neurospheres with 3-5-7 (as odd) or 4-6-8 (as even) cells. Likewise, we have now added further clarifications regarding the way the experiment has been analyzed in the methods.
(5) Live imaging of ACD events would provide more direct evidence.
We agree that live imaging would provide further evidence. Unfortunately, we currently cannot carry out neurosphere cultures to approach those experiments.
(6) Clarification of terminology and statistical markers (e.g., p-values) in Figure 1A would improve clarity.
We thank the reviewer for pointing out this issue. To improve clarity, we have now included a Supplementary Figure (Fig. S1) with the statistical parameters used. Additionally, we have performed a hierarchical clustering of genes showing significant or not-significant changes in their expression levels.
(7) Given the group's expertise, an alternative to mouse xenografts could be a Drosophila genetic model of glioblastoma, which would provide an in vivo validation system aligned with their research approach.
The established Drosophila genetic model of glioblastoma is an excellent model system to get deep insight into different aspects of human GBM. However, the main aim of our study was to determine whether an imbalance in the mode of stem cell division, favoring symmetric divisions, could contribute to the expansion of the tumor. We chose human GBM cell lines-derived neurospheres because in human GBM it has been demonstrated the existence of cancer stem cells (glioblastoma or glioma stem cells -GSCs--). And these GSCs, as all stem cells, can divide symmetric or asymmetrically. In the case of the Drosophila model of GBM, the neoplastic transformation observed after overexpressing the EGF receptor and PI3K signaling is due to the activation of downstream genes that promote cell cycle progression and inhibit cell cycle exit. It has also been suggested that the neoplastic cells in this model come from committed glial progenitors, not from stem-like cells.
With all, it would be difficult to conclude the causes of the potential effects of manipulating the Rap2l levels in this Drosophila system of GBM. We do not discard this analysis in the future (we have all the "set up" in the lab). However, this would probably imply a new project to comprehensively analyze and understand the mechanism by which Rap2l (and other ACD regulators) might be acting in this context, if it is having any effect.
However, as we mentioned in the Discussion, we agree that the results we have obtained in this study must be definitely validated in vivo in the future using xenografts with 3D-primary patient-derived cell lines.
Reviewer #2 (Public review):
This study investigates the role of RAP2A in regulating asymmetric cell division (ACD) in glioblastoma stem cells (GSCs), bridging insights from Drosophila ACD mechanisms to human tumor biology. They focus on RAP2A, a human homolog of Drosophila Rap2l, as a novel ACD regulator in GBM is innovative, given its underexplored role in cancer stem cells (CSCs). The hypothesis that ACD imbalance (favoring symmetric divisions) drives GSC expansion and tumor progression introduces a fresh perspective on differentiation therapy. However, the dual role of ACD in tumor heterogeneity (potentially aiding therapy resistance) requires deeper discussion to clarify the study's unique contributions against existing controversies. Some limitations and questions need to be addressed.
(1) Validation of RAP2A's prognostic relevance using TCGA and Gravendeel cohorts strengthens clinical relevance. However, differential expression analysis across GBM subtypes (e.g., MES, DNA-methylation subtypes ) should be included to confirm specificity.
We have now included a Supplementary figure (Supplementary Figure 2), in which we show the analysis of RAP2A levels in the different GBM subtypes (proneural, mesenchymal and classical) and their prognostic relevance (i.e. the proneural subtype that presents RAP2A levels significantly higher than the others is the subtype that also shows better prognostic).
(2) Rap2l knockdown-induced ACD defects (e.g., mislocalization of Cno/Numb) are well-designed. However, phenotypic penetrance and survival rates of Rap2l mutants should be quantified to confirm consistency.
We have now analyzed two additional and independent RNAi lines of Rap2l along with the original RNAi line. We have validated the results observed with this line and found a similar phenotype in the two additional RNAi lines now analyzed. To determine the phenotypic penetrance, we have substantially increased the number of samples analyzed (both the number of NB lineages and the number of different brains analyzed). With that, we have been able to determine that the penetrance of the phenotype was 100% or almost 100% in the 3 different Rap2l RNAi lines analyzed (n>14 different brains/larvae analyzed in all cases). These results have been added to the text ("Results section", page 6, lines 142-148) and are shown in Supplementary Figure 3 and in the corresponding figure legend.
(3) While GB5 cells were effectively used, justification for selecting this line (e.g., representativeness of GBM heterogeneity) is needed. Experiments in additional GBM lines (especially the addition of 3D primary patient-derived cell lines with known stem cell phenotype) would enhance generalizability.
We tried to explain this point in the paper (Results). As we mentioned, we tested six different GBM cell lines finding similar mRNA levels of RAP2A in all of them, and significantly lower levels than in control Astros (Fig. 3A). We decided to focus on the GBM cell line called GB5 as it grew well (better than the others) in neurosphere cell culture conditions, for further analyses. We agree that the addition of at least some of the analyses performed with the GB5 line using other lines (ideally in primary patientderive cell lines, as the reviewer mentions) would reinforce the results. Unfortunately, we cannot perform experiments in cell lines in the lab currently. We will consider all of this for future experiments.
(4) Indirect metrics (odd/even cell clusters, NUMB asymmetry) are suggestive but insufficient. Live imaging or lineage tracing would directly validate ACD frequency.
We agree that live imaging would provide further evidence. Unfortunately, we cannot approach those experiments in the lab currently.
(5) The initial microarray (n=7 GBM patients) is underpowered. While TCGA data mitigate this, the limitations of small cohorts should be explicitly addressed and need to be discussed.
We completely agree with this comment. We had available the microarray, so we used it as a first approach, just out of curiosity of knowing whether (and how) the levels of expression of those human homologs of Drosophila ACD regulators were affected in this small sample, just as starting point of the study. We were conscious of the limitations of this analysis and that is why we followed up the analysis in the datasets, on a bigger scale. We already mentioned the limitations of the array in the Discussion:
"The microarray we interrogated with GBM patient samples had some limitations. For example, not all the human genes homologs of the Drosophila ACD regulators were present (i.e. the human homologs of the determinant Numb). Likewise, we only tested seven different GBM patient samples. Nevertheless, the output from this analysis was enough to determine that most of the human genes tested in the array presented altered levels of expression"[....] In silico analyses, taking advantage of the existence of established datasets, such as the TCGA, can help to more robustly assess, in a bigger sample size, the relevance of those human genes expression levels in GBM progression, as we observed for the gene RAP2A."
(6) Conclusions rely heavily on neurosphere models. Xenograft experiments or patient-derived orthotopic models are critical to support translational relevance, and such basic research work needs to be included in journals.
We completely agree. As we already mentioned in the Discussion, the results we have obtained in this study must be definitely validated in vivo in the future using xenografts with 3D-primary patient-derived cell lines.
(7) How does RAP2A regulate NUMB asymmetry? Is the Drosophila Rap2l-Cno/aPKC pathway conserved? Rescue experiments (e.g., Cno/aPKC knockdown with RAP2A overexpression) or interaction assays (e.g., Co-IP) are needed to establish molecular mechanisms.
The mechanism by which RAP2A is regulating ACD is beyond the scope of this paper. We do not even know how Rap2l is acting in Drosophila to regulate ACD. In past years, we did analyze the function of another Drosophila small GTPase, Rap1 (homolog to human RAP1A) in ACD, and we determined the mechanism by which Rap1 was regulating ACD (including the localization of Numb): interacting physically with Cno and other small GTPases, such as Ral proteins, and in a complex with additional ACD regulators of the "apical complex" (aPKC and Par-6). Rap2l could be also interacting physically with the "Ras-association" domain of Cno (domain that binds small GTPases, such as Ras and Rap1). We have added some speculations regarding this subject in the Discussion:
"It would be of great interest in the future to determine the specific mechanism by which Rap2l/RAP2A is regulating this process. One possibility is that, as it occurs in the case of the Drosophila ACD regulator Rap1, Rap2l/RAP2A is physically interacting or in a complex with other relevant ACD modulators."
(8) Reduced stemness markers (CD133/SOX2/NESTIN) and proliferation (Ki-67) align with increased ACD. However, alternative explanations (e.g., differentiation or apoptosis) must be ruled out via GFAP/Tuj1 staining or Annexin V assays.
We agree with these possibilities. Regarding differentiation, the potential presence of increased differentiation markers would be in fact a logic consequence of an increase in ACD divisions/reduced stemness markers. Unfortunately, we cannot approach those experiments in the lab currently.
(9) The link between low RAP2A and poor prognosis should be validated in multivariate analyses to exclude confounding factors (e.g., age, treatment history).
We have now added this information in the "Results section" (page 5, lines 114-123).
(10) The broader ACD regulatory network in GBM (e.g., roles of other homologs like NUMB) and potential synergies/independence from known suppressors (e.g., TRIM3) warrant exploration.
The present study was designed as a "proof-of-concept" study to start analyzing the hypothesis that the expression levels of human homologs of known Drosophila ACD regulators might be relevant in human cancers that contain cancer stem cells, if those human homologs were also involved in modulating the mode of (cancer) stem cell division.
To extend the findings of this work to the whole ACD regulatory network would be the logic and ideal path to follow in the future.
We already mentioned this point in the Discussion:
"....it would be interesting to analyze in the future the potential consequences that altered levels of expression of the other human homologs in the array can have in the behavior of the GSCs. In silico analyses, taking advantage of the existence of established datasets, such as the TCGA, can help to more robustly assess, in a bigger sample size, the relevance of those human genes expression levels in GBM progression, as we observed for the gene RAP2A."
(11) The figures should be improved. Statistical significance markers (e.g., p-values) should be added to Figure 1A; timepoints/culture conditions should be clarified for Figure 6A.
Regarding the statistical significance markers, we have now included a Supplementary Figure (Fig. S1) with the statistical parameters used. Additionally, we have performed a hierarchical clustering of genes showing significant or notsignificant changes in their expression levels.
Regarding the experimental conditions corresponding to Figure 6A, those have now been added in more detail in "Materials and Methods" ("Pair assay and Numb segregation analysis" paragraph).
(12) Redundant Drosophila background in the Discussion should be condensed; terminology should be unified (e.g., "neurosphere" vs. "cell cluster").
As we did not mention much about Drosophila ACD and NBs in the "Introduction", we needed to explain in the "Discussion" at least some very basic concepts and information about this, especially for "non-drosophilists". We have reviewed the Discussion to maintain this information to the minimum necessary.
We have also reviewed the terminology that the Reviewer mentions and have unified it.
Reviewer #1 (Recommendations for the authors):
To improve the manuscript's impact and quality, I would recommend:
(1) Expand Cell Line Validation: Include additional GBM cell lines, particularly primary patient-derived 3D cultures, to increase the robustness of the findings.
(2) Mechanistic Exploration: Further examine the conservation of the Rap2lCno/aPKC pathway in human cells using rescue experiments or protein interaction assays.
(3) Direct Evidence of ACD: Implement live imaging or lineage tracing approaches to strengthen conclusions on ACD frequency.
(4) RNAi Specificity Validation: Clarify Rap2l RNAi specificity and its expression in neuroblasts or intermediate neural progenitors.
(5) Quantitative Analysis: Improve quantification of neurosphere size, Ki-67 expression, and apoptosis to normalize findings.
(6) Figure Clarifications: Address inconsistencies in Figures 6A and 6B and refine statistical markers in Figure 1A.
(7) Alternative In Vivo Model: Consider leveraging a Drosophila glioblastoma model as a complementary in vivo validation approach.
Addressing these points will significantly enhance the manuscript's translational relevance and overall contribution to the field.
We have been able to address points 4, 5 and 6. Others are either out of the scope of this work (2) or we do not have the possibility to carry them out at this moment in the lab (1, 3 and 7). However, we will complete these requests/recommendations in other future investigations.
Reviewer #2 (Recommendations for the authors):
Major Revision /insufficient required to address methodological and mechanistic gaps.
(1) Enhance Clinical Relevance
Validate RAP2A's prognostic significance across multiple GBM subtypes (e.g., MES, DNA-methylation subtypes) using datasets like TCGA and Gravendeel to confirm specificity.
Perform multivariate survival analyses to rule out confounding factors (e.g., patient age, treatment history).
(2) Strengthen Mechanistic Insights
Investigate whether the Rap2l-Cno/aPKC pathway is conserved in human GBM through rescue experiments (e.g., RAP2A overexpression with Cno/aPKC knockdown) or interaction assays (e.g., Co-IP).
Use live-cell imaging or lineage tracing to directly validate ACD frequency instead of relying on indirect metrics (odd/even cell clusters, NUMB asymmetry).
(3) Improve Model Systems & Experimental Design
Justify the selection of GB5 cells and include additional GBM cell lines, particularly 3D primary patient-derived cell models, to enhance generalizability.
It is essential to perform xenograft or orthotopic patient-derived models to support translational relevance.
(5) Address Alternative Interpretations
Rule out other potential effects of RAP2A knockdown (e.g., differentiation or apoptosis) using GFAP/Tuj1 staining or Annexin V assays.
Explore the broader ACD regulatory network in GBM, including interactions with NUMB and TRIM3, to contextualize findings within known tumor-suppressive pathways.
