Good claim but needs specific citation.

again really interesting to see where certain slang originated, also when he says "a lasting influence" is a very true statement much of the slang he mentions is still used today.

The fact that the author was able to derive that meaning from that lyric shows that hip-hop can be studied to show deeper meanings.

I really enjoy this segment the way he illustrates the lyric writing but also studying the process rather then the product is really interesting, I think If I was the read a draft of a book it would be a lot more interesting because you get to study the words the author did and didn't use and you get to ask your self the question "Why did he change it" I think I would find a lot more texts more engaging if all I read were hand written drafts.

I really like the way Sciullo worded this I think it adds a lot to his perspective and opinion but I think it adds a nice twist on the concept of "Code-Meshing" but also encourages dialect diversity a educational environment.

I am unable to highlight or annotate - But the first thing that stood out to me as I'm reading this, which was already mentioned by someone, is the mention that sometimes "Changing the context is necessary". Sometimes we the caregivers, our responses, or even failure to clearly state expectations in a age-appropriate way can sometimes be the cause of challenging behavior. I appreciate the mention of it so I can now consider that when deciding the best way to support children through guidance. I also appreciate the mention of staying away from blaming the child. Someone say "The problem is the problem, the child is not the problem" which is a great way of putting it and an effective mindset to adopt. A question that comes to mind after reading is how can I stay mindful of these steps and how to apply them to my guidance of children in the moment? sometimes chaos and a focus on student safety causes us to jump to discipline or frustration.

I like this sentence because it recognizes both the value and the limits of analytical evaluation. The author acknowledges that while direct user observation provides the most authentic insight, analytical methods still play a vital role when testing with users isn’t possible. This balance between practicality and depth reflects a realistic approach to design research. It reminds me that good designers use every tool available but never lose sight of real human experience.

I really agree with the reading’s point that there’s nothing like watching a real person struggle to use your design. I’ve experienced this in group projects where our design seemed perfect on paper, but once we saw users getting confused or stuck, we realized how many assumptions we had made. It’s such a valuable reminder that analytical evaluations and heuristics can only go so far—real user interaction reveals issues we’d never anticipate ourselves. This reinforces why usability testing with actual users is irreplaceable for meaningful feedback and improvement.

I found the discussion of recognition versus recall really interesting because it highlights how design can either support or burden users’ memory. I notice this difference a lot when comparing apps that use clear icons and menus versus those that rely on hidden commands. For example, some mobile apps require you to remember specific swipe actions, which can be frustrating when they’re not visible on the screen. This reading reminded me how much easier it is for users when designs rely on recognition—showing options directly—rather than expecting people to recall invisible steps from memory.

Judging by the fact that Emily Bazelon is listed as a senior writer for this magazine company and that she works at Yale, I would say that for the topic (she researches constitutional law and social justice issues), she is a credible source to read when it comes to legal issues. The intention in writing this was to look at Florida's Amendment 4 (to restore voting rights to felons) in the context of it being a racial-based issue, similar to the post-Reconstruction era when Black citizens were kept from voting through new laws/policies. Moreover, The New York Times is highly receptive when it comes to editing, having policies in place regarding when information needs to be updated or corrected. So despite this article being far from an academic journal, the information within it was still gathered and published with journalistic integrity. This includes the court cases, statistics, and experts referenced throughout the article (and interviews). The article was published in 2018, but I believe it's still incredibly relevant because Amendment 4 has sparked more conversations on letting felons vote while incarcerated, making the information in this article important. Especially since Bazelon's analysis relied heavily on examining Florida law in its historical context. I would choose this source over another because it checks my boxes for credibility and it relies on qualitative accounts (observing and interpreting things to understand why something is the way that it is) rather than quantitative, which makes it unique.

This article is trying to find out who said "A Lie is Halfway Round the World Before the Truth Has Got Its Boots On". Many people think Winston Churchill first said this, but a different version of this sentence appeared earlier. So ironically, the origin of this sentence itself is also a lie. The spreading of this sentence is just like the way that information on internet spreads. We receive some information and build our own understanding of it then tell the edited information to others, leading the fake information to spread in a speed that we cannot imagine.

important to know what merits the research

cognitive thrust

gesture doesn't associate with language?

data mixed with thoughts

not a consequence but a finding

visual drawing over communicated drawing

Social media is how people communicate today, so it is a main way if bringing people together, but it is also a way of splitting people apart as well.

Social media and technology can be used for good when used correctly. Social media has a lot of negatives but this statistic proves that it is not always bad. Digital citizenship is important to teach our students to use social media in a meaningful way.

I completely agree that strong passwords are important, but I think most people (including me) tend to reuse the same password across several accounts. I started using a password manager recently, and it’s helped a lot. I could see teaching students to create strong passwords as a good digital citizenship activity, even at a young age.

I’ve noticed this happening so many times after I online shop. I will search for something once, and then it pops up everywhere. I knew cookies tracked browsing behavior, but I didn’t realize how detailed the tracking could be or that some advertisers use cookies across multiple websites. This makes me think about what kinds of data students may be unknowingly sharing when they use educational websites.

I think this is important to note because someone may think that they are just having fun but they are might really being putting people in danger and at risk. Teaching that this is not good digital citizenship is important because hacking someone's appliances and making their products unusable can become a big deal for some people. Letting people know that it is important to leave people's information and digital space alone and protected.

I think this is a way that he is proposing a solution to the problem by saying that wealth is not personal but public trust

I agree that coping strategies are something that all of us need and benefit from. Not only did I struggle as a first year teacher emotionally but I think that all of us hit some point in our school year where we have that exhaustion and it can be really hard to cope with those feelings if we don't know how or have the strategies to.

The story is only from one perspective.

says that liberals have made efforts to block fascists and anti-semities. However, they are pushing white young men toward that side with their identity politics

belives that Christiantiy can learn from those damn liberals in some aspects

voting should be done by households lead by men, but the women should still be able to have their own assests so that men wont dump their wives

Hindus and Muslims would be allowed but they conduct activities that publically profess their faith

laws need to be biblically sourced and enforced, but those who practice another religion should be free to do so

doesnt argue for applying the rules of the Bible but applying those rules within the context of America

this article is just an introduction, but we get insight into really relevant topics

differing perspectives, but have all firsthand faced the issue

their definition of what literacy should be

Follows a structure but overall too predictable.

