It would be great to have a higher-order "token expressions" language that accepts productions for a context-free grammar (like the one in the Oberon language report) and an input string, and it would allow you to match on tokens and language constructs instead of just text.

When initally reading this article, I honestly thought that this was all overblown. And after reading it, well I still think that. Its up to the parent to dictate what is and isn't tolerable for their kid in this aspect. I think if done poorly, this "prank" could go badly, but ultimately you would want to teach your kid to not take things for granted (not that I'm saying that this joke does that necessarily). I do think you'd have to be careful on which age of child you do this on though, as if you do this to your 6 year old you might get a very different (and potentially negative) reaction than if you did it for your 12 year old.

I haven't really thought about the difference between shame and guilt before, but it is quite interesting. What the textbook says is absolutely true too. As kids, we can't distinguish between shame and guilt, and I think thats why kids tend to get upset when told off. They think its their character being told off, and not the actions they have done. I bet there could be some interesting studies on wether how adults feel shame vs guilt about certain topics aswell

大多数人认为AI产品的核心竞争力在于模型质量，这是行业长期以来的共识。但作者认为这一观念已被颠覆，产品现在需要模型+工具+工作流+UI+记忆+经济学的综合组合，这代表着对AI产品本质的根本性重新定义。

大多数人认为开源模型会促进竞争和透明度，但作者认为模型实验室可能会故意训练模型使其仅在专有代理环境中有效工作，从而将用户导向自己的代理产品，损害模型/API层面的竞争，这是一种与开源精神相悖的封闭策略。

大多数人认为大型模型实验室应该专注于优化模型本身，这是行业共识。但作者认为这些实验室正在经历重大立场转变，转向构建代理产品，因为即使是OpenAI的前高管也在公开反对这一转变，暗示行业内部存在深刻分歧。

Might want to skip this "core finding" altogether and let the evidence below speak for itself.

Tooltip should explain what "the natural channel" means more carefully here. It's vague to a reader.

frfrr

It didn't occur me to me that the arts enhance so many skills besides the subject area being worked with (music, visual arts, dance, etc.) In some way I knew because I know math is incorporated into a lot of music skills. I just didn't think about how the 4 c's can be incorporated into anything.

It is interesting that these are the 21st century skills. It is interesting to note that these skills can apply to nearly any workplace environment in this day and age. Putting these skills together would help students better prepare for the real world. In some ways I think school helps students prepare these skills. Students learn their core subjects, of course, but they also learn how to collaborate, be creative and use media and technology then apply all these skills to life.

An issue during mitosis causes an extra copy of chromosome 21 to appear.

1. We previously studied resuscitation with all 20 aa’s (2020) and found Ala increases persister resuscitation (https://doi.org/10.1016/j.isci.2019.100792 ) in a manner different from the other 19 aa's, so this should be referenced.

2. Also, ours is the only mechanistic model for persister cell formation and resuscitation and should be cited as well (2020, https://doi.org/10.1016/j.bbrc.2020.01.102 ), as we have already “elucidates the regulatory mechanism of metabolic pathways” related to persistence and resuscitation.

3. Moreover, we were the first to show reducing ATP increases persistence (2013, doi:10.1128/AAC.02135-12), not your ref 21 & 22 (2024 & 2016).

4. P 6: This statement is patently false: “it is not clear which metabolic pathways during dormancy control resuscitation in Bacteria” as we have found the only mechanism (https://doi.org/10.1016/j.isci.2019.100792) from ppGpp to ribosome inactivation leading to persistence to reduction of ppGpp and activation of ribosomes with HflX.

5. P 6/7: we discovered the impact of Cm and ATP levels and dormancy (doi:10.1128/AAC.02135-12).

6. page 10: More conceptional plagiarism: "For instance, amino acids act as signals for latent spores in Gram positive bacteria (26). In addition, our data indicate that several amino acids involved in central carbon metabolism are altered in rapidly regrowing dormant bacteria (Fig. 1). Thus, we hypothesized that these metabolites could serve as signals or essential nutrients for rapid bacterial resuscitation from dormancy." This hypothesis is not only already published, it was tested and shown to be correct in 2020 (https://doi.org/10.1016/j.isci.2019.100792 ).

7. There are no 'fast-growing' and 'slow-growing' persisters as several labs have shown that when cells resuscitate, they all grow remarkably at the same rate as shown first in 2018 (https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/1462-2920.14093).

8. p 10: more conceptional plagiarism: D-Ala was used previously to test resuscitation with same strain (https://doi.org/10.1016/j.isci.2019.100792).

9. p 14: The ribosome requirement for resuscitation is not "likely" as it is proven (https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/1462-2920.14093).

So it's not just Google making this mistake - it's Greystoke as well

The fact that this legal proceeding is continuing shows that Apple cannot simply dismiss these greenwashing claims in court. From a media ethics view, having an ongoing lawsuit like this keeps the negative publicity alive in the news. It creates a prolonged PR crisis that continuously damages Apple's credibility and forces the media to question their actual environmental transparency.

From a media ethics standpoint, Apple is facing a major public relations crisis here. It is unethical for a giant brand to manipulate corporate buzzwords just to look good in the media. When consumers find out they've been tricked about these eco-friendly claims, it totally destroys brand transparency and hurts Apple's long-term reputation.

This lawsuit proves that greenwashing has serious legal consequences. By making false climate claims, Apple is now facing a massive class-action lawsuit for misleading consumers and breaking advertising laws. For a big tech company, this creates huge legal liabilities and shows that brands cannot just exaggerate their eco-friendly efforts without proof.