(6) Improve Figures & Clarity
Add statistical significance markers (e.g., p-values) in Figure 1A and clarify timepoints/culture conditions for Figure 6A.
Condense redundant Drosophila background in the discussion and ensure consistent terminology (e.g., "neurosphere" vs. "cell cluster").
We have been able to address points 1, partially 3 and 6. Others are either out of the scope of this work or we do not have the possibility to carry them out at this moment in the lab. However, we are very interested in completing these requests/recommendations and we will approach that type of experiments in other future investigations.
eLife Assessment
This paper describes Unbend - a new method for measuring and correcting motions in cryo-EM images, with a particular emphasis on more challenging in situ samples such as lamella and whole cells. The method, which fits a B-spline model using cross-correlation-based local patch alignment of micrograph frames, represents a valuable tool for the cryo-EM community. The authors elegantly use 2D template matching to provide solid evidence that Unbend outperforms the previously reported method of Unblur by the same authors. The paper would benefit from the inclusion of a similar analysis for established alternative methods, such as MotionCor2.
Reviewer #1 (Public review):
Kong et al.'s work describes a new approach that does exactly what the title states: "Correction of local beam-induced sample motion in cryo-EM images using a 3D spline model." I find the method appropriate, logical, and well-explained. Additionally, the work suggests using 2DTM-related measurements to quantify the improvement of the new method compared to the old one in cisTEM, Unblur. I find this part engaging; it is straightforward, accurate, and, of course, the group has a strong command of 2DTM, presenting a thorough study.
However, everything in the paper (except some correct general references) refers to comparisons with the full-frame approach, Unblur. Still, we have known for more than a decade that local correction approaches perform better than global ones, so I do not find anything truly novel in their proposal of using local methods (the method itself- Unbend- is new, but many others have been described previously). In fact, the use of 2DTM is perhaps a more interesting novelty of the work, and here, a more systematic study comparing different methods with these proposed well-defined metrics would be very valuable. As currently presented, there is no doubt that it is better than an older, well-established approach, and the way to measure "better" is very interesting, but there is no indication of how the situation stands regarding newer methods.
Regarding practical aspects, it seems that the current implementation of the method is significantly slower than other patch-based approaches. If its results are shown to exceed those of existing local methods, then exploring the use of Unbend, possibly optimizing its code first, could be a valuable task. However, without more recent comparisons, the impact of Unbend remains unclear.
Reviewer #2 (Public review):
Summary:
The authors present a new method, Unbend, for measuring motion in cryo-EM images, with a particular emphasis on more challenging in situ samples such as lamella and whole cells<br /> (that can be more prone to overall motion and/or variability in motion across a field of view). Building on their previous approach of full-frame alignment (Unblur), they now perform full-frame alignment followed by patch alignment, and then use these outputs to generate a 3D cubic spline model of the motion. This model allows them to estimate a continuous, per-pixel shift field for each movie frame that aims to better describe complex motions and so ultimately generate improved motion-corrected micrographs. Performance of Unbend is evaluated using the 2D template matching (2DTM) method developed previously by the lab, and results are compared to using full-frame correction alone. Several different in situ samples are used for evaluation, covering a broad range that will be of interest to the rapidly growing in situ cryo-EM community.
Strengths:
The method appears to be an elegant way of describing complex motions in cryo-EM samples, and the authors present convincing data that Unbend generally improves SNR of aligned micrographs as well as increases detection of particles matching the 60S ribosome template when compared to using full-frame correction alone. The authors also give interesting insights into how different areas of a lamella behave with respect to motion by using Unbend on a montage dataset collected previously by the group. There is growing interest in imaging larger areas of in situ samples at high resolution, and these insights contribute valuable knowledge. Additionally, the availability of data collected in this study through the EMPIAR repository will be much appreciated by the field.
Weaknesses:
While the improvements with Unbend vs. Unblur appear clear, it is less obvious whether Unbend provides substantial gains over patch motion correction alone (the current norm in the field). It might be helpful for readers if this comparison were investigated for the in situ datasets. Additionally, the authors are open that in cases where full motion correction already does a good job, the extra degrees of freedom in Unbend can perhaps overfit the motions, making the corrections ultimately worse. I wonder if an adaptive approach could be explored, for example, using the readout from full-frame or patch correction to decide whether a movie should proceed to the full Unbend pipeline, or whether correction should stop at the patch estimation stage.
Reviewer #3 (Public review):
Summary
Kong and coauthors describe and implement a method to correct local deformations due to beam-induced motion in cryo-EM movie frames. This is done by fitting a 3D spline model to a stack of micrograph frames using cross-correlation-based local patch alignment to describe the deformations across the micrograph in each frame, and then computing the value of the deformed micrograph at each pixel by interpolating the undeformed micrograph at the displacement positions given by the spline model. A graphical interface in cisTEM allows the user to visualise the deformations in the sample, and the method has been proven to be successful by showing improvements in 2D template matching (2DTM) results on the corrected micrographs using five in situ samples.
Impact
This method has great potential to further streamline the cryo-EM single particle analysis pipeline by shortening the required processing time as a result of obtaining higher quality particles early in the pipeline, and is applicable to both old and new datasets, therefore being relevant to all cryo-EM users.
Strengths
(1) One key idea of the paper is that local beam induced motion affects frames continuously in space (in the image plane) as well as in time (along the frame stack), so one can obtain improvements in the image quality by correcting such deformations in a continuous way (deformations vary continuously from pixel to pixel and from frame to frame) rather than based on local discrete patches only. 3D splines are used to model the deformations: they are initialised using local patch alignments and further refined using cross-correlation between individual patch frames and the average of the other frames in the same patch stack.
(2) Another strength of the paper is using 2DTM to show that correcting such deformations continuously using the proposed method does indeed lead to improvements. This is shown using five in situ datasets, where local motion is quantified using statistics based on the estimated motions of ribosomes.
Weaknesses
(1) While very interesting, it is not clear how the proposed method using 3D splines for estimating local deformations compares with other existing methods that also aim to correct local beam-induced motion by approximating the deformations throughout the frames using other types of approximation, such as polynomials, as done, for example MotionCor2.
(2) The use of 2DTM is appropriate, and the results of the analysis are enlightening, but one shortcoming is that some relevant technical details are missing. For example, the 2DTM SNR is not defined in the article, and it is not clear how the authors ensured that no false positives were included in the particles counted before and after deformation correction. The Jupyter notebooks where this analysis was performed have not been made publicly available.
(3) It is also not clear how the proposed deformation correction method is affected by CTF defocus in the different samples (are the defocus values used in the different datasets similar or significantly different?) or if there is any effect at all.
eLife Assessment
This study identifies the Periportal Lamellar Complex (PLC), an important new structure revealed by a novel 3D imaging method. However, the evidence supporting its distinct cellular identity and functional role is currently incomplete, as it relies on transcriptomic re-analysis and correlation without direct experimental validation. Addressing the key issues of methodological rigor and providing functional evidence is essential to fully substantiate these significant claims.
Reviewer #1 (Public review):
Summary:
In this manuscript, Chengjian Zhao et al. focused on the interactions between vascular, biliary, and neural networks in the liver microenvironment, addressing the critical bottleneck that the lack of high-resolution 3D visualization has hindered understanding of these interactions in liver disease.
Strengths:
This study developed a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized CUBIC tissue clearing. This method enables the simultaneous 3D visualization of spatial networks of the portal vein, hepatic artery, bile ducts, and central vein in the mouse liver. The authors reported a perivascular structure termed the Periportal Lamellar Complex (PLC), which is identified along the portal vein axis. This study clarifies that the PLC comprises CD34⁺Sca-1⁺ dual-positive endothelial cells with a distinct gene expression profile, and reveals its colocalization with terminal bile duct branches and sympathetic nerve fibers under physiological conditions.
Weaknesses:
This manuscript is well-written, organized, and informative. However, there are some points that need to be clarified.
(1) After MCNP-dye injection, does it remain in the blood vessels, adsorb onto the cell surface, or permeate into the cells? Does the MCNP-dye have cell selectivity?
(2) All MCNP-dyes were injected after the mice were sacrificed, and the mice's livers were fixed with PFA. After the blood flow had ceased, how did the authors ensure that the MCNP-dyes were fully and uniformly perfused into the microcirculation of the liver?
(3) It is advisable to present additional 3D perspective views in the article, as the current images exhibit very weak 3D effects. Furthermore, it would be better to supplement with some videos to demonstrate the 3D effects of the stained blood vessels.
(4) In Figure 1-I, the authors used MCNP-Black to stain the central veins; however, in addition to black, there are also yellow and red stains in the image. The authors need to explain what these stains are in the legend.
(5) There is a typo in the title of Figure 4F; it should be "stem cell".
(6) Nuclear staining is necessary in immunofluorescence staining, especially for Figure 5e. This will help readers distinguish whether the green color in the image corresponds to cells or dye deposits.
Reviewer #2 (Public review):
Summary:
The present manuscript of Xu et al. reports a novel clearing and imaging method focusing on the liver. The authors simultaneously visualized the portal vein, hepatic artery, central vein, and bile duct systems by injecting metal compound nanoparticles (MCNPs) with different colors into the portal vein, heart left ventricle, inferior vena cava, and the extrahepatic bile duct, respectively. The method involves: trans-cardiac perfusion with 4% PFA, the injection of MCNPs with different colors, clearing with the modified CUBIC method, cutting 200 micrometer thick slices by vibratome, and then microscopic imaging. The authors also perform various immunostaining (DAB or TSA signal amplification methods) on the tissue slices from MCNP-perfused tissue blocks. With the application of this methodical approach, the authors report dense and very fine vascular branches along the portal vein. The authors name them as 'periportal lamellar complex (PLC)' and report that PLC fine branches are directly connected to the sinusoids. The authors also claim that these structures co-localize with terminal bile duct branches and sympathetic nerve fibers, and contain endothelial cells with a distinct gene expression profile. Finally, the authors claim that PLC-s proliferate in liver fibrosis (CCl4 model) and act as a scaffold for proliferating bile ducts in ductular reaction and for ectopic parenchymal sympathetic nerve sprouting.
Strengths:
The simultaneous visualization of different hepatic vascular compartments and their combination with immunostaining is a potentially interesting novel methodological approach.
Weaknesses:
This reviewer has several concerns about the validity of the microscopic/morphological findings as well as the transcriptomics results. In this reviewer's opinion, the introduction contains overstatements regarding the potential of the method, there are severe caveats in the method descriptions, and several parts of the Results are not fully supported by the documentation. Thus, the conclusions of the paper may be critically viewed in their present form and may need reconsideration by the authors.
Reviewer #3 (Public review):
Summary:
In the reviewed manuscript, researchers aimed to overcome the obstacles of high-resolution imaging of intact liver tissue. They report successful modification of the existing CUBIC protocol into Liver-CUBIC, a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized liver tissue clearing, significantly reducing clearing time and enabling simultaneous 3D visualization of the portal vein, hepatic artery, bile ducts, and central vein spatial networks in the mouse liver. Using this novel platform, the researchers describe a previously unrecognized perivascular structure they termed Periportal Lamellar Complex (PLC), regularly distributed along the portal vein axis. The PLC originates from the portal vein and is characterized by a unique population of CD34⁺Sca-1⁺ dual-positive endothelial cells. Using available scRNAseq data, the authors assessed the CD34⁺Sca-1⁺ cells' expression profile, highlighting the mRNA presence of genes linked to neurodevelopment, biliary function, and hematopoietic niche potential. Different aspects of this analysis were then addressed by protein staining of selected marker proteins in the mouse liver tissue. Next, the authors addressed how the PLC and biliary system react to CCL4-induced liver fibrosis, implying PLC dynamically extends, acting as a scaffold that guides the migration and expansion of terminal bile ducts and sympathetic nerve fibers into the hepatic parenchyma upon injury.
The work clearly demonstrates the usefulness of the Liver-CUBIC technique and the improvement of both resolution and complexity of the information, gained by simultaneous visualization of multiple vascular and biliary systems of the liver at the same time. The identification of PLC and the interpretation of its function represent an intriguing set of observations that will surely attract the attention of liver biologists as well as hepatologists; however, some claims need more thorough assessment by functional experimental approaches to decipher the functional molecules and the sequence of events before establishing the PLC as the key hub governing the activity of biliary, arterial, and neuronal liver systems. Similarly, the level of detail of the methods section does not appear to be sufficient to exactly recapitulate the performed experiments, which is of concern, given that the new technique is a cornerstone of the manuscript.
Nevertheless, the work does bring a clear new insight into the liver structure and functional units and greatly improves the methodological toolbox to study it even further, and thus fully deserves the attention of readers.
Strengths:
The authors clearly demonstrate an improved technique tailored to the visualization of the liver vasulo-biliary architecture in unprecedented resolution.
This work proposes a new biological framework between the portal vein, hepatic arteries, biliary tree, and intrahepatic innervation, centered at previously underappreciated protrusions of the portal veins - the Periportal Lamellar Complexes (PLCs).
Weaknesses:
Possible overinterpretation of the CD34+Sca1+ findings was built on re-analysis of one scRNAseq dataset.