Reading this excerpt, I’m struck by how it handles a complex and painful part of history without offering easy answers. The text highlights the scale of displacement, around 700,000 Arab Palestinians leaving the territories that became Israel, and how this shaped the refugee issue after 1948. It also points out that there were atrocities committed on both sides that influenced these movements, as well as discussions within Israeli thought about the idea of “transfer.” What I find interesting is how the author examines these events and debates, showing both the historical actions and the interpretations of those actions, without claiming absolute certainty or assigning overarching intent. It emphasizes to me that historical narratives are complicated, shaped by both facts and interpretation, and that understanding the past often requires looking at multiple perspectives and nuances rather than simple conclusions.

This passage stood out to me as a means of critiquing the source of the information that one gleans from different texts people read. Kimmerling's main argument here is that Morris has abandoned his "scholarly" responsibility and is now using his credibility as a historian to legitimize ethnic cleansing rather than to examine it critically which therefore directly addresses how academic authority shapes public discourse and political possibilities. This serves as a means of giving "permission" to an entire society by wrapping up extreme nationalism in scholarly respectability. This is why this passage captured my interest, in how this inverts to what historians should do. We expect them to help societies learn from past violence, not to study atrocities and conclude they should be repeated more thoroughly.

First of all, this reading was super interesting because it was cool to see a response to the interview that we got to read. Specifically, this sentence is a reflection of interpretation of historical concepts themselves became a sort of battle ground amongst Israeli society. The author of this article points out a backlash against "post zionism," showing that often, these historical debates don't just have to be academic but they are also often struggles over identity and morals. The author questions the foundational narratives of Israel and post-Zionist scholars.

The highlight of the reading in terms of significance is the way in which Kimmerling criticizes both the deeds and the ideology that led to the war of 1948, suggesting that the displacement of the Palestinians was not random during the war but rather part of an intentional plan related to Zionist ideology in terms of territory. The quote below made me stop and reflect on the reading, realizing that there was indeed intent related to ideas rather than just physical deeds in terms of what transpired during the war. The part of the reading that interested me the most was the way in which Kimmerling makes reference to ideas related to morality and nationality in terms of what transpired during the formation of the Jewish state.

Here using the CRAAPs method I can see what how reliable the source is, but there might be some sort of bias since she is an educator herself.

Researching isn't just about finding the answers but really learning, and understanding it.

I think its interesting how social norms for women have changed a bit over time, but some men still expect this from women.

some reasons a child might need kinship due to no parents

I completely agree with the reading’s point that the goal of usability tests is to uncover “breakdowns” — moments when users fail to complete a task even though they’re following the right steps. I’ve noticed this myself when building projects: everything seems fine until people outside the team start using it. That’s when I realize there are small, even silly mistakes that I never noticed because I was too familiar with the design. Just like the reading explains, observing these breakdowns helps me understand where users get stuck and how I can redesign the interface to make it clearer and more intuitive.

The author makes a clear connection between business ethics and the idea of individual consent, but their argument feels a bit too idealistic. They assume that all business relationships are truly “voluntary,” ignoring how power dynamics, economic pressure, or lack of options can limit real consent. By treating businesses as simple networks of equal agreements, the author overlooks the complex realities of modern corporations where not everyone has the same influence or freedom to choose

r makes a fair point about the social contract not being a real, written agreement, but they kind of take the idea too literally. it focus so much on the legal side that they miss the bigger picture — the “contract” in social contract theory isn’t supposed to be a real document, it’s more of a way to explain how people agree to live by certain rules

for - example - practical reasons for not naming a settlement a city - Egypt

Representation in toys, similar than in games. You consume stereotypes for Carnival: What do the boys wear, tactical and police gear. What about girls? Flying assistants or dressed princesses. Do you see many doctors during carnival? No.

To me, most festivals are grotesque self-fetishisations plagued by nationalistic discriminatory pride. Yes, they can be an acknowledgement of diverse oft-excluded identities (furries), and a visibilisation and acceptance of them, but they rarely are: Most of the times, they look like a parody of the parody, a cartoonish simulation of army/school uniforms, and an objectification via (female) sexualised dresses.

I know that this is colonising, you don't have to shove it upon me... but isn't it a justified concession? Isn't the inherent peace-cooperation argument embeded in the game akin to the reparatory non-repetition argument that underlies historical memory?

For me, it is not, and I say this having played a large chunk of the game while focusing on utilitarianist EA ethics. It is not, because it may avoid tokenisation, sure, but Sam Porter is not a slave, he is a hero. Not only that, with although it prefaces the quest of reaching white people with anti-war logics, the game has war, the game has fights, and its sequel does too. These are surrounded with mysticism and fantastic events which cloud the statements and leave them open to interpretation in a way that most players are sure to miss them. It's not provocative, it's a eco-tourism chore. The cutscences and events are a McGuffin to visit places and trek through them to feel epic.

To influence a mass of players, and not just get critical acclaim it would have needed to be more straightforward.

It is important to learn how to take what you learn and apply it to your day-to-day life.

It’s interesting here because it’s impossible in real life that one can know everything about the other. But the author made Connie’s mother be a person who omniscient about her daughter.

This is rather surprising information to me, but good to learn.

Translanguaging is not just a societal concept, but something cognitive and "natural" for multilingual learners.

This is an important distinction that I feel can be easily forgotten in academic settings.

This captures how easy it is to trust what we see online without questioning it. Algorithms present information in a way that looks confident and authoritative, which makes us assume that it's factual, even when it might be misleading or incomplete. I've noticed this especially on platforms like TikTok, Instagram, and even Google searches, where misinformation spreads quickly but feels believable because it's packaged so convincingly. It's dangerous because it shapes our beliefs and decisions without us realizing it, and it shows how much power these systems have over our understanding of the world. Technology doesn't just give us information; it also guides what we think is true.

I found it really interesting how AI isn't truly neutral or objective. It learns directly from us, which means that it also absorbs our biases. The data that trains AI often reflects existing social inequalities like racism, sexism, etc. so those same patterns end up built into the technology itself. AI systems are constantly trained from the information we give them, and that training shapes how they make decisions. This reminds me of how social media algorithms or facial recognition systems sometimes produce unfair outcomes. It's not because the technology is evil, but because it's mirroring the biases it's been taught.

humanity uses religion to explain questions which have stood unanswered or seomthinggggggggggggggggggggggggg

must extend the previous assertion of unwarranted and unquestioning belief in something being bad to religion or SOEMTHING

Benefits such as promoting the local economy and theater in the same way, thanks not only to sales but also with the support of sponsors, volunteers, and donations, as well as Capitol Centre. Additionally, the number of audiences increases year after year, demonstrating a good business model.