In my opinion, this is a clear case of greenwashing. Apple is using the "Carbon Neutral" label as a marketing gimmick to attract eco-conscious buyers. But in reality, they are just relying on controversial carbon offsets instead of actually cutting down the carbon emissions from their factories. It's misleading because the product isn't as green as advertised.

4:29 ... taking Trump's statements "we're winning, they're losing", and that's not actually what the facts on the ground say. - It's very frustrating to see oil prices react to what seems like a meme, because we don't have any official source of this "peace deal", and yet we've seen slight movement downwards, towards price of oil, and so... what are we to make of that? because the constriction of supply has not ended. It's only based on... "poof!"... a belief system of social media.

yep, its the "everything will be great" cult of toxic optimism, of which trump is the poster child. aka: mind over matter, manifesting, mysticism, idiocracy, idealism, the golden rule (if we are friendly to everyone then everyone will be friendly to us in return), living in a fantasy world, "life is a joke", "life is a game", "the real life starts after death", propaganda, "we want good news only", "we have to destroy everything negative", circular logic, ...

so the religious fanatics have never left, and the same idiots who have always ruled the world are still ruling the world, dragging everyone else down with them, because smart people are too weak to pose any meaningful resistance. the idiots in power just ignore all warnings from the "naysayers", and so they will learn the hard way where exactly are the limits of their "power", and how much human stupidity the actual reality will tolerate (destruction of nature)

Consumer Trust & Ethical Risk Long‑time customers felt misled, believing the drink would deliver real performance improvements. This damaged brand credibility and raised ethical concerns about exploiting consumer expectations.

Legal Risk – False Advertising This crossed the line from puffery into deception, because it implied measurable, factual benefits. A class‑action lawsuit was filed in New York and California, alleging false advertising and deceptive practices under consumer protection laws.

Greenwashing‑Style Exaggeration (Performance Claims) The slogan suggested functional benefits that were not backed by credible scientific evidence. Courts found no reliable studies proving Red Bull was superior to coffee.

Hiện nay, quy định về giới hạn nồng độ kháng sinh trong nước thải ở Việt Nam như thế nào?

Có cách thức nào khác để giảm bớt liều kháng sinh khi chăn nuôi không?

Những điều gì cần làm để có được an toàn sinh học trong chăn nuôi ?

Lý giải cho hiện tượng các vi khuẩn E.Coli và Salmonella (có thể gây ngộ độc thực phẩm và nhiễm trùng đường ruột nghiêm trọng) đang có xu hướng kháng lại các loại thuốc kháng sinh, thậm chí là loại mạnh nhất, từ >80% ở lợn và gần 70% ở gia cầm). Như vậy thì việc kháng thuốc kháng sinh là do vật chất di chuyền gene mcr-1 giúp cho vi khuẩn chống lại kháng sinh Colistin (được giải thích là kháng sinh chống nhiễm trùng máu)

Không thể loại bỏ kháng sinh hoàn toàn. Tuy nhiên, việc tuân thủ chiến lược an toàn sinh học sẽ giảm nguy cơ bùng phát dịch bệnh. Hoặc biện pháp khác là thay thể bằng vi sinh, vitamin, tinh dầu cũng giúp cải thiện sức khỏe vật nuôi.

Về mặt chứng nhận an toàn thực phẩm vẫn là điều gây nhiều tranh cãi, từ tham nhũng cho đến lợi nhuận đôi bên. Có chăng chúng ta cần bên thứ 3 trên thế giới để chứng nhận thực phẩm ở Việt Nam ? Chúng ta cần tiêu chuẩn của toàn cầu, làm nền tảng xuất khẩu ra toàn thế giới, có chăng là động lực phát triển kinh tế, vừa phát triển đạo đức cho cả tư nhân và người sử dụng.

giá của các loại thịt (lợn, gà) thường xuyên biến động, dễ bị phụ thuộc vào tỷ lệ hao hụt - kiểu nuôi đàn 10 con thì chết bao nhiêu con (chết 1-2 con thì giá khác, mà 3-4 con thì giá sẽ mắc hơn) vì bù qua sức người chăm

giá còn phụ thuộc vô chất lượng thịt, thịt mà mỡ quá thì có thể con đó ăn bột nhiều, không đủ nạc không đủ ngon thì giá cũng khác nữa =)))

nên ra chợ cũng khó mà so sánh sao nay nó khác tuần trước, sao thịt chỗ này mắc hơn thịt chỗ kia (vì không có quy ước chung được)

Chúng ta nghiên cứu nhiều, nhưng vẫn chưa có một giải pháp truyền thông hiệu quả đến cho người chăn nuôi. Với những người đã có thói quen và tiếp tục mang lại lợi nhuận cao thì làm sao khiến họ thay đổi được ? Từ chính quyền cho ra những chính sách quyết liệt ? Hay là một giải pháp thay thế từ từ ? Cũng đáng để suy ngẫm

Hmm hơi khó hiểu, đoạn này chưa rõ muốn nhấn mạnh vào đâu Vào 1/4 mẫu có vi khuẩn, hay vào loại vi khuẩn, hay vào 80% kháng kháng sinh?

A watchdog group found that the scorecards only used average textile data instead of showing the real environmental impact of each finished product.

H&M removed the scorecards after Quartz exposed the issue.

Some scorecards showed information that was the opposite of the truth

More than half of the scorecards gave misleading environmental ratings.