Lack of detail in the materials and methods section greatly limits the usefulness of the new technique to other researchers.
This procedure details the responsibilities for providing technical support to vessels and the responsibilities of the onboard engineering team.
Remove the text
eLife Assessment
This study presents valuable findings on the role of KLF6 in in vitro endothelial cells exposed to altered (high or low) shear stress with a customized microfluidic device to investigate mechanisms of atherosclerosis. The finding that altered shear stress results in endothelial cell ferroptosis through reduced expression of KLF6 is compelling and adds a new layer of complexity to the pathogenesis of atherosclerotic plaques. However, the inclusion of an arterial cell line and re-evaluation of the statistical tests used would strengthen the authors' conclusions.
Reviewer #1 (Public review):
Summary:
The authors used an in vitro microfluidic system where HUVECs are exposed to high, low, or physiologic (normal) shear stress to demonstrate that both high and low shear stress for 24 hours resulted in decreased KLF6 expression, decreased lipid peroxidation, and increased cell death, which was reversible upon treatment with Fer-1, the ferroptosis inhibitor. RNA sequencing (LSS vs normal SS) revealed decreased steroid synthesis and UPR signaling in low shear stress conditions, which they confirmed by showing reduced expression of proteins that mitigate ER stress under both LSS and HSS. Decreased KLF6 expression after exposure to HSS/LSS was associated with decreased expression of regulators of ER stress (PERK, BiP, MVD), which was restored with KLF6 overexpression. Overexpression of KLF6 also restored SLC7A11 expression, Coq10, and reduced c11 bodipy oxidation state- all markers of lipid peroxidation and ferroptosis. The authors then used vascular smooth muscle cells (atherosclerotic model) with HUVECs and monocytes to show that KLF6 overexpression reduces the adhesion of monocytes and lipid accumulation in conditions of low shear stress.
Strengths:
(1) The use of a microfluidic device to simulate shear stress while keeping the pressure constant when varying the shear stress applied is improved and more physiologic compared to traditional cone and shearing devices. Similarly, the utilization of both low and high shear stress in most experiments is a strength.
(2) This study provides a link between disturbed shear stress and ferroptosis, which is novel, and fits nicely with existing knowledge that endothelial cell ferroptosis promotes atherosclerosis. This concept was also recently reported in September 2025, when a publication also demonstrated that LSS triggers ferroptosis in vascular endothelial cells (PMID: 40939914), which partly validates these findings.
Weaknesses:
(1) While HUVECs are commonly used in endothelial in vitro studies, it would be preferable to confirm the findings using an arterial cell line, such as human coronary artery cells, when studying mechanisms of early atherosclerosis. Furthermore, physiologic arterial shear stress is higher than venous shear stress, and different vascular beds have varying responses to altered shear stress; as such, the up- and downregulated pathways in HUVECs should be confirmed in an arterial system.
(2) The authors provide convincing evidence of disturbances in shear stress inducing endothelial ferroptosis with assays for impaired lipid peroxidation and increased cell death that was reversed with a ferroptosis inhibitor. However, more detailed characterization of ferroptosis with iron accumulation assays, as well as evaluating GPX4 activity as a consequence of the impaired mevalonate pathway, and testing for concomitant apoptosis in addition to ferroptosis, would add to the data.
(3) The authors state that KLF2 and KLF4 are not amongst the differentially expressed genes downregulated by reduced shear stress, which is contrary to previous data, where both KLF2 and KLF4 are well studied to be upregulated by physiologic laminar shear stress. While this might be due to the added pressure in their microfluidic system, it also might be due to changes in gene expression over time. In this case, a time course experiment would be needed. It is possible that KLF2, KLF4 and KLF6 are all reduced in low (and high) shear stress and cooperatively regulate the endothelial cell phenotype. Both KLF2 and KLF4 have been shown to be protective against atherosclerosis.
Reviewer #2 (Public review):
Summary:
The manuscript by Cui et al. titled "abnormal shear stress induces ferroptosis in endothelial cells via KLF6 downregulation" investigated in a microfluidic device the effect of 24-hour low, medium, and high shear stress levels upon human vein endothelial cells. The authors found that KLF6 is an important regulator of endothelial cell ferroptosis through the BiP-PERK-Slc7a11 and MVD-ID11-CoQ10 axis under both low and high shear stress, postulating this may explain the spatial preference of atherosclerosis at bifurcations of the arteries.
Strengths:
The main strength of the study is the use of a microfluidic device within which the authors could vary the shear stress (low, medium, high), whilst keeping fluid pressure near the physiological range of 70 mmHg. Deciding to focus on transcription factors that respond to shear stress, the authors found KLF6 in their dataset, for which they provide compelling evidence that endothelial cell ferroptosis is triggered by both excessive and insufficient shear stress, inversely correlating with KLF6 expression. Importantly, it was demonstrated that cell death in endothelial cells during HSS and LSS was prevented through the addition of Fer-1, supporting the role of ferroptosis. Moreso, the importance of KLF6 as an essential regulator was demonstrated through KLF6 overexpression.
Weaknesses:
There are some major concerns with the results:
(1) Inappropriate statistical tests were used (i.e., an unpaired t-test cannot be used to compare more than two groups).<br /> (2) Inconsistencies in western blot normalization as different proteins seem to have been used (GAPDH and B-actin) without specifying which is used when and why this differs.<br /> (3) Absence of transcriptomic analysis on HSS-exposed endothelial cells (which is not explained).
Moreso, the conclusions are predominantly based on an in vitro microfluidic chip model seeded with HUVECs. Although providing mechanistic insight into the effects of shear stress on (venous) endothelial cells, it does not recapitulate the in vivo complexity. The absence of validation (a.o. levels of KLF6) in clinical samples and/or animal models limits the translatability of the reported findings towards atherosclerosis. Among others, assessing the spatial heterogeneity of KLF6 abundance in atherosclerotic plaques depending on its proximity to arterial bifurcations may be interesting.
Points to be addressed:
(1) As a statistical test, the authors report having used unpaired t-tests; however, often three groups are compared for which t-tests are inadequate. This is faulty as, amongst other things, it does not take multiple comparison testing into account.
(2) Both B-Actin and GAPDH seem to have been used for protein-level normalization. Why? The Figure 2HL first panel reports B-actin, whereas the other three report GAPDH. The same applies to Figures 3E-F, where both are shown, and it is not mentioned which of the two has been used. Moreso, uncropped blots seem to be unavailable as supplementary data for proper review. These should be provided as supplementary data.
(3) LSS and MSS were compared based on transcriptomic analysis. Conversely, RNA sequencing was not reported for the HSS. Why is this data missing? It would be valuable to assess transcriptomics following HSS, and also to allow transcriptomic comparison of LSS and HSS.
(4) Actual sample sizes should be reported rather than "three or more". Moreso, it would be beneficial to show individual datapoints in bar graphs rather than only mean with SD if sample sizes are below 10 (e.g., Figures 1B-H, Figure 2G, etc.).
(5) The authors claim that by modifying the thickness of the middle layer, shear stress could be modified, whilst claiming to keep on-site pressure within physiological ranges (approx. 70 mmHg) as a hallmark of their microfluidic devices. Has it been experimentally verified that pressures indeed remain around 70 mmHg?
(6) A coculture model (VSMC, EC, monocytes) is mentioned in the last part of the results section without any further information. Information on this model should be provided in the methods section (seeding, cell numbers, etc.). Moreover, comparison of LSS vs LSS+KLF6 OE and HSS vs HSS+KLF6 OE is shown. It would benefit the interpretation of the outcomes if MSS were also shown. I twould also be beneficial to demonstrate differences between LSS, MSS, and HSS in this coculture model (without KLF6 OE).
(7) The experiments were solely performed with a venous endothelial cell line (HUVECs). Was the use of an arterial endothelial cell line considered? It may translate better towards atherosclerosis, which occurs within arteries. HUVECs are not accustomed to the claimed near-physiological pressures.
My tax status with the government is as an “auteur/artiste”—an author/artist. With this declaration, I have one very practical thing: health insurance.
Registering with l'Urssaf as an "artiste-auteur" (or micro-entrepreneur), which aligns one with a specific régime for the payment of social charges, automate registration with CPAM, the administrative arm of the French universal healthcare system.
eLife Assessment
This important study provides new insights into the synchronization of ripple oscillations in the hippocampus, both within and across hemispheres. Using carefully designed statistical methods, it presents compelling evidence that synchrony is significantly higher within a hemisphere than across. This study will be of interest to neuroscientists studying the hippocampus and memory.
Reviewer #2 (Public review):
Summary
The authors completed a statistically rigorous analysis of the synchronization of sharp-wave ripples in the hippocampal CA1 across and within hemispheres. They used a publicly available dataset (collected in the Buzsaki lab) from 4 rats (8 sessions) recorded with silicon probes in both hemispheres. Each session contained approximately 8 hours of activity recorded during rest. The authors found that the characteristics of ripples did not differ between hemispheres, and that most ripples occurred almost simultaneously on all probe shanks within a hemisphere as well as across hemispheres. The differences in amplitude and exact timing of ripples between recording sites increased slightly with distance between recording sites. However, the phase coupling of ripples (in the 100-250 Hz range), changed dramatically with distance between recording sites. Ripples in opposite hemispheres were about 90% less coupled than ripples on nearby tetrodes in the same hemisphere. Phase coupling also decreased with distance within the hemisphere. Finally, pyramidal cell and interneuron spikes were coupled to the local ripple phase and less so to ripples at distant sites or the opposite hemisphere.
The authors also analyzed the changes in ripple coupling in relation to a couple of behavioral variables. Interestingly, while exposure to a novel track increased ripple abundance by ~5%, it did not change any form of ripple coupling within or between hemispheres.
Strengths
The analysis was well-designed and rigorous. The authors used statistical tests well suited to the hypotheses being tested, and clearly explained these tests. The paper is very clearly written, making it easy to understand and reproduce the analysis. The authors included an excellent review of the literature to explain the motivation for their study.
Weaknesses
The authors have addressed all of my concerns and recommendations.
This paper presents an important and unique analysis of ripple coupling. The same method could be used in the future to analyze the effects of other behavioral variables, such as satiety versus hunger, sleep deprivation, or enrichment, to address potential functions and causes of ripple coupling.
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
Summary:
In this manuscript, the authors analyze electrophysiological data recorded bilaterally from the rat hippocampus to investigate the coupling of ripple oscillations across the hemispheres. Commensurate with the majority of previous research, the authors report that ripples tend to co-occur across both hemispheres. Specifically, the amplitude of ripples across hemispheres is correlated but their phase is not. These data corroborate existing models of ripple generation suggesting that CA3 inputs (coordinated across hemispheres via the commisural fibers) drive the sharp-wave component while the individual ripple waves are the result of local interactions between pyramidal cells and interneurons in CA1.
Strengths:
The manuscript is well-written, the analyses well-executed and the claims are supported by the data.
Weaknesses:
One question left unanswered by this study is whether information encoded by the right and left hippocampi is correlated.
Thank you for raising this important point. While our study demonstrates ripple co-occurrence across hemispheres, we did not directly assess whether the information encoded in each hippocampus is correlated. Addressing this question would require analyses of coordinated activity patterns, such as neuronal assemblies formed during novelty exposure, which falls beyond the scope of the present study. However, we agree this is an important avenue for future work, and we now acknowledge this limitation and outlined it as a future direction in the Conclusion section (lines 796–802).
Reviewer #2 (Public review):
Summary:
The authors completed a statistically rigorous analysis of the synchronization of sharp-wave ripples in the hippocampal CA1 across and within hemispheres. They used a publicly available dataset (collected in the Buzsaki lab) from 4 rats (8 sessions) recorded with silicon probes in both hemispheres. Each session contained approximately 8 hours of activity recorded during rest. The authors found that the characteristics of ripples did not differ between hemispheres, and that most ripples occurred almost simultaneously on all probe shanks within a hemisphere as well as across hemispheres. The differences in amplitude and exact timing of ripples between recording sites increased slightly with the distance between recording sites. However, the phase coupling of ripples (in the 100-250 Hz range), changed dramatically with the distance between recording sites. Ripples in opposite hemispheres were about 90% less coupled than ripples on nearby tetrodes in the same hemisphere. Phase coupling also decreased with distance within the hemisphere. Finally, pyramidal cell and interneuron spikes were coupled to the local ripple phase and less so to ripples at distant sites or the opposite hemisphere.
Strengths:
The analysis was well-designed and rigorous. The authors used statistical tests well suited to the hypotheses being tested, and clearly explained these tests. The paper is very clearly written, making it easy to understand and reproduce the analysis. The authors included an excellent review of the literature to explain the motivation for their study.
Weaknesses:
The authors state that their findings (highly coincident ripples between hemispheres), contradict other findings in the literature (in particular the study by Villalobos, Maldonado, and Valdes, 2017), but fail to explain why this large difference exists. They seem to imply that the previous study was flawed, without examining the differences between the studies.