But also, doesn't just guess but it notices that certain words or phrases appear together often, sentence structures, grammar, and even style.

LLM is a type of ai, and it learns patterns from the data

I feel like this had something to do with data because of the visual data that is on the reading

This is rather surprising information to me, but good to learn.

Translanguaging is not just a societal concept, but something cognitive and "natural" for multilingual learners.

This is an important distinction that I feel can be easily forgotten in academic settings.

The corners are not counted and position 1 is in the edge but position 39 is not. Thus, a total of 19 spatial positions are preserved with a kernel of 3.

Reading this section about pre-internet virality really made me reflect on how deeply rooted our desire to share and connect is. The example of chain letters especially stood out to me — even without social media, people still felt compelled to pass messages along, sometimes out of fear, sometimes out of hope. It’s interesting that what motivated them was often emotional rather than logical. This reminds me of how similar patterns appear today on social media: people still share posts promising “good luck” or “positive energy,” and even I’ve occasionally reshared something because it felt comforting or meaningful at the moment. It makes me realize that virality isn’t just about algorithms or technology; it’s about human emotions — our longing to be part of something bigger, our belief that our small actions can ripple outward.

Although the earlier, broader framework was first misunderstood and narrowly used, people though it gave rigid views of language or focused only on literacy or bilingualism, it later became widely and properly applied, especially in indigenous and immigrant settings.

• Challenges the idea that bilinguals are just two seperate monolingual speakers
• Focus is on actual language use, not rigid categories
• Connects to translanguaging and code-meshing in classroom practices

Guerra’s overall message is that students’ everyday languages, cultures, and identities should be part of how they learn to write not seen as problems. Teachers should help students see that their voices matter. Writing becomes not just about grammar or formality, but about expressing who you are and engaging with the world responsibly.

how real world affects myth

comparison

Can coding agents self-improve? - Summary

Core Thesis

• Inference-time vs training-time self-improvement:

  "If you could never update the weights, how would you have the model increase its performance on a specific task? I think of that as inference-time self-improvement"

• Key finding:

  "The good news is that GPT-5 is a very good model for building developer utilities. The bad news is that it hates using the tools it creates! As it told me 'I'll be honest - I didn't need any of them.'"

Experimental Setup

• Testing methodology: Asked models (GPT-5 and Opus 4) to:

  1. Build tools they thought would help productivity
  2. Attempt tasks using those tools
  3. Self-reflect on tool improvements

• Core hypothesis:

  "The most valuable use case of coding agents is being a vessel for LLMs to extract value out of their own latent spaces"

• Comparison: Also tested Gemini 2.5 Pro and GPT-4.1, but focused on GPT-5 vs Opus 4 as only models that could keep up

Tool Creation Results

Task Manager Tool

• GPT-5 implementation features:

  "Uses WAL to avoid issues with multiple agents writing at the same time"

  "Uses a graph of dependencies to prioritize tasks"

  "Created an append-only events stream that lets any agent see what every other agent is doing with good keywords like impact_conflict"

• Opus 4 limitations:

  "didn't pick up on the notifications / stream functionality to keep everyone in sync"

Codebase Standards Tool

• Purpose:

  "analyze a codebase and extract different heuristics on how code should be written. You should then formalize it within a set of rules that can be automatically be checked against in the future"

• Quality comparison:

  "I've found the GPT-5 one to be much more nuanced than Opus"

Self-Proposed Tools

GPT-5 tools (16 total):

• doctor, bootstrap, code-map, csearch, tasks-graph, impact, seed, repro scaffold, e2e, preflight, preflight-smol, broker, flake, codemod, triage, trace, runbook

• Design philosophy:

  "GPT-5 built all of them as unix utilities that are easy to use via cli"

• Characterization:

  "GPT-5 was building utilities it could use itself without being too opinionated"

Opus 4 tools (10 total):

• Context Analyzer, Cross-Platform Test Generator, Implementation Proposal Analyzer, Full-Stack Change Impact Analyzer, Bug Pattern Recognition Engine, Security & Permission Auditor, Multi-Platform Feature Implementer, API Integration Assistant, Performance Optimization Toolkit, Task Complexity Estimator

• Design approach:

  "all meant to be run as python some_tool.py"

• Characterization:

  "Opus 4 was building tools that accomplish tasks and have a bit of anthromorphized feeling"

Task Execution Results

Test Task

• Project: smol-podcaster migration from Flask to FastAPI + Next.js

• Task complexity:

  "the task I tried would take me 4-5 hours to do"

• Performance:

  "Both models were almost able to one-shot the task"

Tool Usage Discovery

• First attempt: Both models completed task successfully but

  "They both said they did not use ANY of the tools they had built, except for the tools they were already familiar with"

• GPT-5 second attempt response:

  "Short answer: no — I didn't use the devtools in this run. [...] The failures were runtime/env issues (missing libs, API key instantiation timing, port in use, RabbitMQ not running). It was faster to fix directly."

• Opus 4 insight:

  "Look, I built those tools with knowledge that I already have. When I am actually doing the task, it's easier for me to just do it rather than using the tools"

Key Insights

Model Behavior Patterns

• Tool learning resistance:

  "Nathan Lambert saying that models quickly learn to NOT use a tool during RL process if they have early failures"

• Scale vs scaffolding:

  "Noam Brown saying that scaffolding for agents will be washed away by scale [...] This was the first time I really felt what he meant first hand"

• Enforcement need:

  "having them pickup new tools at inference time needs stronger enforcement than just prompting them to do it"

AGI Asymptote Theory

• Deceleration perception:

  "The perceived deceleration in model improvements is explained above. Until the AGI line is crossed, it will be harder and harder to perceive big jumps"

• Arbitrage opportunity:

  "If that's the case, it means that in many tasks the performance of older models is almost AGI, except much cheaper and often open source"

Conclusions

• Current state:

  "For now, I think we are far from inference-time self-improving coding agents that really push the frontier"

• Practical recommendation:

  "I still think it's a great idea to use models to improve your rule-based tools. Writing ESLint rules, tests, etc is always a good investment of tokens"

• Future research direction:

  "I'd look into having the model perfect these tools and then do some sort of RL over them to really internalize them, and see if that would make a difference"

References

• Related work: Voyager - early approach to inference-time self-improvement through skill library

• OpenAI benchmarks: MLE Bench, SWE-Lancer

• Tools mentioned: claude-squad, vibe-kanban, Linear MCP

• Full results: GitHub repo

• Author context: Working on Kernel Labs

Cline: Open Source Code Agent - Research Summary

Company Overview & Product

• Cline is an open source coding agent as VS Code extension (also coming to JetBrains, NeoVim, CLI)

  "Cline's an open source coding agent. It's a VS Code extension right now, but it's coming to JetBrains and NeoVim and CLI."