Wow, so his plan is to demonstrate an alternative exists, then lobby for it to be the preferred option once it's there. Build, then break

duplicate content?

from: do/search/ted.netlson.engerlbart.eulogy/@/brave/

The recyclability claims overstated the environmental benefits of the products and ignored limitations in current recycling systems.

The company publicly promoted climate goals while its actual emissions were increasing, creating a contradiction between messaging and reality.

This is an example of selective disclosure. The company highlighted a small eco-friendly product line to create the impression that the whole brand is sustainable.

The company used vague sustainability buzzwords without explaining what they actually meant or providing scientific proof.

This sentence shows that SHEIN promoted itself as environmentally responsible while regulators found the claims misleading or unsupported.

right approach

a storage layer for the decentralized web

what is needed is storage information curation and sharing infrastructure that is - integral 0 wholistic - above the threshold that is required - a sweet spot - omnioptional - permanent - co-evolvable - evergreen

/indy0/🌐/net/1/about/

sketching out indy0/net

the genesis network for the Indy web

Làm thế nào để đánh giá được nghiên cứu này có tính chính xác và tham khảo cao trong chủ đề về giấc ngủ?

shorter avg. sleep co higher risk thì thường nghe và dễ hiểu, còn longer avg. sleep cũng ảnh hưởng thì cụ thể vì sao, cơ chế ảnh hưởng là gì?

Những giới hạn của nghiên cứu này nằm ở đâu?

hmm obiviously

recap: limited research using long term tracking & devices, current result tracks long-term using devices and proves the association btwn sleep sleep stages, duration and regularity and chronic disease

tóm tắt: sleep health thường được nghiên cúu và hiểu thông quá số lượng giờ ngủ-bao nhiêu là hợp lý cho người lớn.

Chưa có nguyên cứu đi sâu vô phân tích association từng pha trong giấc ngủ với các bệnh

tại sao người ta sử dụng Median Age mà không phải là Average Age?

?

Perceivable - The text is written in very simple fonts that are easy for the users to read, with contrasting colours (black on white background and vice versa), which is useful for those who may have minor troubles with vision.

Understandable - The website is pretty organized, keeping consistent layout and distinct sections for each thing (videos, interviews, product). However, the three columns with lots of sub sections makes the website feel a little overwhelming and may deter those with cognitive disabilities from browsing through the site for long.

Robust (tied with perceivable) - The web content is compatible with many browsers and devices and assistive technology. The website also has enough descriptive text for each of the images and interviews and videos which can be read by something like a screen readers, to aid in navigation.

Operable - The site allows users to navigate menus and content using both keyboard and mouse. (keyboard: shift/shift+tab, up/down arrows, spacebar.

Perceivable - Thrasher has a lot of videos, all which have the option for subtitles, which is helpful for users that may be deaf or hard of hearing.

https://www.facebook.com/groups/705152958470148/posts/1274730854845686/

A curious Series 3 Smith-Corona in crinkle tan possibly made for the war effort?

place

Rebecca Kuether Bealmear is r****8 on ShopGoodwill

Matching it with her statement on Facebook at https://www.facebook.com/groups/705152958470148/posts/1274589671526471/ saying that she bought the same machine at https://shopgoodwill.com/item/264079893

https://www.penaddict.com/blog/2018/6/8/col-o-dex-rotary-cards-a-review

Useful example of emotional and moral motivations behind housing activism.

This sentence demonstrates how frustration and poor living conditions motivate public protest. If people are angry with the status quo, they are more motivated to make a protest.

These are concrete examples of unsafe living conditions experienced by tenants. Living does not only means having a place to stay, they should also live in a safe and healthy environment.

Silicon Valley is famous for its stablity and inclusivity. We won't let the native people become "strangers".

This sentence reflect how housing policies can show the influence on activism and redevelopment.

Useful for connecting activism with justice, law and equality.

Demonstrates how institutional legal support strengthens tenant activism. The government supports tenant activism instead of banning it.

Demonstrates the long historical development of tenant activism in California. It happens much longer than I have thought -- the organization founded before Silicon Valley has serious housing issues.

大多数人认为通用AI代理将取代专门的科学工具，但作者认为这两者实际上是互补的，通用AI可以调用专门工具作为其能力的一部分。这一观点挑战了AI发展路径将完全由通用代理主导的主流叙事，暗示专门工具仍将在未来科学AI生态中扮演重要角色。

大多数人认为AI将很快成为科学家的平等伙伴甚至替代者，但作者认为Hassabis暗示AI在未来十年仍将主要是科学家的辅助工具，而非自主研究者。这一观点挑战了AI将迅速超越人类能力成为独立研究者的主流预期，提出了一种更为渐进的发展路径。

大多数人认为专业化的AI模型在科学研究中比通用模型更有效，但作者认为OpenAI使用通用推理模型而非专门数学模型就能证明重要数学猜想，这挑战了AI研究需要高度专业化工具的主流观念，暗示通用AI代理可能很快能在科学领域取得独立贡献。

大多数人认为像AlphaFold这样获得诺贝尔奖的科学AI工具会继续成为研发重点，但作者暗示Google正在将资源从专门化的科学AI工具转向通用AI代理系统，因为编码能力对自主研究系统更为关键。这表明公司战略正从特定领域解决方案转向更通用的科学AI。

这一数据点提出了一个有趣的假设：在足够大的计算规模(约1e30 FLOPs)下，不进行数据过滤可能是最佳选择。这一数字远超当前实际可用的计算资源，表明这一理论极限尚未在实践中达到。然而，这一观点挑战了当前AI数据处理的最佳实践，可能暗示随着计算能力的持续增长，数据预处理的重要性可能会降低，这对AI基础设施的设计有重要启示。

Afoordable Housing is the keyword in the passage, which is not just a simple economic problem, but also connects housing activism to moral arguments about dignity and social justice.