The paper fails to mention the context in which the data was collected (the behavior the animals performed before and after the analyzed data), which may in fact have a large impact on the results and explain the differences between the current study and that by Villalobos et al. The Buzsaki lab data includes mice running laps in a novel environment in the middle of two rest sessions. Given that ripple occurrence is influenced by behavior, and that the neurons spiking during ripples are highly related to the prior behavioral task, it is likely that exposure to novelty changed the statistics of ripples. Thus, the authors should analyze the pre-behavior rest and post-behavior rest sessions separately. The Villalobos et al. data, in contrast, was collected without any intervening behavioral task or novelty (to my knowledge). Therefore, I predict that the opposing results are a result of the difference in recent experiences of the studied rats, and can actually give us insight into the memory function of ripples.
We appreciate this thoughtful hypothesis and have now addressed it explicitly. Our main analysis was conducted on 1-hour concatenated SWS epochs recorded before any novel environment exposure (baseline sleep). This was not clearly stated in the original manuscript, so we have now added a clarifying paragraph (lines 131–143). The main findings therefore remain unchanged.
To directly test the reviewer’s hypothesis, we performed the suggested comparison between pre- and post-maze rest sessions, including maze-type as a factor. These new analyses are now presented in a dedicated Results subsection (lines 475 - 493) and in Supplementary Figure 5.1. While we observed a modest increase in ripple abundance after the maze sessions — consistent with known experienced-dependent changes in ripple occurrence — the key findings of interhemispheric synchrony remained unchanged. Both pre- and post-maze sleep sessions showed robust bilateral time-locking of ripple events and similar dissociations between phase and amplitude coupling across hemispheres.
In one figure (5), the authors show data separated by session, rather than pooled. They should do this for other figures as well. There is a wide spread between sessions, which further suggests that the results are not as widely applicable as the authors seem to think. Do the sessions with small differences between phase coupling and amplitude coupling have low inter-hemispheric amplitude coupling, or high phase coupling? What is the difference between the sessions with low and high differences in phase vs. amplitude coupling? I noticed that the Buzsaki dataset contains data from rats running either on linear tracks (back and forth), or on circular tracks (unidirectionally). This could create a difference in inter-hemisphere coupling, because rats running on linear tracks would have the same sensory inputs to both hemispheres (when running in opposite directions), while rats running on a circular track would have different sensory inputs coming from the right and left (one side would include stimuli in the middle of the track, and the other would include closer views of the walls of the room). The synchronization between hemispheres might be impacted by how much overlap there was in sensory stimuli processed during the behavior epoch.
Thank you for this insightful suggestion. In our new analyses comparing pre- and post-maze sessions, we have also addressed this question. Supplementary Figures 4.1 and 5.1 (E-F) present coupling metrics averaged per session and include coding for maze type. Additionally, we have incorporated the reviewer’s hypothesis regarding sensory input differences and their potential impact on inter-hemispheric synchronization into a new Results subsection (lines 475–493).
The paper would be a lot stronger if the authors analyzed some of the differences between datasets, sessions, and epochs based on the task design, and wrote more about these issues. There may be more publicly available bi-hemispheric datasets to validate their results.
To further validate our findings, we have analyzed another publicly available dataset that includes bilateral CA1 recordings (https://crcns.org/data-sets/hc/hc-18). We have added a description of this dataset and our analysis approach in the Methods section (lines 119–125 and 144-145), and present the corresponding results in a new Supplementary Figure (Supplementary Figure 4.2). These new analyses replicated our main findings, confirming robust interhemispheric time-locking of ripple events and a greater dissociation between phase and amplitude coupling in ipsilateral versus contralateral recordings.
Reviewer #1 (Recommendations for the authors):
My only suggestion is that the introduction can be shortened. The authors discuss in great length literature linking ripples and memory, although the findings in the paper are not linked to memory. In addition, ripples have been implicated in non-mnemonic functions such as sleep and metabolic homeostasis.
The reviewer`s suggestion is valid and aligns with the main message of our paper. However, we believe that the relationship between ripples and memory has been extensively discussed in the literature, sometimes overshadowing other important functional roles (based on the reviewer’s comment, we now also refer to non-mnemonic functions of ripples in the revised introduction [lines 87–89]). Thus, we find it important to retain this context because highlighting the publication bias towards mnemonic interpretations helps frame the need for studies like ours that revisit still incompletely understood basic ripple mechanisms.
We also note that, based on a suggestion from reviewer 2, we have supplemented our manuscript with a new figure demonstrating ripple abundance increases during SWS following novel environment exposure (Supplementary Figure 5.1), linking it to memory and replicating the findings of Eschenko et al. (2008), though we present this result as a covariate, aimed at controlling for potential sources of variation in ripple synchronization.
Reviewer #2 (Recommendations for the authors):
It would be useful to include more information about the analyzed dataset in the methods section, e.g. how long were the recordings, how many datasets per rat, did the authors analyze the entire recording epoch or sub-divide it in any way, how many ripples were detected per recording (approximately).
We have now included more detailed information in the Methods section (lines 104 - 145).
A few of the references to sub-figures are mislabeled (e.g. lines 327-328).
Thank you for noticing these inconsistencies. We have carefully reviewed and corrected all figure sub-panel labels and references throughout the manuscript.
In Figure 7 C&D, are the neurons on the left sorted by contralateral ripple phase? It doesn't look like it. It would be easier to compare to ipsilateral if they were.
In Figures 7C and 7D, neurons are sorted by their ipsilateral peak ripple phase, with the contralateral data plotted using the same ordering to facilitate comparison. To avoid confusion, we have clarified this explicitly in the figure legend and corresponding main text (lines 544–550).
In Figure 6, using both bin sizes 50 and 100 doesn't contribute much.
We used both 50 ms and 100 ms bin sizes to directly compare with previous studies (Villalobos et al. 2017 used 5 ms and 100 ms; Csicsvari et al. 2000 used 5–50 ms). Because the proportion of coincident ripples is a non-decreasing function of the window size, larger bins can inflate coincidence measures. Including a mid-range bin of 50 ms allowed us to show that high coincidence levels are reached well before the 100 ms upper bound, supporting that the 100 ms window is not an overshoot. We have added clarification on this point in the Methods section on ripple coincidence (lines 204–212).
eLife Assessment
This important study combines EEG, neural networks and multivariate pattern analysis to show that real-world size, retinal size and real-world depth are represented at different latencies. The evidence presented is convincing and the work will be of broader interest to the experimental and computational vision community.
Reviewer #1 (Public review):
Lu & Golomb combined EEG, artificial neural networks, and multivariate pattern analyses to examine how different visual variables are processed in the brain. The conclusions of the paper are mostly well supported.
The authors find that not only real-world size is represented in the brain (which was known), but both retinal size and real-world depth is represented, at different time points or latencies, which may reflect different stages of processing. Prior work has not been able to answer the question of real-world depth due to stimuli used. The authors made this possible by assess real-world depth and testing it with appropriate methodology, accounting for retinal and real-world size. The methodological approach combining behavior, RSA, and ANNs is creative and well thought out to appropriately assess the research questions, and the findings may be very compelling if backed up with some clarifications and further analyses.
The work will be of interest to experimental and computational vision scientists, as well as the broader computational cognitive neuroscience community as the methodology is of interest and the code is or will be made available. The work is important as it is currently not clear what the correspondence between many deep neural network models are and the brain are, and this work pushes our knowledge forward on this front. Furthermore, the availability of methods and data will be useful for the scientific community.
Reviewer #3 (Public review):
The authors used an open EEG dataset of observers viewing real-world objects. Each object had a real-world size value (from human rankings), a retinal size value (measured from each image), and a scene depth value (inferred from the above). The authors combined the EEG and object measurements with extant, pre-trained models (a deep convolutional neural network, a multimodal ANN, and Word2vec) to assess the time course of processing object size (retinal and real-world) and depth. They found that depth was processed first, followed by retinal size, and then real-world size. The depth time course roughly corresponded to the visual ANNs, while the real-world size time course roughly corresponded to the more semantic models.
The time course result for the three object attributes is very clear and a novel contribution to the literature. The authors have revised the ANN motivations to increase clarity. Additionally, the authors have appropriately toned down some of the language about novelty, and the addition of a noise ceiling has helped the robustness of the work.
While I appreciate the addition of Cornet in the Supplement, I am less compelled by the authors' argument for Word2Vec over LLMs for "pure" semantic embeddings. While I'm not digging in on this point, this choice may prematurely age this work.
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
Lu & Golomb combined EEG, artificial neural networks, and multivariate pattern analyses to examine how different visual variables are processed in the brain. The conclusions of the paper are mostly well supported, but some aspects of methods and data analysis would benefit from clarification and potential extensions.
The authors find that not only real-world size is represented in the brain (which was known), but both retinal size and real-world depth are represented, at different time points or latencies, which may reflect different stages of processing. Prior work has not been able to answer the question of real-world depth due to the stimuli used. The authors made this possible by assessing real-world depth and testing it with appropriate methodology, accounting for retinal and real-world size. The methodological approach combining behavior, RSA, and ANNs is creative and well thought out to appropriately assess the research questions, and the findings may be very compelling if backed up with some clarifications and further analyses.
The work will be of interest to experimental and computational vision scientists, as well as the broader computational cognitive neuroscience community as the methodology is of interest and the code is or will be made available. The work is important as it is currently not clear what the correspondence between many deep neural network models and the brain is, and this work pushes our knowledge forward on this front. Furthermore, the availability of methods and data will be useful for the scientific community.
Reviewer #2 (Public Review):
Summary:
This paper aims to test if neural representations of images of objects in the human brain contain a 'pure' dimension of real-world size that is independent of retinal size or perceived depth. To this end, they apply representational similarity analysis on EEG responses in 10 human subjects to a set of 200 images from a publicly available database (THINGS-EEG2), correlating pairwise distinctions in evoked activity between images with pairwise differences in human ratings of real-world size (from THINGS+). By partialling out correlations with metrics of retinal size and perceived depth from the resulting EEG correlation time courses, the paper claims to identify an independent representation of real-world size starting at 170 ms in the EEG signal. Further comparisons with artificial neural networks and language embeddings lead the authors to claim this correlation reflects a relatively 'high-level' and 'stable' neural representation.
Strengths:
The paper features insightful figures/illustrations and clear figures.
The limitations of prior work motivating the current study are clearly explained and seem reasonable (although the rationale for why using 'ecological' stimuli with backgrounds matters when studying real-world size could be made clearer; one could also argue the opposite, that to get a 'pure' representation of the real-world size of an 'object concept', one should actually show objects in isolation).
The partial correlation analysis convincingly demonstrates how correlations between feature spaces can affect their correlations with EEG responses (and how taking into account these correlations can disentangle them better).
The RSA analysis and associated statistical methods appear solid.
Weaknesses:
The claim of methodological novelty is overblown. Comparing image metrics, behavioral measurements, and ANN activations against EEG using RSA is a commonly used approach to study neural object representations. The dataset size (200 test images from THINGS) is not particularly large, and neither is comparing pre-trained DNNs and language models, or using partial correlations.
Thanks for your feedback. We agree that the methods used in our study – such as RSA, partial correlations, and the use of pretrained ANN and language models – are indeed well-established in the literature. We therefore revised the manuscript to more carefully frame our contribution: rather than emphasizing methodological novelty in isolation, we now highlight the combination of techniques, the application to human EEG data with naturalistic images, and the explicit dissociation of real-world size, retinal size, and depth representations as the primary strengths of our approach. Corresponding language in the Abstract, Introduction, and Discussion has been adjusted to reflect this more precise positioning:
(Abstract, line 34 to 37) “our study combines human EEG and representational similarity analysis to disentangle neural representations of object real-world size from retinal size and perceived depth, leveraging recent datasets and modeling approaches to address challenges not fully resolved in previous work.”
(Introduction, line 104 to 106) “we overcome these challenges by combining human EEG recordings, naturalistic stimulus images, artificial neural networks, and computational modeling approaches including representational similarity analysis (RSA) and partial correlation analysis …”
(Introduction, line 108) “We applied our integrated computational approach to an open EEG dataset…”
(Introduction, line 142 to 143) “The integrated computational approach by cross-modal representational comparisons we take with the current study…”
(Discussion, line 550 to 552) “our study goes beyond the contributions of prior studies in several key ways, offering both theoretical and methodological advances: …”
The claims also seem too broad given the fairly small set of RDMs that are used here (3 size metrics, 4 ANN layers, 1 Word2Vec RDM): there are many aspects of object processing not studied here, so it's not correct to say this study provides a 'detailed and clear characterization of the object processing process'.
Thanks for pointing this out. We softened language in our manuscript to reflect that our findings provide a temporally resolved characterization of selected object features, rather than a comprehensive account of object processing:
(line 34 to 37) “our study combines human EEG and representational similarity analysis to disentangle neural representations of object real-world size from retinal size and perceived depth, leveraging recent datasets and modeling approaches to address challenges not fully resolved in previous work.”
(line 46 to 48) “Our research provides a temporally resolved characterization of how certain key object properties – such as object real-world size, depth, and retinal size – are represented in the brain, …”
The paper lacks an analysis demonstrating the validity of the real-world depth measure, which is here computed from the other two metrics by simply dividing them. The rationale and logic of this metric is not clearly explained. Is it intended to reflect the hypothesized egocentric distance to the object in the image if the person had in fact been 'inside' the image? How do we know this is valid? It would be helpful if the authors provided a validation of this metric.