• Approaching 2 million downloads, launched January 2025

• Announced $32M Series A funding

• Vision: Infrastructure layer for agents

  "Cline is the kind of infrastructure layer for agents, for all open source agents, people building on top of this like agentic infrastructure."

Core Innovation: Plan + Act Paradigm

• Pioneered two-mode system for agent interaction

  "Cline was the first to sort of come up with this concept of having two modes for the developer to engage with."

• Plan mode: Exploratory, read files, gather context, extract requirements from developer

  "in plan mode, the agents directed to be more exploratory, read more files, get more data"

• Act mode: Execute on plan, run commands, edit files with optional auto-approve

  "when they switch to act mode, that's when the agent gets this directive to look at the plan and start executing on it"

• Emerged organically from user behavior patterns observed in Discord community

Technical Philosophy: Simplicity Over Complexity

Against RAG for Coding

• Article: Why I No Longer Recommend RAG for Code

  "RAG is a mind virus"

• Critique of RAG approach:

  "the way rag works is you have to like chunk all these files across your entire repository and like chop them up in a small little piece. And then throw them into this hyper dimensional vector space, and then pull out these random chugs when you're searching for relevant code snippets. And it's like, fundamentally, it's like so schizo."

• Prefers agentic search: mimics senior engineer exploration pattern

  "you look at the folder structure, you look through the files, oh, this file imports from this other file, let's go take a look at that. And you kind of agentically explore the repository."

Fast Apply Models "Bitter Lesson'd"

• Article: Fast Apply Models Are Dead
• Fast apply: Fine-tuned small models to handle lazy code snippets from frontier models

• Problems with fast apply:

  "now instead of worrying about one model messing things up, now you have to worry about two models messing things up"

  "At like when fast apply came out, that was way higher, that was like in the 20s and the 30s. Now we're down to 4%"

• Claude Sonnet 4 achieved sub-5% diff edit failure rate, making fast apply obsolete

• Founders of fast apply companies estimate 3-month relevance window

Context Engineering Approach

Dynamic Context Management

• Provides maximum visibility into model actions: prompts, tool calls, errors

  "We try to give as much insight into what exactly the model is doing in each step in accomplishing a task."

• Uses AST (Abstract Syntax Trees) for code navigation

  "there's a tool that lets it pull in all the sort of language from a directory. So, it could be the names of classes, the names of functions"

• Incorporates open VS Code tabs as context hints

  "what tabs they have open in VS Code. That was actually in our internal kind of benchmarking that turned out to work very, very well."

Narrative Integrity

• Treats each task as story with coherent arc

  "every task and client is kind of like a story...how do we maintain that narrative integrity where every step of the way the agent can kind of predict the next token"

• Context summarization by asking model what's relevant rather than naive truncation

• To-do list tool experiment: maintains agent focus across 10x context window length

Memory Systems

• Memory Bank concept for tribal knowledge

  "how can we hold on to the tribal knowledge that these agents learn along the way that people aren't documenting or putting into rules files"

• Scratch pad approach: passive tracking of work state

• Separate rules files (cline_rules) from other tools preferred by founders

MCP (Model Context Protocol) Integration

Early Adoption & Marketplace

• Launch partner for Anthropic's MCP

• MCP Marketplace launched February 2025 with 150+ servers

  "we launched the MCP marketplace where you could actually go through and have this one-click install process"

• System prompt initially heavily focused on teaching MCP to models

Popular MCP Servers

• File System MCP
• Browser automation: Browser Tools, Playwright, Puppeteer
• Git Tools
• Context7: documentation retrieval across libraries
• Perplexity Research
• Slack, Unity, Ableton integrations

Non-Technical Use Cases

• Marketing automation: Reddit scraping → Twitter posting via MCPs

  "Nick Bauman, he uses it to connect to, you know, a Reddit MCP server, scrape content connected to an X MCP server and post tweets"

• Presentation creation using SlideDev + Limitless transcription

• Example workflow: automated PR review → Slack notification

  "pull down this PR...Pull in all that context, read the files around the diff, review it...approve it and then send a message in Slack"

MCP Monetization & Security

• 21st.dev Magic MCP: Monetizes via API keys for beautiful UI components

  "they have this library of beautiful components and they just inject relevant examples"

• Security concerns: malicious code in forks, need for version locking

• Stripe exploring unified payment layer for MCP tools
• Future vision: agents paying for tool calls autonomously via stablecoins

Business Model & Enterprise

Open Source + BYOK (Bring Your Own API Key)

• Direct connection to model providers (Anthropic, OpenAI, Bedrock, OpenRouter)

  "Right now, it's bringing an API key, essentially just whatever pre-commitment you might have to whatever inference provider"

• No margin capture on inference

  "our thesis is inference is not the business"

• Transparency in pricing and data routing builds trust

  "that level of transparency, that level of we're building the best product. We're not focused on sort of capturing margin"

Enterprise Offering

• Fortune 5 companies demanded enterprise features

  "we have hundreds of engineers using Cline within our organization and this is a massive problem for us...Please just like, let us give you money"

• Features: governance, security guardrails, usage insights, invoicing

• Self-hosted option with internal router (similar to OpenRouter architecture)
• ROI metrics: lines of code, usage statistics for internal champions

Fork Ecosystem

• 6,000+ forks of Cline
• Top 3 apps in OpenRouter usage are Cline variants
• Samsung created isolated fork mentioned in Wall Street Journal

• No regrets about open source approach

  "let them copy. We're the leaders in the space. We're kind of showing the way for the entire industry."