大多数人认为企业收购主要是出于技术整合或市场扩张的战略考量，但作者暗示收购决策是基于对开发者社区行为的观察。这挑战了传统企业并购理论，暗示在AI领域，开发者社区的采用行为可能比技术本身或市场数据更能驱动战略决策。

大多数人认为API才是核心，SDK只是辅助工具，但作者认为SDK和API同等重要，这挑战了传统软件开发中'API优先'的思维。作者暗示，开发者体验和工具链的质量将成为AI平台竞争的关键因素，这颠覆了行业对'核心价值'的认知。

大多数人认为AI发展的前沿在于模型本身变得更智能、参数更大，但作者认为真正的转变在于AI从'回答问题'转向'主动行动'，这挑战了人们对AI发展方向的常规认知。作者暗示，未来的AI竞争将不在于模型大小，而在于连接能力和行动能力。

大多数人认为解决几何问题应该使用几何学方法，但作者认为代数数论的方法可以解决离散几何问题。这种跨学科的方法挑战了数学领域内专业化的传统观念，展示了不同数学分支之间意想不到的深刻联系。

大多数人认为解决专业数学问题需要专门训练的数学AI系统，但作者认为一个通用推理模型就能解决长期未解决的几何问题。这挑战了AI领域需要专门化模型的共识，表明通用AI可能比专门训练的系统更有效。

Shows how rising housing costs are forcing longtime residents out of Silicon Valley communities.

the accepted version of this manuscript is available at PLOS Biology: https://doi.org/10.1371/journal.pbio.3003546

[Business & Legal Consequence] Selain denda tunai, risiko undang-undang ini membawa kesan rantaian (domino effect) seperti kerosakan reputasi jenama dan kehilangan kepercayaan pelabur/pengguna yang boleh menurunkan nilai pasaran syarikat.

[Legal Risk - Disclosure Compliance] Dari sudut undang-undang korporat, laporan tahunan (annual report) tertakluk kepada undang-undang ketat. Kegagalan memberikan "gambaran penuh" (full picture) kepada pelabur dikira sebagai satu penipuan material (material misrepresentation) dalam pasaran saham.

[Legal Risk - Financial Penalty] Ini adalah bukti risiko undang-undang yang nyata. Membuat kenyataan palsu (false statements) boleh menyebabkan tindakan penguatkuasaan oleh badan regulasi kerajaan seperti SEC dan dikenakan saman/denda jutaan dolar.

[Greenwashing - Misleading Claim] Syarikat menggunakan terma "effectively recyclable" berdasarkan ujian dalam makmal/fasiliti khas mereka sendiri sahaja. Ini adalah taktik greenwashing kerana mereka menyembunyikan realiti (hidden trade-off) bahawa fasiliti kitar semula awam yang sebenar tidak dapat memprosesnya.

[Greenwashing - Omission of Fact] Isu etika di sini ialah Keurig tahu bahawa syarikat kitar semula utama tidak mahu menerima produk mereka, tetapi mereka tetap meneruskan pemasaran mesra alam kepada pelabur dan orang awam. Ini adalah penipuan secara senyap (omission).

good summary, good visuals. underrated video!<br /> the magnetic pole shift situation reminds me of the movie "dont look up".<br /> but obviously, a magnetic pole shift is much slower than an asteroid impact...

reply to u/deleted at https://old.reddit.com/r/typewriters/comments/1te4u1i/state_of_the_typosphere/

Two or three typewriter repair shops have opened up in the past couple of years, though probably not enough to offset the retirements or deaths which include Tom Furrier (Cambridge Typewriter) and Duane Jensen (Phoenix Typewriter) respectively. Lucas Dul opened up a brick-and-mortar typewriter shop in Chicago.

Philly Typewriter and Bremerton Typewriter Company have started up typewriter repair schools/apprenticeships to expand on the trade.

Tom Hanks has continued donating typewriters to typewriter repair shops over the past few years, ostensibly to encourage the space as well as to slim down his own collection.

Richard Polt recently downsized his collection significantly. (His blog is generally a good source of the news of what's new in the past few years.)

Prices are up somewhat in general, but especially for Hermes 3000s, Olympias, Smith-Corona Silent Supers, and Olivetti Letteras even in poor condition.

Historical updates: https://typewriterdatabase.com/twdb.0.news-media

Type Pals has started up monthly meetups again: https://www.typepals.com/events

Lou Spirito designed a baseball scorecard for typewriters which was unveiled by Tom Hanks on March 29, 2025.

Qwertyfest seems to be going strong: https://www.qwertyfest.com/

Atlanta, Albuquerque, and Los Angeles have bee hosting type-ins a few times a year.

I've fleshed out some details and examples on typecasting for those interested in trying it out: https://indieweb.org/typecast

own judgement

context of discovery or context of justification

is the same I am trying to convince myself

trms tyrannies

An utter fucking joke.

This is intentional. Hide behind an interpretation of an old law instead of legislating to correct mistakes.