We appreciate the comment regarding the real-world depth metric. Specifically, this metric was computed as the ratio of real-world size (obtained via behavioral ratings) to measured retinal size. The rationale behind this computation is grounded in the basic principles of perspective projection: for two objects subtending the same retinal size, the physically larger object is presumed to be farther away. This ratio thus serves as a proxy for perceived egocentric depth under the simplifying assumption of consistent viewing geometry across images.
We acknowledge that this is a derived estimate and not a direct measurement of perceived depth. While it provides a useful approximation that allows us to analytically dissociate the contributions of real-world size and depth in our RSA framework, we agree that future work would benefit from independent perceptual depth ratings to validate or refine this metric. We added more discussions about this to our revised manuscript:
(line 652 to 657) “Additionally, we acknowledge that our metric for real-world depth was derived indirectly as the ratio of perceived real-world size to retinal size. While this formulation is grounded in geometric principles of perspective projection and served the purpose of analytically dissociating depth from size in our RSA framework, it remains a proxy rather than a direct measure of perceived egocentric distance. Future work incorporating behavioral or psychophysical depth ratings would be valuable for validating and refining this metric.”
Given that there is only 1 image/concept here, the factor of real-world size may be confounded with other things, such as semantic category (e.g. buildings vs. tools). While the comparison of the real-world size metric appears to be effectively disentangled from retinal size and (the author's metric of) depth here, there are still many other object properties that are likely correlated with real-world size and therefore will confound identifying a 'pure' representation of real-world size in EEG. This could be addressed by adding more hypothesis RDMs reflecting different aspects of the images that may correlate with real-world size.
We thank the reviewer for this thoughtful and important point. We agree that semantic category and real-world size may be correlated, and that semantic structure is one of the plausible sources of variance contributing to real-world size representations. However, we would like to clarify that our original goal was to isolate real-world size from two key physical image features — retinal size and inferred real-world depth — which have been major confounds in prior work on this topic. We acknowledge that although our analysis disentangled real-world size from depth and retinal size, this does not imply a fully “pure” representation; therefore, we now refer to the real-world size representations as “partially disentangled” throughout the manuscript to reflect this nuance.
Interestingly, after controlling for these physical features, we still found a robust and statistically isolated representation of real-world size in the EEG signal. This motivated the idea that realworld size may be more than a purely perceptual or image-based property — it may be at least partially semantic. Supporting this interpretation, both the late layers of ANN models and the non-visual semantic model (Word2Vec) also captured real-world size structure. Rather than treating semantic information as an unwanted confound, we propose that semantic structure may be an inherent component of how the brain encodes real-world size.
To directly address the your concern, we conducted an additional variance partitioning analysis, in which we decomposed the variance in EEG RDMs explained by four RDMs: real-world depth, retinal size, real-world size, and semantic information (from Word2Vec). Specifically, for each EEG timepoint, we quantified (1) the unique variance of real-world size, after controlling for semantic similarity, depth, and retinal size; (2) the unique variance of semantic information, after controlling for real-world size, depth, and retinal size; (3) the shared variance jointly explained by real-world size and semantic similarity, controlling for depth and retinal size. This analysis revealed that real-world size explained unique variance in EEG even after accounting for semantic similarity. And there was also a substantial shared variance, indicating partial overlap between semantic structure and size. Semantic information also contributed unique explanatory power, as expected. These results suggest that real-world size is indeed partially semantic in nature, but also has independent neural representation not fully explained by general semantic similarity. This strengthens our conclusion that real-world size functions as a meaningful, higher-level dimension in object representation space.
We now include this new analysis and a corresponding figure (Figure S8) in the revised manuscript:
(line 532 to 539) “Second, we conducted a variance partitioning analysis, in which we decomposed the variance in EEG RDMs explained by three hypothesis-based RDMs and the semantic RDM (Word2Vec RDM), and we still found that real-world size explained unique variance in EEG even after accounting for semantic similarity (Figure S9). And we also observed a substantial shared variance jointly explained by real-world size and semantic similarity and a unique variance of semantic information. These results suggest that real-world size is indeed partially semantic in nature, but also has independent neural representation not fully explained by general semantic similarity.”
The choice of ANNs lacks a clear motivation. Why these two particular networks? Why pick only 2 somewhat arbitrary layers? If the goal is to identify more semantic representations using CLIP, the comparison between CLIP and vision-only ResNet should be done with models trained on the same training datasets (to exclude the effect of training dataset size & quality; cf Wang et al., 2023). This is necessary to substantiate the claims on page 19 which attributed the differences between models in terms of their EEG correlations to one of them being a 'visual model' vs. 'visual-semantic model'.
We argee that the choice and comparison of models should be better contextualized.
First, our motivation for selecting ResNet-50 and CLIP ResNet-50 was not to make a definitive comparison between model classes, but rather to include two widely used representatives of their respective categories—one trained purely on visual information (ResNet-50 on ImageNet) and one trained with joint visual and linguistic supervision (CLIP ResNet-50 on image–text pairs). These models are both highly influential and commonly used in computational and cognitive neuroscience, allowing for relevant comparisons with existing work (line 181-187).
Second, we recognize that limiting the EEG × ANN correlation analyses to only early and late layers may be viewed as insufficiently comprehensive. To address this point, we have computed the EEG correlations with multiple layers in both ResNet and CLIP models (ResNet: ResNet.maxpool, ResNet.layer1, ResNet.layer2, ResNet.layer3, ResNet.layer4, ResNet.avgpool; CLIP: CLIP.visual.avgpool, CLIP.visual.layer1, CLIP.visual.layer2, CLIP.visual.layer3, CLIP.visual.layer4, CLIP.visual.attnpool). The results, now included in Figure S4, show a consistent trend: early layers exhibit higher similarity to early EEG time points, and deeper layers show increased similarity to later EEG stages. We chose to highlight early and late layers in the main text to simplify interpretation.
Third, we appreciate the reviewer’s point that differences in training datasets (ImageNet vs. CLIP's dataset) may confound any attribution of differences in brain alignment to the models' architectural or learning differences. We agree that the comparisons between models trained on matched datasets (e.g., vision-only vs. multimodal models trained on the same image–text corpus) would allow for more rigorous conclusions. Thus, we explicitly acknowledged this limitation in the text:
(line 443 to 445) “However, it is also possible that these differences between ResNet and CLIP reflect differences in training data scale and domain.”
The first part of the claim on page 22 based on Figure 4 'The above results reveal that realworld size emerges with later peak neural latencies and in the later layers of ANNs, regardless of image background information' is not valid since no EEG results for images without backgrounds are shown (only ANNs).
We revised the sentence to clarify that this is a hypothesis based on the ANN results, not an empirical EEG finding:
(line 491 to 495) “These results show that real-world size emerges in the later layers of ANNs regardless of image background information, and – based on our prior EEG results – although we could not test object-only images in the EEG data, we hypothesize that a similar temporal profile would be observed in the brain, even for object-only images.”
While we only had the EEG data of human subjects viewing naturalistic images, the ANN results suggest that real-world size representations may still emerge at later processing stages even in the absence of background, consistent with what we observed in EEG under with-background conditions.
The paper is likely to impact the field by showcasing how using partial correlations in RSA is useful, rather than providing conclusive evidence regarding neural representations of objects and their sizes.
Additional context important to consider when interpreting this work:
Page 20, the authors point out similarities of peak correlations between models ('Interestingly, the peaks of significant time windows for the EEG × HYP RSA also correspond with the peaks of the EEG × ANN RSA timecourse (Figure 3D,F)'. Although not explicitly stated, this seems to imply that they infer from this that the ANN-EEG correlation might be driven by their representation of the hypothesized feature spaces. However this does not follow: in EEG-image metric model comparisons it is very typical to see multiple peaks, for any type of model, this simply reflects specific time points in EEG at which visual inputs (images) yield distinctive EEG amplitudes (perhaps due to stereotypical waves of neural processing?), but one cannot infer the information being processed is the same. To investigate this, one could for example conduct variance partitioning or commonality analysis to see if there is variance at these specific timepoints that is shared by a specific combination of the hypothesis and ANN feature spaces.
Thanks for your thoughtful observation! Upon reflection, we agree that the sentence – "Interestingly, the peaks of significant time windows for the EEG × HYP RSA also correspond with the peaks of the EEG × ANN RSA timecourse" – was speculative and risked implying a causal link that our data do not warrant. As you rightly points out, observing coincident peak latencies across different models does not necessarily imply shared representational content, given the stereotypical dynamics of evoked EEG responses. And we think even variance partitioning analysis would still not suffice to infer that ANN-EEG correlations are driven specifically by hypothesized feature spaces. Accordingly, we have removed this sentence from the manuscript to avoid overinterpretation.
Page 22 mentions 'The significant time-window (90-300ms) of similarity between Word2Vec RDM and EEG RDMs (Figure 5B) contained the significant time-window of EEG x real-world size representational similarity (Figure 3B)'. This is not particularly meaningful given that the Word2Vec correlation is significant for the entire EEG epoch (from the time-point of the signal 'arriving' in visual cortex around ~90 ms) and is thus much less temporally specific than the realworld size EEG correlation. Again a stronger test of whether Word2Vec indeed captures neural representations of real-world size could be to identify EEG time-points at which there are unique Word2Vec correlations that are not explained by either ResNet or CLIP, and see if those timepoints share variance with the real-world size hypothesized RDM.
We appreciate your insightful comment. Upon reflection, we agree that the sentence – "'The significant time-window (90-300ms) of similarity between Word2Vec RDM and EEG RDMs (Figure 5B) contained the significant time-window of EEG x real-world size representational similarity (Figure 3B)" – was speculative. And we have removed this sentence from the manuscript to avoid overinterpretation.
Additionally, we conducted two analyses as you suggested in the supplement. First, we calculated the partial correlation between EEG RDMs and the Word2Vec RDM while controlling for four ANN RDMs (ResNet early/late and CLIP early/late) (Figure S8). Even after regressing out these ANN-derived features, we observed significant correlations between Word2Vec and EEG RDMs in the 100–190 ms and 250–300 ms time windows. This result suggests that
Word2Vec captures semantic structure in the neural signal that is not accounted for by ResNet or CLIP. Second, we conducted an additional variance partitioning analysis, in which we decomposed the variance in EEG RDMs explained by four RDMs: real-world depth, retinal size, real-world size, and semantic information (from Word2Vec) (Figure S9). And we found significant shared variance between Word2Vec and real-world size at 130–150 ms and 180–250 ms. These results indicate a partially overlapping representational structure between semantic content and real-world size in the brain.
We also added these in our revised manuscript:
(line 525 to 539) “To further probe the relationship between real-world size and semantic information, and to examine whether Word2Vec captures variances in EEG signals beyond that explained by visual models, we conducted two additional analyses. First, we performed a partial correlation between EEG RDMs and the Word2Vec RDM, while regressing out four ANN RDMs (early and late layers of both ResNet and CLIP) (Figure S8). We found that semantic similarity remained significantly correlated with EEG signals across sustained time windows (100-190ms and 250-300ms), indicating that Word2Vec captures neural variance not fully explained by visual or visual-language models. Second, we conducted a variance partitioning analysis, in which we decomposed the variance in EEG RDMs explained by three hypothesis-based RDMs and the semantic RDM (Word2Vec RDM), and we still found that real-world size explained unique variance in EEG even after accounting for semantic similarity (Figure S9). And we also observed a substantial shared variance jointly explained by realworld size and semantic similarity and a unique variance of semantic information. These results suggest that real-world size is indeed partially semantic in nature, but also has independent neural representation not fully explained by general semantic similarity.”
Reviewer #3 (Public Review):
The authors used an open EEG dataset of observers viewing real-world objects. Each object had a real-world size value (from human rankings), a retinal size value (measured from each image), and a scene depth value (inferred from the above). The authors combined the EEG and object measurements with extant, pre-trained models (a deep convolutional neural network, a multimodal ANN, and Word2vec) to assess the time course of processing object size (retinal and real-world) and depth. They found that depth was processed first, followed by retinal size, and then real-world size. The depth time course roughly corresponded to the visual ANNs, while the real-world size time course roughly corresponded to the more semantic models.
The time course result for the three object attributes is very clear and a novel contribution to the literature. However, the motivations for the ANNs could be better developed, the manuscript could better link to existing theories and literature, and the ANN analysis could be modernized. I have some suggestions for improving specific methods.
(1) Manuscript motivations
The authors motivate the paper in several places by asking " whether biological and artificial systems represent object real-world size". This seems odd for a couple of reasons. Firstly, the brain must represent real-world size somehow, given that we can reason about this question. Second, given the large behavioral and fMRI literature on the topic, combined with the growing ANN literature, this seems like a foregone conclusion and undermines the novelty of this contribution.