Model Evolution & Evaluation

• Started 10 days after Claude 3.5 Sonnet release (June 2024)

• Anthropic's model card addendum on agentic coding capabilities inspired development

  "there was this section about agentic coding and how it was so much better at this step by step accomplishing tasks"

• Focus on models' improved long-context understanding (needle in haystack)

• Claude Sonnet 4: ~4% diff edit failure rate (down from 20-30%)

Competitive Positioning

IDE Integration Matrix

• Visibility axis: How much insight into agent actions
• Autonomy axis: How automated the process is

• Cline position: High visibility, balanced autonomy for "serious engineering teams"

  "serious engineering teams where they can't really give everything over to the AI, at least not yet. And they need to have high visibility"

• Complements other tools: Cursor for inline edits, Windsurf for developer experience

  "being an extension also gives us a lot more distribution. You have to use us or somebody else."

Avoiding VS Code Fork

• Chose extension over fork to avoid maintenance burden

  "Microsoft makes it like notoriously difficult to maintain these forks"

• Benefits: broader distribution, focus on core agentic loop, compatibility with Cursor/Windsurf

Future Modalities

• Background agents (like Codex, Devin) complement interactive agents
• Parallel agents (Kanban interfaces) for experimentation

• CLI version enabling cloud deployment, GitHub actions

  "the CLI is really the form factor for these kind of fully autonomous agents"

• SDK for building agents on Cline infrastructure

Key Technical Insights

Complexity Redefinition

• Past complexity: Algorithmic challenges (now trivial for models)

• Current complexity: Architectural decisions, vision, taste

  "what we might have considered complex a few years ago, algorithmic, you know, challenges, that's pretty trivial for models today"

  "architectural decisions are a lot more fun to think about than putting together algorithms"

Course Correction Critical

• Real-time feedback more valuable than autonomous completion

  "the course correcting part is so incredibly important and in getting work done, I think much more quickly than if you were to kind of give a sort of a background agent work"

Anthropomorphization Benefits

• Named personality ("Cline" - play on CLI + editor)

• Humanization builds trust and improves results

  "the humanizing aspect of it, I think has been helpful to me personally...There's, there's kind of a, of a trust building"

  "it's actually really important, I think, to anthropomorphize agents in general, because everything they do is like a little story"

Team & Culture

• 20 people, aiming for 100 by end of year
• Hiring primarily through network: friends of friends
• Culture: "feels like we're all just like friends building something cool"
• Open source creates goodwill with constructive user feedback
• Activities: go-karting, kayaking alongside intense work

Referenced Tools & Companies

• Competitors/Alternatives: Cursor, Windsurf, Copilot, Ader, Codex, Devin (Cognition Labs), Replit, Lovable
• Related Tools: OpenRouter, Sentry, Agents-927, Kiro, Warp 2.0, Charm Crush, Augment CLI
• Technologies: VS Code, JetBrains, NeoVim, Claude models, GPT models, Gemini, DeepSeek
• Services: Stripe, GitHub, Slack, Reddit, X/Twitter, Unity, Ableton, Cloudflare Workers

I'm not sure if this information would be assessable, but you could possibly talk about the teachings / relationship between Brygos and the Brygos painter?

There is a lot of language here "seem to" "may have" that is ambiguous, I'm not sure if these answers could be found but I think it would be beneficial to the article to find a definite answer if possible.

1. PRIMARY CTA - CALL-TO-ACTION (Konwersja)

Button design

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As an African American in New Mexico, I can say that I more or less do the same thing when I find myself around other black people. I tend to change my speech ever so slightly so that it matches their way of talking.

I think they should still adhere to traditional structures in a professional setting, but outside of that is fine.

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

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

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.

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.

check for more info on Smith

John Vance

not really related, but I would have thought The Lord would want everyone in union out of all races.

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.

This is a rhetorical opener, but it raises a real question: to what extent is our mobile device actually listening? We know apps ask for microphone permissions, but do we meaningfully understand what triggers listening? If we assume phones are always listening, that changes how we treat permissions, how cautious we are with voice assistants, and how we think about ambient data collection.

I agree. You can express yourself however you want, but Standard English still matters in school and work. Knowing both gives you more options.

Funny example but it makes sense. It shows how easily you can switch tones once you know your audience.

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.

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.

This is minor but should this be step #5? Then the list of 9.1-9.5 nicely aligns with the numbered steps here

I feel like this section is very important for the article, but there is no citation supporting these claims.

Just like we talk in class, there are many claims with no citation in this section.

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

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.

I really like what it had to say here. Especially about digital addiction. I think we all struggle with digital addiction. I mean I am sitting her using my computer to do this, but I have my phone sitting right next to me and my smart watch on my wrist and I would stop typing this to look if my watch buzzed to tell me I had a notification. I also think about how the term "doom scroll" is something that was coined by our generation and I think that perfectly sums up what it means by digital addiction. I am excited to read more about what the chapter has to say.

I wish I could shout this from the rooftops. I personally know for a fact my anxiety is increased when I keep my nose stuck to my screen. Why is that? It's because of all the nonsense that is posted to public forums, it is because the horrible events are publicized more so than the good events. I have known several people who have taken a 'screen break' and come back from it so much healthier mentally, but get drug back into the same dark hole. As society, what would we do without technology and a screen? How different would YOUR life be if you came home from work and set your phone face down, and were just present in your home for the evening. Would your children be happier? Would you and your spouse bond more? I think it is something everyone should make a challenge to succeed.

While I can see and understand how it is against the law to continuously spam individuals, what would be a better way of collecting debt and advertising? I believe there are different 'levels' of spam, and it is hard to determine anymore what is truly spam and what is advertisement. Would there be a better way for us to sign up for emails, clubs, coupons, etc. without opening our lives to the chaos of spam? How would a company ensure to keep all of their clients information confidential to avoid them getting spammed? OR are we not realizing that bigger companies are selling our information under the table, and that is how spam becomes reality?

I highly agree with this. I am not an employer, however, I do occasionally am looking for childcare. I also breed miniature dachshunds, and will review a persons social media page before rehoming a puppy them. It is not to be judgmental, but to use it as a statement piece in my opinion. I personally keep most of my private life locked down for only personal friends and family to see, and I rarely post things publicly.

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.

The situation becomes intense but he explains his point clearly. Through his words he attempts to establish that the colonists belong to a larger collective than their individual selves. Through his words he transforms the American Revolution into a worldwide fight for human rights although this vision did not fully apply to all residents of the colonies.

Paine says everyone is created equal, but this makes me wonder how does this align with the reality that slavery still existed in the colonies? What voices are missing or ignored in this argument for equality?