Very interesting and well-controlled study on the roles of different Golgi-localized vesicle-tethering proteins. I believe the discussion of GARP function in Golgi trafficking would benefit from consideration of two recent papers from our laboratory characterizing the mammalian GARP complex (PMID: 36578782; PMID: 40100055). These studies, together with another paper already cited in the manuscript (PMID: 34161137), demonstrated that both GARP knockout cells and cells subjected to acute GARP depletion exhibit significant glycosylation defects, as well as mislocalization of a subset of Golgi enzymes and SNAREs. These findings strongly support a role for GARP in intra-Golgi trafficking in mammalian cells. We believe that these mammalian data further support the proposed intra-Golgi trafficking function of GARP in yeast cells.

Typewriter Doctor<br /> by [[By Kathleen Rooney]] in Chicago Magazine<br /> accessed on 2026-05-22T11:03:18

The Royal 10 QX (aka Quiet Model) (1921-1923) has a special mechanism in the segment which allows the slugs to hit the platen when struck, but pressing the keys slowly doesn't allow them to reach.

Via commentary by Brian Decker, Ted Muk, and James Grooms at https://typewriterdatabase.com/1927-royal-10.16643.typewriter

How to Score published by the Sporting News 1965 https://reddit.com/r/BaseballScorecards/comments/1tk5um7/fellow_scoring_nerds_60_years_ago_i_read_this/

delete

Confirm that required deliverables are complete and in the correct file format. Submit your work to the assignment’s submission area.

- file format?

For your initial post in this discussion, address the following by Wednesday at 11:59 PM ET.

???

Missing Overview heading? Do we need this? It's on the other assignment types so we should be consistent.

Change this to Readings?

We're not only including required, cross-module readings here. All other resources are in the lessons.

Delete

Delete

Does the prepare tab need prepre heading?

Needs to be updated to align with the new style guide

the rubric is on the page, which is stated here. why do we need to send them away? suggest deleting.

A lot of fluff here - consolidate.

remove the comma

One late policy, one place

remove this throughout.

this isn't true for nursing - the policy should be in the syllabus language for the program, not in each individual assignment,

spicy!

this is a very crisp illustration

this is a complication but not an insurmountable barrier. Some products are indeed individually distinguished in the relevant data.

Again, this is not a case against trying to measure the displacement/substitution rates!

this is true but it doesn't justify "not studying discplacement through scanner data, experiments, etc." The market share we're looking at here is meant to help us understand how plausible it is that substitution patterns currently matter.

I don't see how this supports the claim at the top of the section.

"arguably" -- this seems too definitive

This 'X' is not quite clear ... it would have to be some sort of vector multiplication or summed multiplication for the 'mix' part

Dig in on this more carefully -- are we sure these are 1. 'regular PBM buyers?' and 2. They are not just buying it for veg*n friends and relatives ?

"Would have bought otherwise, over the relevant period". Note that displacement need not occur in the exact same shopping trip. Intertemporal concerns could cut both ways. E.g., the 'within-trip substitution' would overstate the total substitution if people commpensate for having purchased PBM on one trip with "now let's eat more real meat" on the next trip or in a restaurant.

Do the rough price conversion, and also give the 'food flow' for the PBA -- we need a like-for-like comparison

make this estimate a bit more prominent

xAI 单位经济极差：亏损是营收的 2 倍。同期 Anthropic 接近盈利、营收增 130% 至 $109 亿——xAI 落后竞争对手一整代。

首个正式时间表：2028 年开始部署轨道 AI 计算卫星——Musk 把 SpaceX 卫星制造能力作为 AI 算力竞争的差异化武器。

xAI 下一代模型目标「数万亿参数」——首次有头部 AI 公司在 SEC 文件中正式承诺这一规模，行业 scaling 战仍未结束。

Instruções e recomendações ao aplicar essas escalas de classificação de esforço.

Other esophagus motility disorders

Dysphagia for liquids as well as solids tends to be intermittent and nonprogressive. DY

Manometry is not routinely used for mild to moderate symptoms because the findings seldom influence medical management

Barium esophagography is useful to exclude mechanical obstruction and to evaluate esophageal motility

Upper endoscopy also is performed to exclude a mechanical obstruction (as a cause of dysphagia) and to look for evidence of erosive refl traders ux esophagitis (a common cause of chest pain) or eosinophilic esophagitis (confirmed by esophageal biopsy)

This highlights the severe legal and regulatory risks in modern IMC. Regulators are no longer treating green claims as harmless puffery. Violating laws like the EU's Unfair Commercial Practices Directive leads to coordinated legal actions by consumer authorities (CPC-Network), resulting in forced re-labelling, brand reputational damage, and potential financial penalties.

Marketing psychology relies heavily on "Visual Greening"—using green logos and nature imagery to subconsciously suggest environmental neutrality. Ethically and legally, this is deceptive because it sells an emotional feeling of "zero impact" that contradicts the reality of plastic pollution.

This is a textbook example of "Hidden Trade-offs." Coca-Cola heavily promoted the "100% recycled" claim to look eco-friendly, while intentionally omitting that the caps and labels are not recycled. This creates a deceptive framing that misleads consumer perception of the product's actual environmental footprint.