Thanks for your helpful comment. We agree that asking whether the brain represents real-world size is not a novel question, given the existing behavioral and neuroimaging evidence supporting this. Our intended focus was not on the existence of real-world size representations per se, but the nature of these representations, particularly the relationship between the temporal dynamics and potential mechanisms of representations of real-world size versus other related perceptual properties (e.g., retinal size and real-world depth). We revised the relevant sentence to better reflect our focue, shifting from a binary framing (“whether or not size is represented”) to a more mechanistic and time-resolved inquiry (“how and when such representations emerge”):
(line 144 to 149) “Unraveling the internal representations of object size and depth features in both human brains and ANNs enables us to investigate how distinct spatial properties—retinal size, realworld depth, and real-world size—are encoded across systems, and to uncover the representational mechanisms and temporal dynamics through which real-world size emerges as a potentially higherlevel, semantically grounded feature.”
While the introduction further promises to "also investigate possible mechanisms of object realworld size representations.", I was left wishing for more in this department. The authors report correlations between neural activity and object attributes, as well as between neural activity and ANNs. It would be nice to link the results to theories of object processing (e.g., a feedforward sweep, such as DiCarlo and colleagues have suggested, versus a reverse hierarchy, such as suggested by Hochstein, among others). What is semantic about real-world size, and where might this information come from? (Although you may have to expand beyond the posterior electrodes to do this analysis).
We thank the reviewer for this insightful comment. We agree that understanding the mechanisms underlying real-world size representations is a critical question. While our current study does not directly test specific theoretical frameworks such as the feedforward sweep model or the reverse hierarchy theory, our results do offer several relevant insights: The temporal dynamics revealed by EEG—where real-world size emerges later than retinal size and depth—suggest that such representations likely arise beyond early visual feedforward stages, potentially involving higherlevel semantic processing. This interpretation is further supported by the fact that real-world size is strongly captured by late layers of ANNs and by a purely semantic model (Word2Vec), suggesting its dependence on learned conceptual knowledge.
While we acknowledge that our analyses were limited to posterior electrodes and thus cannot directly localize the cortical sources of these effects, we view this work as a first step toward bridging low-level perceptual features and higher-level semantic representations. We hope future work combining broader spatial sampling (e.g., anterior EEG sensors or source localization) and multimodal recordings (e.g., MEG, fMRI) can build on these findings to directly test competing models of object processing and representation hierarchy.
We also added these to the Discussion section:
(line 619 to 638) “Although our study does not directly test specific models of visual object processing, the observed temporal dynamics provide important constraints for theoretical interpretations. In particular, we find that real-world size representations emerge significantly later than low-level visual features such as retinal size and depth. This temporal profile is difficult to reconcile with a purely feedforward account of visual processing (e.g., DiCarlo et al., 2012), which posits that object properties are rapidly computed in a sequential hierarchy of increasingly complex visual features. Instead, our results are more consistent with frameworks that emphasize recurrent or top-down processing, such as the reverse hierarchy theory (Hochstein & Ahissar, 2002), which suggests that high-level conceptual information may emerge later and involve feedback to earlier visual areas. This interpretation is further supported by representational similarities with late-stage artificial neural network layers and with a semantic word embedding model (Word2Vec), both of which reflect learned, abstract knowledge rather than low-level visual features. Taken together, these findings suggest that real-world size is not merely a perceptual attribute, but one that draws on conceptual or semantic-level representations acquired through experience. While our EEG analyses focused on posterior electrodes and thus cannot definitively localize cortical sources, we see this study as a step toward linking low-level visual input with higher-level semantic knowledge. Future work incorporating broader spatial coverage (e.g., anterior sensors), source localization, or complementary modalities such as MEG and fMRI will be critical to adjudicate between alternative models of object representation and to more precisely trace the origin and flow of real-world size information in the brain.”
Finally, several places in the manuscript tout the "novel computational approach". This seems odd because the computational framework and pipeline have been the most common approach in cognitive computational neuroscience in the past 5-10 years.
We have revised relevant statements throughout the manuscript to avoid overstating novelty and to better reflect the contribution of our study.
(2) Suggestion: modernize the approach
I was surprised that the computational models used in this manuscript were all 8-10 years old. Specifically, because there are now deep nets that more explicitly model the human brain (e.g., Cornet) as well as more sophisticated models of semantics (e.g., LLMs), I was left hoping that the authors had used more state-of-the-art models in the work. Moreover, the use of a single dCNN, a single multi-modal model, and a single word embedding model makes it difficult to generalize about visual, multimodal, and semantic features in general.
Thanks for your suggestion. Indeed, our choice of ResNet and CLIP was motivated by their widespread use in the cognitive and computational neuroscience area. These models have served as standard benchmarks in many studies exploring correspondence between ANNs and human brain activity. To address you concern, we have now added additional results from the more biologically inspired model, CORnet, in the supplementary (Figure S10). The results for CORnet show similar patterns to those observed for ResNet and CLIP, providing converging evidence across models.
Regarding semantic modeling, we intentionally chose Word2Vec rather than large language models (LLMs), because our goal was to examine concept-level, context-free semantic representations. Word2Vec remains the most widely adopted approach for obtaining noncontextualized embeddings that reflect core conceptual similarity, as opposed to the contextdependent embeddings produced by LLMs, which are less directly suited for capturing stable concept-level structure across stimuli.
(3) Methodological considerations
(a) Validity of the real-world size measurement
I was concerned about a few aspects of the real-world size rankings. First, I am trying to understand why the scale goes from 100-519. This seems very arbitrary; please clarify. Second, are we to assume that this scale is linear? Is this appropriate when real-world object size is best expressed on a log scale? Third, the authors provide "sand" as an example of the smallest realworld object. This is tricky because sand is more "stuff" than "thing", so I imagine it leaves observers wondering whether the experimenter intends a grain of sand or a sandy scene region. What is the variability in real-world size ratings? Might the variability also provide additional insights in this experiment?
We now clarify the origin, scaling, and interpretation of the real-world size values obtained from the THINGS+ dataset.
In their experiment, participants first rated the size of a single object concept (word shown on the screen) by clicking on a continuous slider of 520 units, which was anchored by nine familiar real-world reference objects (e.g., “grain of sand,” “microwave oven,” “aircraft carrier”) that spanned the full expected size range on a logarithmic scale. Importantly, participants were not shown any numerical values on the scale—they were guided purely by the semantic meaning and relative size of the anchor objects. After the initial response, the scale zoomed in around the selected region (covering 160 units of the 520-point scale) and presented finer anchor points between the previous reference objects. Participants then refined their rating by dragging from the lower to upper end of the typical size range for that object. If the object was standardized in size (e.g., “soccer ball”), a single click sufficed. These size judgments were collected across at least 50 participants per object, and final scores were derived from the central tendency of these responses. Although the final size values numerically range from 0 to 519 (after scaling), this range is not known to participants and is only applied post hoc to construct the size RDMs.
Regarding the term “sand”: the THINGS+ dataset distinguished between object meanings when ambiguity was present. For “sand,” participants were instructed to treat it as “a grain of sand”— consistent with the intended meaning of a discrete, minimal-size reference object.
Finally, we acknowledge that real-world size ratings may carry some degree of variability across individuals. However, the dataset includes ratings from 2010 participants across 1854 object concepts, with each object receiving at least 50 independent ratings. Given this large and diverse sample, the mean size estimates are expected to be stable and robust across subjects. While we did not include variability metrics in our main analysis, we believe the aggregated ratings provide a reliable estimate of perceived real-world size.
We added these details in the Materials and Method section:
(line 219 to 230) “In the THINGS+ dataset, 2010 participants (different from the subjects in THINGS EEG2) did an online size rating task and completed a total of 13024 trials corresponding to 1854 object concepts using a two-step procedure. In their experiment, first, each object was rated on a 520unit continuous slider anchored by familiar reference objects (e.g., “grain of sand,” “microwave oven,” “aircraft carrier”) representing a logarithmic size range. Participants were not shown numerical values but used semantic anchors as guides. In the second step, the scale zoomed in around the selected region to allow for finer-grained refinement of the size judgment. Final size values were derived from aggregated behavioral data and rescaled to a range of 0–519 for consistency across objects, with the actual mean ratings across subjects ranging from 100.03 (‘grain of sand’) to 423.09 (‘subway’).”
(b) This work has no noise ceiling to establish how strong the model fits are, relative to the intrinsic noise of the data. I strongly suggest that these are included.
We have now computed noise ceiling estimates for the EEG RDMs across time. The noise ceiling was calculated by correlating each participant’s EEG RDM with the average EEG RDM across the remaining participants (leave-one-subject-out), at each time point. This provides an upper-bound estimate of the explainable variance, reflecting the maximum similarity that any model—no matter how complex—could potentially achieve, given the intrinsic variability in the EEG data.
Importantly, the observed EEG–model similarity values are substantially below this upper bound. This outcome is fully expected: Each of our model RDMs (e.g., real-world size, ANN layers) captures only a specific aspect of the neural representational structure, rather than attempting to account for the totality of the EEG signal. Our goal is not to optimize model performance or maximize fit, but to probe which components of object information are reflected in the spatiotemporal dynamics of the brain’s responses.
For clarity and accessibility of the main findings, we present the noise ceiling time courses separately in the supplementary materials (Figure S7). Including them directly in the EEG × HYP or EEG × ANN plots would conflate distinct interpretive goals: the model RDMs are hypothesis-driven probes of specific representational content, whereas the noise ceiling offers a normative upper bound for total explainable variance. Keeping these separate ensures each visualization remains focused and interpretable.
Reviewer #1 (Recommendations For The Authors)::
Some analyses are incomplete, which would be improved if the authors showed analyses with other layers of the networks and various additional partial correlation analyses.
Clarity
(1) Partial correlations methods incomplete - it is not clear what is being partialled out in each analysis. It is possible to guess sometimes, but it is not entirely clear for each analysis. This is important as it is difficult to assess if the partial correlations are sensible/correct in each case. Also, the Figure 1 caption is short and unclear.
For example, ANN-EEG partial correlations - "Finally, we directly compared the timepoint-bytimepoint EEG neural RDMs and the ANN RDMs (Figure 3F). The early layer representations of both ResNet and CLIP were significantly correlated with early representations in the human brain" What is being partialled out? Figure 3F says partial correlation
We apologize for the confusion. We made several key clarifications and corrections in the revised version.
First, we identified and corrected a labeling error in both Figure 1 and Figure 3F. Specifically, our EEG × ANN analysis used Spearman correlation, not partial correlation as mistakenly indicated in the original figure label and text. We conducted parital correlations for EEG × HYP and ANN × HYP. But for EEG × ANN, we directly calculated the correlation between EEG RDMs and ANN RDM corresponding to different layers respectively. We corrected these errors: (1) In Figure 1, we removed the erroneous “partial” label from the EEG × ANN path and updated the caption to clearly outline which comparisons used partial correlation. (2) In Figure 3F, we corrected the Y-axis label to “(correlation)”.
Second, to improve clarity, we have now revised the Materials and Methods section to explicitly describe what is partialled out in each parital correlation analysis:
(line 284 to 286) “In EEG × HYP partial correlation (Figure 3D), we correlated EEG RDMs with one hypothesis-based RDM (e.g., real-world size), while controlling for the other two (retinal size and real-world depth).”
(line 303 to 305) “In ANN (or W2V) × HYP partial correlation (Figure 3E and Figure 5A), we correlated ANN (or W2V) RDMs with one hypothesis-based RDM (e.g., real-world size), while partialling out the other two.”
Finally, the caption of Figure 1 has been expanded to clarify the full analysis pipeline and explicitly specify the partial correlation or correlation in each comparison.
(line 327 to 332) “Figure 1 Overview of our analysis pipeline including constructing three types of RDMs and conducting comparisons between them. We computed RDMs from three sources: neural data (EEG), hypothesized object features (real-world size, retinal size, and real-world depth), and artificial models (ResNet, CLIP, and Word2Vec). Then we conducted cross-modal representational similarity analyses between: EEG × HYP (partial correlation, controlling for other two HYP features), ANN (or W2V) × HYP (partial correlation, controlling for other two HYP features), and EEG × ANN (correlation).”
We believe these revisions now make all analytic comparisons and correlation types full clear and interpretable.
Issues / open questions
(2) Semantic representations vs hypothesized (hyp) RDMs (real-world size, etc) - are the representations explained by variables in hyp RDMs or are there semantic representations over and above these? E.g., For ANN correlation with the brain, you could partial out hyp RDMs - and assess whether there is still semantic information left over, or is the variance explained by the hyp RDMs?
Thank for this suggestion. As you suggested, we conducted the partial correlation analysis between EEG RDMs and ANN RDMs, controlling for the three hypothesis-based RDMs. The results (Figure S6) revealed that the EEG×ANN representational similarity remained largely unchanged, indicating that ANN representations capture much more additional representational structure not accounted for by the current hypothesized features. This is also consistent with the observation that EEG×HYP partial correlations were themselves small, but EEG×ANN correlations were much greater.
We also added this statement to the main text:
(line 446 to 451) “To contextualize how much of the shared variance between EEG and ANN representations is driven by the specific visual object features we tested above, we conducted a partial correlation analysis between EEG RDMs and ANN RDMs controlling for the three hypothesis-based RDMs (Figure S6). The EEG×ANN similarity results remained largely unchanged, suggesting that ANN representations capture much more additional rich representational structure beyond these features. ”
(3) Why only early and late layers? I can see how it's clearer to present the EEG results. However, the many layers in these networks are an opportunity - we can see how simple/complex linear/non-linear the transformation is over layers in these models. It would be very interesting and informative to see if the correlations do in fact linearly increase from early to later layers, or if the story is a bit more complex. If not in the main text, then at least in the supplement.