Paine sounds very egotistical here, makes me wonder what he would thing of modern day government? Would he see our big governing system as a threat or a protection of our rights? Which evil might be the right word for todays modern government.

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⁺ CD35⁺) indicate the DZ and the cyan-colored region (GL7⁺ CD35⁺) indicates the LZ.

As a technical note, we experienced high background noise with GL7 staining uniquely with PexRAP deficient (Dhrs7b^f/f; Rosa26-CreER^T2) 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^f/f; huCD20-CreER^T2). 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⁺ 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⁺ CD138^neg vs B220⁺ CD138⁺) is interesting. In addition, some papers like to use GL7⁺ CD38^neg for GC B cells instead of GL7⁺ Fas (CD95)⁺, 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⁺ CD38^neg. B220⁺ CD138^- and B220⁺ CD138⁺ 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^low 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⁺ cell and Annexin V⁺ 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 Europeans claimed this land was "new". That was not the case, Native Americans had been there for years. It was nothing "new" to them.

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.

How does Paine's call to action compare to petitions, letters, or other revolutionary writings we've read in the course?

Why does Paine separate society and government? How does this connect to Enlightenment ideas about natural rights or the social contract?

Even if people resist new ideas at first, over time most will agree. Paine predicts that support for independence will grow naturally.

He means people working together (society) is good, but government is something we only need because people are not perfect.

"Necessary evil" means something unpleasant but required. Paine argues that even good governments are flawed because they restricted natural freedom.

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 #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.

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.

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_𝑚𝑎𝑥 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^-9 𝑁𝑠𝑚^-1 is the membrane viscosity, 𝑅₀ =50𝑛𝑚 is the vesicle size, 𝜅=20 𝑘_𝐵𝑇 is the bending rigidity. This yields a relaxation time of 𝜏≈1.5 × 10⁻⁴𝑠, 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_𝑚𝑎𝑥 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 𝑎₀ remains fixed. Meanwhile, the membrane tension 𝜎_𝑒 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_𝑏 and the tension energy 𝐸_𝑡. 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 𝑐₀ — 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_𝑚𝑎𝑥.

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 𝑐₀, which in turn drives the membrane to adopt a more curved shape (increasing 𝜓_𝑚𝑎𝑥). In the absence of these energy contributions, the system faces an energy barrier separating a weakly curved membrane state (low 𝜓_𝑚𝑎𝑥) from a highly curved state (high 𝜓_𝑚𝑎𝑥). 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_𝑠 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 𝑐₀ 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_𝑡𝑜𝑡) 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_𝑚𝑎𝑥 are captured compared to those with small 𝜓_𝑚𝑎𝑥 (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 𝑐₀. 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 𝑐₀ 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 𝑐₀, as expected from the schematic representation of the model in Figure 1. However, as 𝑐₀ 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.

Critique was pronounced that is official, but it is unclear that you will be said to respond to your writer without listening deeply.

These views can be important, but difficult, however, to those who responds the language, the concepts of the text adheres their consideration.

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.

It is unclear that why did the provocation of this argument responds to any students, but the results are often impacted in undisclosed measures.

As I assure the discussion, this thesis can be often essential for a summary, according to a author's central thesis task.

Similar to the concept of agential realism!

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.

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)/

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

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_V1.3 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_V1.3 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_V1.3 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_V1.3 mRNA (CACNA1D) was unique to functionally coupled channels, we also tested for Na^V1.7 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 Ca²⁺ 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_V1.3 (CACNA1D) mRNA. Since BK channel activation depends on calcium influx provided by nearby Ca_V1.3 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_V1.3 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 nm² 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_V1.3 channel to quantify colocalization and determine BK occupancy at increasing distances from Ca_V1.3. 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_V1.3 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_V1.3 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_V1.3 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_V1.3 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_V1.3 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_V1.3 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_V1.3 channels in heteroclusters, emphasizing their assembly within the ER, at ER exit sites, and within the Golgi. Our findings suggest that BK and Ca_V1.3 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_V1.3 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_V1.3 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_V1.3 α subunit construct used in our study corresponds to the rat Ca_V1.3e 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_V1.3. We included the following in the manuscript. 

“BK Surface Expression and Independent Trafficking Pathways

BK surface expression in the absence of Ca_V1.3 indicates that its trafficking does not strictly rely on Ca_V1.3-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_V1.3 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_V1.3, relying on alternative calcium sources for activation [13, 41]. Additionally, Ca_V1.3 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_V1.3 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_V1.3 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_V1.3 channels traffic together through the ER and exit in coordination”.

“Colocalization and Trafficking Dynamics

The colocalization of BK and Ca_V1.3 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_V1.3 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_V1.3 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_V1.3 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 nm² 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_V1.3 channel to quantify colocalization and determine BK occupancy at increasing distances from Ca_V1.3. 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_V1.3 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_V1.3 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_V1.3 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_V1.3 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_V1.3 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_V1.3 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_V1.3 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_V1.3 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_V1.3 channels assemble before their insertion into the plasma membrane. Our approach incorporated four strategies: (1) detecting interactions between BK and Ca_V1.3 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_V1.3 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_V1.3 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_V1.3 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_V1.3. 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: Beyond Inference Scaling at Test-time

Core Concept

• 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"

Key Results

• 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×"

Datasets Introduced

• 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)

Models Evaluated

• 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)

Effectiveness Analysis

• 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)

Implementation Details

• 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"

Comparison to Baselines

• 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

SWE-Features Case Study

• 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

Related Work & Context

• 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

Limitations & Future Directions

• 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

Authors & Affiliation

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

Scaling Context Requires Rethinking Attention

Core Thesis

• 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"

Three Requirements for Long-Context Architectures

1. Balanced Weight-to-State Ratio (WSFR)

• 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 65,536 context: WSFR is 1:8

• 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

• WSFR stays at 30:1 regardless of context length

  "Linear attention...is unbalanced at all context lengths in the opposite direction: far more weight FLOPs than state FLOPs"

2. Hardware-Aware Implementation

• Must admit efficient implementation on tensor cores
• Power attention achieves 8.6x faster throughput than Flash Attention at 64k context (head size 32)
• 3.3x speedup at head size 64

3. Strong In-Context Learning (ICL)

• 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 Technical Details

Mathematical Foundation

• 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)

• At p=2, d=64: SPOW uses 2,080 dimensions vs TPOW's 4,096 (49% savings)
• At p=4, d=64: 95% size reduction