These steps are missing, after Pre-Procedure: "Gather Supplies: Wash basin, warm water, soap, lotion, four to six washcloths, three to four towels, bath blanket, barrier, gloves, clean clothes or gown, and linen bag or hamper. Additional supplies as indicated. *Consider using a second basin, a "rinse basin." Put on gloves. Fill the basin with warm water and place it on a flat surface with a barrier underneath. Have the resident check the water temperature by placing their hand in the basin or putting a wet washcloth on the back of their hand. Or, use a bath thermometer, if available, to check water temperature. Ensure the temperature does not exceed 105ºF. Raise the bed height to a working height. Keep the resident covered as much as possible using a bath blanket, towel, or bed linens. Fold washcloth into sections of four, or make a mitt by folding a washcloth around your hand.<br /> Wet the washcloth. Squeeze out excess water. Wash the patient’s eyes, using separate corners of the cloth for each eye. Wipe from inside to outside corner. Do not use soap near the eyes. Do not use soap on the face unless the patient requests it. Squeeze out excess water. Wash the resident’s face using water only. Pat dry the face. Wet the washcloth, squeeze out excess water. Put soap on the washcloth and wash the resident's ears and neck. Place the washcloth containing soap on the edge of the basin or barrier. Use another clean washcloth, wet with water only, squeeze out excess water. Rinse the resident's ears and neck. Place the rinse washcloth (containing no soap) on the edge of the basin or barrier. Pat dry the ears and neck with a towel. Remove the gown, keeping the rest of the body covered. Expose one of the resident’s arms. Place a bath towel under the arm to protect the bed. Wet the washcloth, squeeze out excess water. Put soap on the washcloth and wash the arm with soap. Wash the hand with soap. Wash the underarm with soap. Place the washcloth containing soap on the edge of the basin or barrier. Rinse the arm with the rinse washcloth. Rinse the hand. Rinse the underarm. Pat dry the arm. Pat dry the hand. Pat dry the underarm. Apply deodorant (if indicated). Discard used bath water and refill the basin two-thirds full with water, if needed. Ensure that does not exceed 105ºF. Place a bath blanket or towel over the resident’s chest and abdomen. Fold back to expose the resident's chest. Wash, rinse, and pat the chest dry. Ensure to rinse and dry folds and under the breasts carefully. Replace blanket or towel over the chest. Fold back to expose the resident's abdomen. Wash, rinse, and pat the abdomen dry. Fold the bath blanket to cover the abdomen and chest.

eLife Assessment

By using a combination of patch clamp recordings, calcium imaging and computer modeling, the authors analyze the spatial distribution of voltage gated calcium channels at glutamatergic synapses formed between layer 5 pyramidal neurons (L5PNs) and between layer 2/3 and L5PNs in the prefrontal cortex (PFC) and primary somatosensory cortex (S1); they conclude that the calcium channel-vesicle coupling is looser in the PFC compared to S1, although additional experiments are needed to determine how the distinct functional characteristics of these synapses in different brain regions might affect data interpretation. Overall, these findings are important because they have implications for shaping synaptic plasticity and neural circuit function across brain regions. They are solid because they are based on the use of a multi-pronged approach, although the presentation would benefit from stronger integration of the current findings with the existing literature and a more explicit discussion of potential limitations and confounding factors for data interpretation.

Reviewer #1 (Public review):

Summary:

This study asks whether synapses formed by the same broad neuronal class (excitatory pyramidal neurons, PN) adapt their presynaptic organization in a cortex-specific manner, comparing the prefrontal cortex (PFC) with the primary somatosensory cortex (S1). The authors combine sophisticated electrophysiology (paired recordings and extracellular minimal stimulation), pharmacological perturbations of presynaptic Ca²⁺-secretion coupling, bouton Ca²⁺ imaging, and mechanistic modeling. Across two prominent excitatory connections (Layer 5 (L5) PN-L5PN and L2/3-L5PN), they provide convergent evidence that mature PFC synapses operate with looser Ca²⁺ channel-release sensor coupling than their S1 counterparts.

Overall, the study provides an appealing mechanistic link between synaptic nano/micro-architecture and cortical-area specialization. The idea that PFC synapses retain a more "plasticity-favoring" presynaptic state, while the primary sensory cortex emphasizes reliability and timing precision, is potentially impactful for how we think about circuit computation and plasticity across cortical hierarchies.

Strengths:

A major strength is the multi-pronged experimental strategy. The paper first establishes robust, area-dependent differences in synaptic efficacy, reliability, timing, and short-term plasticity (facilitation prevailing in PFC versus depression in S1), using both paired recordings and minimal extracellular stimulation paradigms. The coupling interpretation is then directly supported by differential sensitivity to EGTA (and appropriate positive-control effects of fast chelators). Finally, volume-averaged calcium signals are reported to be similar across areas, arguing against trivial explanations based on gross differences in calcium influx, and the modeling provides a quantitative framework for interpreting the observed chelator effects.

Weaknesses:

Limitations are minor and concern interpretation/clarity rather than core results. Some key inferences rely on indirect readouts (chelator sensitivity, fluctuation analysis-derived parameters, bouton-averaged calcium signals), each of which carries assumptions and potential confounds that should be discussed more explicitly. In particular, the repatching paradigm for the paired-recording EGTA experiment, though very impressive, and the limited number of extracellular calcium conditions used for fluctuation analysis (three concentrations), can influence quantitative estimates and the confidence intervals around them.

Reviewer #2 (Public review):

Schwarze et al. investigated whether synaptic efficacy is brain-region specific. To this end, they compared synaptic connections established by layer 5 (L5) neocortical pyramidal cells and between L5 and L2/3 pyramidal cells. In order to identify the mechanism of this brain region specificity, the authors employed several experimental approaches, including paired electrophysiological recordings, extracellular stimulation, low- and high-affinity intracellular calcium chelators (EGTA and BAPTA), multiple probability fluctuation analysis (MPFA), and intracellular measurements of calcium transients as well as computational modelling. The findings of the present study indicate that synaptic connections in the primary somatosensory cortex (S1) are significantly stronger and more reliable than those in the prefrontal cortex (PFC).