Thank you for the thoughtful suggestion. To address this point, we have computed the EEG correlations with multiple layers in both ResNet and CLIP models (ResNet: ResNet.maxpool, ResNet.layer1, ResNet.layer2, ResNet.layer3, ResNet.layer4, ResNet.avgpool; CLIP:CLIP.visual.avgpool, CLIP.visual.layer1, CLIP.visual.layer2, CLIP.visual.layer3, CLIP.visual.layer4, CLIP.visual.attnpool). The results, now included in Figure S4 and S5, show a consistent trend: early layers exhibit higher similarity to early EEG time points, and deeper layers show increased similarity to later EEG stages. We chose to highlight early and late layers in the main text to simplify interpretation, but now provide the full layerwise profile for completeness.
(4) Peak latency analysis - Estimating peaks per ppt is presumably noisy, so it seems important to show how reliable this is. One option is to find the bootstrapped mean latencies per subject.
Thanks for your suggestion. To estimate the robustness of peak latency values, we implemented a bootstrap procedure by resampling the pairwise entries of the EEG RDM with replacement. For each bootstrap sample, we computed a new EEG RDM and recalculated the partial correlation time course with the hypothesis RDMs. We then extracted the peak latency within the predefined significant time window. Repeating this process 1000 times allowed us to get the bootstrapped mean latencies per subject as the more stable peak latency result. Notably, the bootstrapped results showed minimal deviation from the original latency estimates, confirming the robustness of our findings. Accordingly, we updated the Figure 3D and added these in the Materials and Methods section:
(line 289 to 298) “To assess the stability of peak latency estimates for each subject, we performed a bootstrap procedure across stimulus pairs. At each time point, the EEG RDM was vectorized by extracting the lower triangle (excluding the diagonal), resulting in 19,900 unique pairwise values. For each bootstrap sample, we resampled these 19,900 pairwise entries with replacement to generate a new pseudo-RDM of the same size. We then computed the partial correlation between the EEG pseudo-RDM and a given hypothesis RDM (e.g., real-world size), controlling for other feature RDMs, and obtained a time course of partial correlations. Repeating this procedure 1000 times and extracting the peak latency within the significant time window yielded a distribution of bootstrapped latencies, from which we got the bootstrapped mean latencies per subject.”
(5) "Due to our calculations being at the object level, if there were more than one of the same objects in an image, we cropped the most complete one to get a more accurate retinal size. " Did EEG experimenters make sure everyone sat the same distance from the screen? and remain the same distance? This would also affect real-world depth measures.
Yes, the EEG dataset we used (THINGS EEG2; Gifford et al., 2022) was collected under carefully controlled experimental conditions. We have confirmed that all participants were seated at a fixed distance of 0.6 meters from the screen throughout the experiment. We also added this information in the method (line 156 to 157).
Minor issues/questions - note that these are not raised in the Public Review
(6) Title - less about rigor/quality of the work but I feel like the title could be improved/extended. The work tells us not only about real object size, but also retinal size and depth. In fact, isn't the most novel part of this the real-world depth aspect? Furthermore, it feels like the current title restricts its relevance and impact... Also doesn't touch on the temporal aspect, or processing stages, which is also very interesting. There may be something better, but simply adding something like"...disentangled features of real-world size, depth, and retinal size over time OR processing stages".
Thanks for your suggestion! We changed our title – “Human EEG and artificial neural networks reveal disentangled representations and processing timelines of object real-world size and depth in natural images”.
(7) "Each subject viewed 16740 images of objects on a natural background for 1854 object concepts from the THINGS dataset (Hebart et al., 2019). For the current study, we used the 'test' dataset portion, which includes 16000 trials per subject corresponding to 200 images." Why test images? Worth explaining.
We chose to use the “test set” of the THINGS EEG2 dataset for the following two reasons:
(1) Higher trial count per condition: In the test set, each of the 200 object images was presented 80 times per subject, whereas in the training set, each image was shown only 4 times. This much higher trial count per condition in the test set allows for substantially higher signal-tonoise ratio in the EEG data.
(2) Improved decoding reliability: Our analysis relies on constructing EEG RDMs based on pairwise decoding accuracy using linear SVM classifiers. Reliable decoding estimates require a sufficient number of trials per condition. The test set design is thus better suited to support high-fidelity decoding and robust representational similarity analysis.
We also added these explainations to our revised manuscript (line 161 to 164).
(8) "For Real-World Size RDM, we obtained human behavioral real-world size ratings of each object concept from the THINGS+ dataset (Stoinski et al., 2022).... The range of possible size ratings was from 0 to 519 in their online size rating task..." How were the ratings made? What is this scale - do people know the numbers? Was it on a continuous slider?
We should clarify how the real-world size values were obtained from the THINGS+ dataset.
In their experiment, participants first rated the size of a single object concept (word shown on the screen) by clicking on a continuous slider of 520 units, which was anchored by nine familiar real-world reference objects (e.g., “grain of sand,” “microwave oven,” “aircraft carrier”) that spanned the full expected size range on a logarithmic scale. Importantly, participants were not shown any numerical values on the scale—they were guided purely by the semantic meaning and relative size of the anchor objects. After the initial response, the scale zoomed in around the selected region (covering 160 units of the 520-point scale) and presented finer anchor points between the previous reference objects. Participants then refined their rating by dragging from the lower to upper end of the typical size range for that object. If the object was standardized in size (e.g., “soccer ball”), a single click sufficed. These size judgments were collected across at least 50 participants per object, and final scores were derived from the central tendency of these responses. Although the final size values numerically range from 0 to 519 (after scaling), this range is not known to participants and is only applied post hoc to construct the size RDMs.
We added these details in the Materials and Method section:
(line 219 to 230) “In the THINGS+ dataset, 2010 participants (different from the subjects in THINGS EEG2) did an online size rating task and completed a total of 13024 trials corresponding to 1854 object concepts using a two-step procedure. In their experiment, first, each object was rated on a 520unit continuous slider anchored by familiar reference objects (e.g., “grain of sand,” “microwave oven,” “aircraft carrier”) representing a logarithmic size range. Participants were not shown numerical values but used semantic anchors as guides. In the second step, the scale zoomed in around the selected region to allow for finer-grained refinement of the size judgment. Final size values were derived from aggregated behavioral data and rescaled to a range of 0–519 for consistency across objects, with the actual mean ratings across subjects ranging from 100.03 (‘grain of sand’) to 423.09 (‘subway’).”
(9) "For Retinal Size RDM, we applied Adobe Photoshop (Adobe Inc., 2019) to crop objects corresponding to object labels from images manually... " Was this by one person? Worth noting, and worth sharing these values per image if not already for other researchers as it could be a valuable resource (and increase citations).
Yes, all object cropping were performed consistently by one of the authors to ensure uniformity across images. We agree that this dataset could be a useful resource to the community. We have now made the cropped object images publicly available https://github.com/ZitongLu1996/RWsize.
We also updated the manuscript accordingly to note this (line 236 to 239).
(10) "Neural RDMs. From the EEG signal, we constructed timepoint-by-timepoint neural RDMs for each subject with decoding accuracy as the dissimilarity index " Decoding accuracy is presumably a similarity index. Maybe 1-accuracy (proportion correct) for dissimilarity?
Decoding accuracy is a dissimilarity index instead of a similarity index, as higher decoding accuracy between two conditions indicates that they are more distinguishable – i.e., less similar – in the neural response space. This approach aligns with prior work using classification-based representational dissimilarity measures (Grootswagers et al., 2017; Xie et al., 2020), where better decoding implies greater dissimilarity between conditions. Therefore, there is no need to invert the decoding accuracy values (e.g., using 1 - accuracy).
Grootswagers, T., Wardle, S. G., & Carlson, T. A. (2017). Decoding dynamic brain patterns from evoked responses: A tutorial on multivariate pattern analysis applied to time series neuroimaging data. Journal of Cognitive Neuroscience, 29(4), 677-697.
Xie, S., Kaiser, D., & Cichy, R. M. (2020). Visual imagery and perception share neural representations in the alpha frequency band. Current Biology, 30(13), 2621-2627.
(11) Figure 1 caption is very short - Could do with a more complete caption. Unclear what the partial correlations are (what is being partialled out in each case), what are the comparisons "between them" - both in the figure and the caption. Details should at least be in the main text.
Related to your comment (1). We revised the caption and the corresponding text.
Reviewer #2 (Recommendations For The Authors):
(1) Intro:
Quek et al., (2023) is referred to as a behavioral study, but it has EEG analyses.
We corrected this – “…, one recent study (Quek et al., 2023) …”
The phrase 'high temporal resolution EEG' is a bit strange - isn't all EEG high temporal resolution? Especially when down-sampling to 100 Hz (40 time points/epoch) this does not qualify as particularly high-res.
We removed this phrasing in our manuscript.
(2) Methods:
It would be good to provide more details on the EEG preprocessing. Were the data low-pass filtered, for example?
We added more details to the manuscript:
(line 167 to 174) “The EEG data were originally sampled at 1000Hz and online-filtered between 0.1 Hz and 100 Hz during acquisition, with recordings referenced to the Fz electrode. For preprocessing, no additional filtering was applied. Baseline correction was performed by subtracting the mean signal during the 100 ms pre-stimulus interval from each trial and channel separately. We used already preprocessed data from 17 channels with labels beginning with “O” or “P” (O1, Oz, O2, PO7, PO3, POz, PO4, PO8, P7, P5, P3, P1, Pz, P2) ensuring full coverage of posterior regions typically involved in visual object processing. The epoched data were then down-sampled to 100 Hz.”
It is important to provide more motivation about the specific ANN layers chosen. Were these layers cherry-picked, or did they truly represent a gradual shift over the course of layers?
We appreciate the reviewer’s concern and fully agree that it is important to ensure transparency in how ANN layers were selected. The early and late layers reported in the main text were not cherry-picked to maximize effects, but rather intended to serve as illustrative examples representing the lower and higher ends of the network hierarchy. To address this point directly, we have computed the EEG correlations with multiple layers in both ResNet and CLIP models (ResNet: ResNet.maxpool, ResNet.layer1, ResNet.layer2, ResNet.layer3, ResNet.layer4, ResNet.avgpool; CLIP: CLIP.visual.avgpool, CLIP.visual.layer1, CLIP.visual.layer2, CLIP.visual.layer3, CLIP.visual.layer4, CLIP.visual.attnpool). The results, now included in Figure S4, show a consistent trend: early layers exhibit higher similarity to early EEG time points, and deeper layers show increased similarity to later EEG stages.
It is important to provide more specific information about the specific ANN layers chosen. 'Second convolutional layer': is this block 2, the ReLu layer, the maxpool layer? What is the 'last visual layer'?
Apologize for the confusing! We added more details about the layer chosen:
(line 255 to 257) “The early layer in ResNet refers to ResNet.maxpool layer, and the late layer in ResNet refers to ResNet.avgpool layer. The early layer in CLIP refers to CLIP.visual.avgpool layer, and the late layer in CLIP refers to CLIP.visual.attnpool layer.”
Again the claim 'novel' is a bit overblown here since the real-world size ratings were also already collected as part of THINGS+, so all data used here is available.
We removed this phrasing in our manuscript.
Real-world size ratings ranged 'from 0 - 519'; it seems unlikely this was the actual scale presented to subjects, I assume it was some sort of slider?
You are correct. We should clarify how the real-world size values were obtained from the THINGS+ dataset.
In their experiment, participants first rated the size of a single object concept (word shown on the screen) by clicking on a continuous slider of 520 units, which was anchored by nine familiar real-world reference objects (e.g., “grain of sand,” “microwave oven,” “aircraft carrier”) that spanned the full expected size range on a logarithmic scale. Importantly, participants were not shown any numerical values on the scale—they were guided purely by the semantic meaning and relative size of the anchor objects. After the initial response, the scale zoomed in around the selected region (covering 160 units of the 520-point scale) and presented finer anchor points between the previous reference objects. Participants then refined their rating by dragging from the lower to upper end of the typical size range for that object. If the object was standardized in size (e.g., “soccer ball”), a single click sufficed. These size judgments were collected across at least 50 participants per object, and final scores were derived from the central tendency of these responses. Although the final size values numerically range from 0 to 519 (after scaling), this range is not known to participants and is only applied post hoc to construct the size RDMs.
We added these details in the Materials and Method section:
(line 219 to 230) “In the THINGS+ dataset, 2010 participants (different from the subjects in THINGS EEG2) did an online size rating task and completed a total of 13024 trials corresponding to 1854 object concepts using a two-step procedure. In their experiment, first, each object was rated on a 520unit continuous slider anchored by familiar reference objects (e.g., “grain of sand,” “microwave oven,” “aircraft carrier”) representing a logarithmic size range. Participants were not shown numerical values but used semantic anchors as guides. In the second step, the scale zoomed in around the selected region to allow for finer-grained refinement of the size judgment. Final size values were derived from aggregated behavioral data and rescaled to a range of 0–519 for consistency across objects, with the actual mean ratings across subjects ranging from 100.03 (‘grain of sand’) to 423.09 (‘subway’).”