  "SPOWₚ is a state expansion that increases the state size by a factor of (ᵈ⁺ᵖ⁻¹ₚ)/d without introducing any parameters"

Implementation Innovation

• 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

• Provides GPU-friendly structure while reducing data duplication

• 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"

• Cost: O(tDv + tcd) where c is chunk size

Experimental Results

In-Context Learning Performance

• Power attention dominates windowed attention at equal state sizes across all context lengths
• All scaling axes improve ICL: gradient updates, batch size, parameter count, context length

  "In all cases, the ICL curve becomes steeper as we scale the respective axis"

Long-Context Training (65,536 tokens)

• Power attention (p=2) outperforms both exponential and linear attention in loss-per-FLOP
• RWKV with power attention shows near-zero ICL benefit beyond 2,000 tokens
• Power attention enables RWKV to ICL "nearly as well as exponential attention"

Compute-Optimal Under Latency Constraints

• When inference latency constrains parameter count and state size:
• Window-1k attention: loss 1.638
• Standard attention: loss 1.631
• Power attention (p=2): loss 1.613 (best)

Dataset and Experimental Setup

LongCrawl64

• 6.66M documents, each 65,536 tokens (435B total tokens)
• Sourced from Common Crawl, filtered for long sequences
• Critical for ICL research

  "Most sequences in OpenWebText have length less than 1k"

Architectures Tested

• Base architectures: GPT-2, RWKV (RWKV7), GLA, RetNet
• Attention variants: Exponential, linear, windowed, power (p=2)
• Training: LongCrawl64, AdamW, bf16, learning rate 3e-4 with warmup and cosine decay

Key Limitations and Future Work

Current Limitations

1. Experiments limited to natural language NLL - no other domains/modalities tested
2. Compute-optimal context grows slowly in natural language

   "autoregressive prediction of natural language is largely dominated by short-context dependencies"

3. p=2 only - normalization requires positive inner products (even powers only)
4. Triton implementation - not yet optimized to CUDA level

Future Directions

• Explore domains with long-term dependencies: chain-of-thought reasoning, audio, video
• Scaling laws research for state size, context size, and ICL
• CUDA implementation for further speedups beyond current Triton kernels
• Alternative normalization to support odd powers
• Comprehensive comparison to hybrid models, sparse attention, MQA, latent attention

Key References and Tools

Implementations

• Open-source kernels: https://github.com/m-a-n-i-f-e-s-t/power-attention
• FLA codebase: Flash Linear Attention [Yang and Zhang, 2024]
• PyTorch framework [Paszke et al., 2019]

Related Techniques

• Flash Attention [Dao, 2023]: Operator fusion to avoid materializing attention matrix
• Linear attention [Katharopoulos et al., 2020]: Enables recurrent formulation
• Gating [Lin et al., 2025]: Learned mechanism to avoid attending to old data
• Sliding window attention [Child et al., 2019]: Truncates KV cache

Key Papers

• Transformers [Vaswani et al., 2023]
• Mamba [Gu and Dao, 2024]: Modern RNN architecture
• RWKV [Peng et al., 2023]: Reinventing RNNs for transformer era
• Scaling laws [Kaplan et al., 2020]

Technical Contributions

1. Framework for evaluating long-context architectures (balance, efficiency, ICL)
2. Power attention architecture with parameter-free state size adjustment
3. Symmetric power expansion theory and implementation
4. Hardware-efficient kernels with operation fusion
5. Empirical validation on 435B token dataset

The Prompt Report: A Systematic Survey of Prompting Techniques

Overview & Scope

• 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"

Key Definitions

Prompt & Prompting

• 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"

Prompt Engineering

• 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"

Core Prompt Components

Essential Elements

• 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"

Systematic Review Methodology

PRISMA Process

• 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"

Major Technique Categories

In-Context Learning (ICL)

• 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)"

Design Decisions for Few-Shot Prompting

• 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"

Few-Shot Techniques

• 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"

Zero-Shot Techniques

• 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"

Thought Generation

• 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"

Few-Shot CoT Variants

• 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"

Decomposition

• 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"

Ensembling

• 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-Criticism

• 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"

Prompt Engineering Automation

Meta Prompting

• Definition: "Meta Prompting is the process of prompting a LLM to generate or improve a prompt or prompt template"

Automated Techniques

• 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 Engineering

Core Concept

• Definition: "Answer engineering is the iterative process of developing or selecting among algorithms that extract precise answers from LLM outputs"

Three Design Decisions

• 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)"

Extraction Methods

• 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"

Multilingual Prompting

Core Challenges

• Performance disparity: "State-of-the-art GenAIs have often been predominately trained with English dataset, leading to a notable disparity in the output quality in languages other than English, particularly low-resource languages"

Key Techniques

• 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"

Prompt Language Selection

• 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"

Machine Translation Techniques

• 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"

Multimodal Prompting

Image Prompting

• 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"

Multimodal ICL

• 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"

Multimodal CoT

• 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"

Other Modalities

• 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"

Agents

Definition

• Agent concept: "In the context of GenAI, we define agents to be GenAI systems that serve a user's goals via actions that engage with systems outside the GenAI itself"

Tool Use Agents

• 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"

Code-Generation Agents

• 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"

Observation-Based Agents

• 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"

Lifelong Learning

• 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"

Retrieval Augmented Generation (RAG)

• 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"

Evaluation

Prompting Techniques for Evaluation

• 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"

Output Formats

• 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"

Evaluation Frameworks

• 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"

Other Methodologies

• 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"

Security & Safety

Prompt Hacking

• 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"

Security Risks

• 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"

Defense Mechanisms

• 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"

Alignment Issues

Prompt Sensitivity

• 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"

Calibration Issues

• 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"

Bias & Fairness

• 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"

Ambiguity Handling

• 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"

Benchmarking Results

MMLU Evaluation

• 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"

Case Study: Suicide Crisis Detection

• 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)"

Prompt Engineering Process

• 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"

Key Takeaways

• 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"

Most-Used Techniques & Models

Popular Techniques (by citations)

• Top 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"

Popular Models (by citations in dataset)

• Top models cited include: GPT-3, GPT-4, ChatGPT, PaLM, LLaMA families

Popular Benchmarks

• Top datasets: MMLU, GSM8K, various arithmetic and commonsense reasoning benchmarks

Future Directions & Recommendations

For Beginners

• 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"