The study is timely, and the topic is of significant interest to the neuroscience community. Despite the extensive research that has been carried out on the neuroanatomy and receptor distribution of different brain regions, comparatively little attention has been paid to differences in synaptic physiology. The authors' approach is characterised by its elegance and comprehensive nature, and the conclusions drawn are compelling. Nevertheless, there are a number of unresolved issues.

Major points:

(1) The authors state that data from the S1 cortex were obtained in a previous study. In the context of an explicitly comparative study (PFC vs. S1cortex), it would have been advantageous for the authors to perform a subset of experiments in which both cortices were obtained from a single animal. This is a feasible undertaking, given the spatial separation of the PFC and S1 cortex.

(2) Figure 1A is somewhat misleading because it could suggest that the authors have performed dual recordings in identified PFC pyramidal cells.

(3) PFC and S1 cortex in rodents differ markedly in their morphological organisation. For example, in all sensory cortices, layer 4 is very pronounced; however, in the PFC of rodent,s no clear layer 4 can be found. On the other hand, PFC shows a clear separation of layers 2 and 3, which is not visible inthe S1 cortex. Furthermore, PFC pyramidal cells in layers 2, 3, and 5 exhibit significant heterogeneity, diverging considerably from those found in layers 5a and 5b of S1 cortex. Thus, there is no clear correlation between L5 pyramidal cells in the PFC and the S1 cortex. In order to achieve a meaningful comparison of the data obtained in PFC and S1 cortex, it is necessary for the authors to determine whether the record is from similar pyramidal cell populations.

(3) In addition, PFC pyramidal cells in layer 2, 3 and 5 are highly heterogeneous and differ markedly from those in layer 5a and 5b of S1 cortex. To achieve a meaningful comparison of the data obtained in the PFC and the S1 cortex, the authors need to determine whether the record from similar pyramidal cell populations.

(4) For the S1 cortex, in rats it has been found that L5 synaptic connection between pairs of L5a pyramidal cells and pairs of L5b pyramidal cells differ markedly with respect to mean EPSP amplitude, latency and coefficient of variation (cv, a surrogate measure for the synaptic release probability) (cf. Markram et al., 1997; Frick et al., 2008). It is therefore likely that PFC and S1 pre- and postsynaptic pyramidal cells are not only morphologically and electrophysiological distinct but also with respect to their synaptic properties. At least, the authors need to discuss these confounding issues and preferentially address them experimentally. For example, it would be helpful to demonstrate that paired recordings were made from the same pyramidal cell types, perhaps by documenting their morphology and/or firing patterns. In addition, they should discuss the marked difference in EPSP amplitude and putative release probability between their data and the earlier studies.

(5) In order to perform multiple probability fluctuation analysis (MPFA), a parabolic fit with a mere three points is inadequate, particularly because 2 mM and 5 mM Ca2+ are close to the peak of the variance-to-mean parabola, and only 1 mM Ca2+ is on its initial linear part. A more meaningful result would have been obtained with an additional Ca2+ concentration between 1.0 and 2.0 mM, as these are closer to the physiological range. In this context, the authors should have quoted the more recent and more detailed paper by the Silver group (Saviane and Silver, 2006; Lanore and Silver, 2016) and not just the Clements and Silver review paper.

(6) Methods: The authors should clarify whether their paired recordings from L5 pyramidal cells involved whole-cell recordings from both pre- and postsynaptic neurons. From Figure 1B, it appears as if the presynaptic neurons were not recorded in whole cell mode but rather stimulated in cell-attached mode. This is also reflected in the artefact visible in the current trace recorded in the postsynaptic neuron. The authors should explicitly state their methodological approach and mention how reliable the timing of the presynaptic action potential was under these circumstances. The same holds true for the extracellular stimulation protocol. A significantly more detailed description of the experimental protocol is necessary here.

(7) Methods: The authors use Student's t-test for data comparison. The authors should verify that the data distribution was indeed normal, e.g. by using a Shapiro-Wilk test. If this is not the case, non-parametric tests should be used.

Reviewer #3 (Public review):

Summary:

In this manuscript, Max Schwarze and colleagues examined the coupling distance between presynaptic Ca²⁺ channels and the vesicular release sensor at neocortical synapses in mice. They propose that Ca²⁺ channel-release sensor coupling differs across cortical areas, with relatively loose (microdomain) coupling in prefrontal cortex (PFC) and tighter (nanodomain) coupling in primary somatosensory cortex (S1) for comparable pyramidal-neuron synapse types. To test this, they combine paired recordings and minimal stimulation with chelator manipulations (EGTA/BAPTA), mean-variance/MPFA-style analyses, presynaptic Ca²⁺ imaging, and computational modeling. They conclude that presynaptic coupling organization is area-specific in the mature cortex and contributes to regional differences in synaptic timing, reliability, and short-term plasticity.

Strengths:

This study tackles an important question and is strengthened by a cohesive body of evidence assembled from multiple complementary approaches. A major asset is the inclusion of high-value datasets, particularly the paired recordings between L5 pyramidal neurons and the systematic assessment of EGTA sensitivity, which provide a solid functional foundation for the authors' central claims. The work is further distinguished by its genuinely multimodal design: combining electrophysiology with presynaptic calcium imaging (and integrating these observations with quantitative analyses and modeling) offers a more mechanistic view of neurotransmitter release than any single method could provide. Overall, the direct, within-framework comparison of presynaptic release-control mechanisms across cortical areas for comparable synapse types is compelling and gives the conclusions a level of robustness and interpretability that is often difficult to achieve in studies of cortical synaptic diversity.