Why is conducting a one-tailed (p<0.05) test valid for EEG-ANN comparisons? Shouldn't this be two-tailed?
Our use of one-tailed tests was based on the directional hypothesis that representational similarity between EEG and ANN RDMs would be positive, as supported by prior literature showing correspondence between hierarchical neural networks and human brain representations (e.g., Cichy et al., 2016; Kuzovkin et al., 2014). This is consistent with a large number of RSA studies which conduct one-tailed tests (i.e., testing the hypothesis that coefficients were greater than zero: e.g., Kuzovkin et al., 2018; Nili et al., 2014; Hebart et al., 2018; Kaiser et al., 2019; Kaiser et al., 2020; Kaiser et al., 2022). Thus, we specifically tested whether the similarity was significantly greater than zero.
Cichy, R. M., Khosla, A., Pantazis, D., Torralba, A., & Oliva, A. (2016). Comparison of deep neural networks to spatio-temporal cortical dynamics of human visual object recognition reveals hierarchical correspondence. Scientific reports, 6(1), 27755.
Kuzovkin, I., Vicente, R., Petton, M., Lachaux, J. P., Baciu, M., Kahane, P., ... & Aru, J. (2018). Activations of deep convolutional neural networks are aligned with gamma band activity of human visual cortex. Communications biology, 1(1), 107.
Nili, H., Wingfield, C., Walther, A., Su, L., Marslen-Wilson, W., & Kriegeskorte, N. (2014). A toolbox for representational similarity analysis. PLoS computational biology, 10(4), e1003553.
Hebart, M. N., Bankson, B. B., Harel, A., Baker, C. I., & Cichy, R. M. (2018). The representational dynamics of task and object processing in humans. Elife, 7, e32816.
Kaiser, D., Turini, J., & Cichy, R. M. (2019). A neural mechanism for contextualizing fragmented inputs during naturalistic vision. elife, 8, e48182.
Kaiser, D., Inciuraite, G., & Cichy, R. M. (2020). Rapid contextualization of fragmented scene information in the human visual system. Neuroimage, 219, 117045.
Kaiser, D., Jacobs, A. M., & Cichy, R. M. (2022). Modelling brain representations of abstract concepts. PLoS Computational Biology, 18(2), e1009837.
Importantly, we note that using a two-tailed test instead would not change the significance of our results. However, we believe the one-tailed test remains more appropriate given our theoretical prediction of positive similarity between ANN and brain representations.
The sentence on the partial correlation description (page 11 'we calculated partial correlations with one-tailed test against the alternative hypothesis that the partial correlation was positive (greater than zero)') didn't make sense to me; are you referring to the null hypothesis here?
We revised this sentence to clarify that we tested against the null hypothesis that the partial correlation was less than or equal to zero, using a one-tailed test to assess whether the correlation was significantly greater than zero.
(line 281 to 284) “…, we calculated partial correlations and used a one-tailed test against the null hypothesis that the partial correlation was less than or equal to zero, testing whether the partial correlation was significantly greater than zero.”
(3) Results:
I would prevent the use of the word 'pure', your measurement is one specific operationalization of this concept of real-world size that is not guaranteed to result in unconfounded representations. This is in fact impossible whenever one is using a finite set of natural stimuli and calculating metrics on those - there can always be a factor or metric that was not considered that could explain some of the variance in your measurement. It is overconfident to claim to have achieved some form of Platonic ideal here and to have taken into account all confounds.
Your point is well taken. Our original use of the term “pure” was intended to reflect statistical control for known confounding factors, but we recognize that this wording may imply a stronger claim than warranted. In response, we revised all relevant language in the manuscript to instead describe the statistically isolated or relatively unconfounded representation of real-world size, clarifying that our findings pertain to the unique contribution of real-world size after accounting for retinal size and real-world depth.
Figure 2C: It's not clear why peak latencies are computed on the 'full' correlations rather than the partial ones.
No. The peak latency results in Figure 2C were computed on the partial correlation results – we mentioned this in the figure caption – “Temporal latencies for peak similarity (partial Spearman correlations) between EEG and the 3 types of object information.”
SEM = SEM across the 10 subjects?
Yes. We added this in the figure caption.
Figure 3F y-axis says it's partial correlations but not clear what is partialled out here.
We identified and corrected a labeling error in both Figure 1 and Figure 3F. Specifically, our EEG × ANN analysis used Spearman correlation, not partial correlation as mistakenly indicated in the original figure label and text. We conducted parital correlations for EEG × HYP and ANN × HYP. But for EEG × ANN, we directly calculated the correlation between EEG RDMs and ANN RDM corresponding to different layers respectively. We corrected these errors: (1) In Figure 1, we removed the erroneous “partial” label from the EEG × ANN path and updated the caption to clearly outline which comparisons used partial correlation. (2) In Figure 3F, we corrected the Y-axis label to “(correlation)”.
Reviewer #3 (Recommendations For The Authors):
(1) Several methodologies should be clarified:
(a) It's stated that EEG was sampled at 100 Hz. I assume this was downsampled? From what original frequency?
Yes. We added more detailed about EEG data:
(line 167 to 174) “The EEG data were originally sampled at 1000Hz and online-filtered between 0.1 Hz and 100 Hz during acquisition, with recordings referenced to the Fz electrode. For preprocessing, no additional filtering was applied. Baseline correction was performed by subtracting the mean signal during the 100 ms pre-stimulus interval from each trial and channel separately. We used already preprocessed data from 17 channels with labels beginning with “O” or “P” (O1, Oz, O2, PO7, PO3, POz, PO4, PO8, P7, P5, P3, P1, Pz, P2) ensuring full coverage of posterior regions typically involved in visual object processing. The epoched data were then down-sampled to 100 Hz.”
(b) Why was decoding accuracy used as the human RDM method rather than the EEG data themselves?
Thanks for your question! We would like to address why we used decoding accuracy for EEG RDMs rather than correlation. While fMRI RDMs are typically calculated using 1 minus correlation coefficient, decoding accuracy is more commonly used for EEG RDMs (Grootswager et al., 2017; Xie et al., 2020). The primary reason is that EEG signals are more susceptible to noise than fMRI data. Correlation-based methods are particularly sensitive to noise and may not reliably capture the functional differences between EEG patterns for different conditions. Decoding accuracy, by training classifiers to focus on task-relevant features, can effectively mitigate the impact of noisy signals and capture the representational difference between two conditions.
Grootswagers, T., Wardle, S. G., & Carlson, T. A. (2017). Decoding dynamic brain patterns from evoked responses: A tutorial on multivariate pattern analysis applied to time series neuroimaging data. Journal of Cognitive Neuroscience, 29(4), 677-697.
Xie, S., Kaiser, D., & Cichy, R. M. (2020). Visual imagery and perception share neural representations in the alpha frequency band. Current Biology, 30(13), 2621-2627.
We added this explanation to the manuscript:
(line 204 to 209) “Since EEG has a low SNR and includes rapid transient artifacts, Pearson correlations computed over very short time windows yield unstable dissimilarity estimates (Kappenman & Luck, 2010; Luck, 2014) and may thus fail to reliably detect differences between images. In contrast, decoding accuracy - by training classifiers to focus on task-relevant features - better mitigates noise and highlights representational differences.”
(c) How were the specific posterior electrodes selected?
The 17 posterior electrodes used in our analyses were pre-selected and provided in the THINGS EEG2 dataset, and corresponding to standard occipital and parietal sites based on the 10-10 EEG system. Specifically, we included all 17 electrodes with labels beginning with “O” or “P”, ensuring full coverage of posterior regions typically involved in visual object processing (Page 7).
(d) The specific layers should be named rather than the vague ("last visual")
Apologize for the confusing! We added more details about the layer information:
(line 255 to 257) “The early layer in ResNet refers to ResNet.maxpool layer, and the late layer in ResNet refers to ResNet.avgpool layer. The early layer in CLIP refers to CLIP.visual.avgpool layer, and the late layer in CLIP refers to CLIP.visual.attnpool layer.”
(line 420 to 434) “As shown in Figure 3F, the early layer representations of both ResNet and CLIP (ResNet.maxpool layer and CLIP.visual.avgpool) showed significant correlations with early EEG time windows (early layer of ResNet: 40-280ms, early layer of CLIP: 50-130ms and 160-260ms), while the late layers (ResNet.avgpool layer and CLIP.visual.attnpool layer) showed correlations extending into later time windows (late layer of ResNet: 80-300ms, late layer of CLIP: 70-300ms). Although there is substantial temporal overlap between early and late model layers, the overall pattern suggests a rough correspondence between model hierarchy and neural processing stages.
We further extended this analysis across intermediate layers of both ResNet and CLIP models (from early to late, ResNet: ResNet.maxpool, ResNet.layer1, ResNet.layer2, ResNet.layer3, ResNet.layer4, ResNet.avgpool; from early to late, CLIP: CLIP.visual.avgpool, CLIP.visual.layer1, CLIP.visual.layer2, CLIP.visual.layer3, CLIP.visual.layer4, CLIP.visual.attnpool).”
(e) p19: please change the reporting of t-statistics to standard APA format.
Thanks for the suggestion. We changed the reporting format accordingly:
(line 392 to 394) “The representation of real-word size had a significantly later peak latency than that of both retinal size, t(9)=4.30, p=.002, and real-world depth, t(9)=18.58, p<.001. And retinal size representation had a significantly later peak latency than real-world depth, t(9)=3.72, p=.005.”
(2) "early layer of CLIP: 50-130ms and 160-260ms), while the late layer representations of twoANNs were significantly correlated with later representations in the human brain (late layer of ResNet: 80-300ms, late layer of CLIP: 70-300ms)."
This seems a little strong, given the large amount of overlap between these models.
We agree that our original wording may have overstated the distinction between early and late layers, given the substantial temporal overlap in their EEG correlations. We revised this sentence to soften the language to reflect the graded nature of the correspondence, and now describe the pattern as a general trend rather than a strict dissociation:
(line 420 to 427) “As shown in Figure 3F, the early layer representations of both ResNet and CLIP (ResNet.maxpool layer and CLIP.visual.avgpool) showed significant correlations with early EEG time windows (early layer of ResNet: 40-280ms, early layer of CLIP: 50-130ms and 160-260ms), while the late layers (ResNet.avgpool layer and CLIP.visual.attnpool layer) showed correlations extending into later time windows (late layer of ResNet: 80-300ms, late layer of CLIP: 70-300ms). Although there is substantial temporal overlap between early and late model layers, the overall pattern suggests a rough correspondence between model hierarchy and neural processing stages.”
(3) "Also, human brain representations showed a higher similarity to the early layer representation of the visual model (ResNet) than to the visual-semantic model (CLIP) at an early stage. "
This has been previously reported by Greene & Hansen, 2020 J Neuro.
Thanks! We added this reference.
(4) "ANN (and Word2Vec) model RDMs"
Why not just "model RDMs"? Might provide more clarity.
We chose to use the phrasing “ANN (and Word2Vec) model RDMs” to maintain clarity and avoid ambiguity. In the literature, the term “model RDMs” is sometimes used more broadly to include hypothesis-based feature spaces or conceptual models, and we wanted to clearly distinguish our use of RDMs derived from artificial neural networks and language models. Additionally, explicitly referring to ANN or Word2Vec RDMs improves clarity by specifying the model source of each RDM. We hope this clarification justifies our choice to retain the original phrasing for clarity.
The installer consists of several tabs with lots of configurable parameters
A subcommand is missing from the manual: nssm edit <servicename>, which allow us to show service editing GUI.
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Schools are obligated to help students learn and understand their media-saturated world. Ignoring this pointis detrimental to the continued progression of learning
I agree with this. Teachers today need to do more than just teach from books. Students are already learning via memes, influencers, and YouTube. Schools should teach kids how to think critically about what they read and see online. Media literacy lessons can help teens tell the difference between facts and lies and be better digital citizens.
. All media messages are “constructed.”2. Media messages are constructed using a creative language with its own rules.3. Different people experience the same media message differently.4. The media have embedded values and points of view.5. Media messages are constructed to gain profit and/or power.
These ideas are strong because they show that media is never neutral. Everything, from news broadcasts to TikTok videos, has a reason for being, a target audience, and a bias. I enjoy how this relates to how marketing and algorithms today affect what we see online. It makes me think about how media literacy is about figuring out what those hidden motives are and how to spot persuasion and manipulation in ordinary media.
Media literacy involves critical thinking. To think that it does not would make the study of medialiteracy a passive undertaking, rather than an engaged dynamic.
This is a good reminder that being "media literate" isn't simply understanding how to use media; it's also about questioning and examining it. A lot of individuals look at social media or watch the news without thinking about bias or purpose. Being media literate entails asking why something was made, who profits from it, and what it says. It's not just about consuming; it's an active and thoughtful process.
hile today, films that focus on activism are common, back then, such movies were outliers and not typically successful at the box office.
for - Social change media - Participant Media - question - Did the success of Participant Media lead to its own obsolescence?