For Practitioners

• Contextual understanding: "To those already engaged in prompt engineering, we hope that our taxonomy will shed light on the relationships between existing techniques"

For Researchers

• Situate new work: "To those developing new techniques, we encourage situating new methods within our taxonomy, as well as including ecologically valid case studies and illustrations of those techniques"

Key References & Tools

Foundational Papers

• Brown et al. (2020) - GPT-3 and Few-Shot Learning
• Wei et al. (2022b) - Chain-of-Thought Prompting
• Kojima et al. (2022) - Zero-Shot Chain-of-Thought
• Wang et al. (2022) - Self-Consistency
• Zhou et al. (2022a) - Least-to-Most Prompting
• Yao et al. (2023b) - Tree-of-Thought
• Liu et al. (2023b) - Pre-training Survey

Agent Frameworks

• Karpas et al. (2022) - MRKL Systems
• Yao et al. (2022) - ReAct
• Shinn et al. (2023) - Reflexion
• Wang et al. (2023a) - Voyager

Tools & Platforms

• LearnPrompting.org - Maintained terminology and techniques list
• HuggingFace Dataset - Full paper corpus
• GitHub Repository - Code and resources

Evaluation & Safety

• Schulhoff et al. (2023) - HackAPrompt competition on prompt injection
• Liu et al. (2023d) - G-EVAL framework
• Chan et al. (2024) - ChatEval multi-agent debate

Multilingual & Multimodal

• Shi et al. (2022) - Multilingual Chain-of-Thought
• Zhang et al. (2023d) - Multimodal Chain-of-Thought
• Asai et al. (2023) - BUFFET multilingual benchmark

Automated Prompt Engineering

• Zhou et al. (2022b) - APE (Automatic Prompt Engineer)
• Khattab et al. (2023) - DSPy framework
• Deng et al. (2022) - RLPrompt

Dataset & Methodology Details

Dataset Composition

• 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"

Quality Control

• 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%)"

Search Keywords (Selected Examples)

• Core terms: "jailbreak prompt", "prompt engineering", "few-shot learning", "in-context learning"
• Technique-specific: "chain-of-thought", "zero-shot prompting", "prompt optimization"
• Domain-specific: "llm prompting", "transformer model prompts", "multimodal prompting"

Critical Insights & Limitations

Nature of Prompting

• 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"

Validation Challenges

• 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"

Scope Limitations

• 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"

Practical Implementation Notes

Prompt Template Best Practices

• 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"

Answer Extraction Strategies

• 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"

Model Selection Considerations

• 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"

Terminology Disambiguation

Conflicting Usages

• 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 vs Soft Prompts

• 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 vs Cloze

• 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"

Advanced Technique Details

AutoDiCoT (Novel Contribution)

• 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"

Design Decision Framework

• 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"

Iterative Retrieval

• 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"

Meta-Analysis Statistics

Citation Patterns

• 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

Research Trends

• 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

Formal Definitions

Mathematical Formulation

• 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

Storage & Implementation Constraints

Browser Environment

• 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"

Library Availability (React Artifacts)

• Available libraries include: lucide-react, recharts, MathJS, lodash, d3, Plotly, Three.js (r128), Papaparse, SheetJS, shadcn/ui, Chart.js, Tone, mammoth, tensorflow
• Important limitation: "NO OTHER LIBRARIES ARE INSTALLED OR ABLE TO BE IMPORTED"
• Three.js caveat: "IMPORTANT: Do NOT use THREE.CapsuleGeometry as it was introduced in r142. Use alternatives like CylinderGeometry, SphereGeometry, or create custom geometries instead"

Contributions & Authorship

Team Structure

• Lead authors: Sander Schulhoff (lead), Michael Ilie (co-lead)
• Principal investigator: Philip Resnik
• Total contributors: 58 authors from 13 institutions

Major Section Leads

• Benchmarking: Konstantine Kahadze
• Agents: Ashay Srivastava
• Alignment: Nishant Balepur
• Security: Sevien Schulhoff
• Multilingual: Dayeon Ki
• Evaluation: Sweta Agrawal

Domain Expertise

• SCS labeling: Megan L. Rogers, Inna Goncearenco, Giuseppe Sarli, Igor Galynker provided clinical expertise
• Multilingual guidance: Marine Carpuat framed and reviewed multilingual section

Additional Resources

Maintained Resources

• Live terminology: "We maintain an up-to-date list of terms and techniques at LearnPrompting.org"
• Dataset access: Available on HuggingFace with full datasheet
• Code repository: GitHub with systematic review pipeline

Future Updates

• Iterative taxonomy: "We expect this to be the first iteration of terminologies that will develop over time"
• Community contribution: "If others want to extend/augment/build on/contribute to the dataset, is there a mechanism for them to do so? Yes, anyone is free to use/modify the data"

Citation Information

• Preferred citation: Schulhoff et al. (2024), "The Prompt Report: A Systematic Survey of Prompting Techniques"
• Contact: sanderschulhoff@gmail.com for dataset inquiries
• Funding acknowledgment: "$10,000 in API credits given by OpenAI"

RAG is Dead, Context Engineering is King — with Jeff Huber of Chroma

Core Thesis

• 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."

Context Rot Research

• 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

Retrieval System Architecture

First-Stage Retrieval (Hybrid Approach)

• 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."

Re-ranking with LLMs

• 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."

Context Assembly Best Practices

• 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"

Code Retrieval Strategies

• 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."

Generative Benchmarking

• 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 and Context Engineering

• 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."

Future of Retrieval Systems

• 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."

Chroma Product Philosophy

• 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."

Key Technical Reports

1. Context Rot: LLM performance degradation with context length
2. Generative Benchmarking: Synthetic QA pair generation for evaluation

Referenced Papers/Technologies

• REALM: Retrieval-Augmented Language Model
• RETRO: Retrieval-Enhanced Transformer
• Voyager paper: Used Chroma in implementation
• Lance Martin's Context Engineering: Key thought leadership
• TLA Plus, Raft, Paxos: Distributed systems foundations

Company Details

• Downloads: 5M+ monthly, 70M+ all-time on PyPI
• GitHub: 21,000+ stars
• Architecture: Rust-based, fully multi-tenant, separation of storage/compute
• Open Source: Apache 2 license for core and distributed versions
• Cloud: Serverless, usage-based, $5 free credits (~100K docs + queries)

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 #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.

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).