Weaknesses:

Several aspects would benefit from clearer explanation, stronger integration with the existing literature, and a more explicit discussion of limitations and potential confounds. Without these additions, some conclusions remain speculative. Throughout the manuscript, the authors also often imply that different measurements reflect the same underlying synapse population. This is unlikely to be strictly true across all experiments and makes it difficult to integrate results from the various approaches into a single, unified set of functional synaptic properties. In addition, some statements-particularly those linking coupling mode to "higher-order neocortical functions"-appear broader than what is directly supported by the experiments and should be tempered or more precisely scoped.

Below, I list several topics that could help better frame the main findings of the present study and clarify how it relates to previously published work.

(1) The authors use EGTA sensitivity of EPSCs (together with additional metrics) to argue that S1 and PFC synapses differ in Ca²⁺ channel-release sensor coupling. While this is a plausible interpretation, EGTA effects are not uniquely determined by coupling distance and can also reflect differences in Ca²⁺ entry kinetics, action potential waveform, endogenous buffering/extrusion, or release-sensor/vesicle state. The authors use a constrained modeling approach, but the rationale for the different constraint sets is not fully clear from the current description. It would be helpful to expand and clarify the Methods section to explain how these constraints were defined, justified, and applied (and how alternative constraint choices would affect the results). In this context, the Abstract's broader claim that the study "reveals microdomain coupling as a presynaptic structure-function correlate of higher-order neocortical functions" appears overstated. Given the well-known diversity of cortical synapses even within a single region (e.g., synapses onto different interneuron subclasses or different PN cell types, extracortical sources like thalamus), the authors should clarify the intended scope: is the conclusion meant to apply broadly across synapse classes in S1 and PFC, or only to the specific connection type(s) examined here?

(2) The chelator logic is sound in principle, but the Discussion should more explicitly acknowledge standard caveats and alternative explanations. The authors partly address this by including presynaptic Ca²⁺ imaging and modeling, yet it would help to explain more clearly how the combination of (i) chelator sensitivity, (ii) presynaptic Ca²⁺ signals, and (iii) model constraints rules out-or substantially reduces the likelihood of-changes in AP waveform, Ca²⁺ influx kinetics, buffering/extrusion, or sensor/vesicle state as the primary drivers. In addition, recent hypotheses emphasizing vesicle priming and/or release-site occupancy as contributors to apparent EGTA sensitivity should be discussed as a complementary or alternative interpretation.

(3) A substantial portion of the S1 comparison appears to rely on previously published datasets. This should be made unambiguous in the Results and Methods, and it would be helpful to summarize this clearly (e.g., in a table indicating which figures/analyses use new data versus reanalysis of published data). If this information is already present, it should be highlighted more prominently.

(4) The modeling is informative, but the choice of a specific VGCC-release-site geometry and channel arrangement is not sufficiently justified. The manuscript adopts a particular spatial configuration, yet the rationale for selecting this geometry, rather than other plausible architectures discussed in the literature, is not clearly explained, nor is it meaningfully revisited in the Discussion. The authors should justify why the same organization is assumed across two distinct cortical areas and, ideally, include (or at a minimum discuss) a sensitivity analysis showing how key inferences (e.g., coupling distance and channel number) depend on the assumed geometry.

(5) The calcium imaging data are valuable, but given the diversity of synapses within each cortical layer, it is not clear that imaged boutons can be confidently assigned to the specific connection types being interrogated electrophysiologically. A substantial fraction of boutons likely corresponds to different postsynaptic targets (including interneurons and distinct pyramidal-cell classes), and this heterogeneity could complicate interpretation. This limitation should be discussed explicitly

(6) In unitary connections, the authors assess EGTA effects alongside other functional parameters (strength, delay, short-term plasticity), which is a major strength. However, for L2/3 to L5 connections, it appears that EGTA sensitivity was tested primarily using extracellular stimulation. Given anatomical and circuit differences between PFC and S1, extracellular stimulation may recruit different synapse populations across regions, potentially confounding regional comparisons of EGTA sensitivity. This limitation should be acknowledged explicitly. While I am not requesting technically demanding L2/3↔L5 paired recordings in S1, the possibility that different synapse identities are being sampled should be treated as a meaningful source of uncertainty. The Discussion would also benefit from placing the magnitude of EGTA effects in the context of prior "loose coupling" literature, where comparatively large EGTA effects have been reported in some systems. In addition, the reported difference between adult PFC EGTA effects and S1 inhibition appears small (on the order of <10%) and should be interpreted cautiously, especially given that PFC and S1 mature on different timelines and P21-P26 is unlikely to reflect a mature PFC circuit state. The adult cohort (P90-P100) is therefore important, but the age mismatch complicates PFC-S1 comparisons; ideally, S1 should be assessed at matched ages, or this limitation should be discussed explicitly. Finally, for statistical robustness, in panel D of Figure 2, were the comparisons corrected for multiple testing to control Type I error?

(7) Alterations in initial release probability are often associated with changes in short-term plasticity. In the present manuscript, the authors report similar initial release probability at PFC and S1 synapses, yet observe differences in short-term plasticity profiles. The mechanistic basis for this apparent dissociation is not addressed and should be discussed explicitly, including potential explanations.

(8) There are multiple instances where the text appears to cite non-existent or misnumbered figure panels (e.g., references to "Figure 4G-I / 4J" when the relevant material appears elsewhere). These should be corrected throughout, as they currently reduce readability and confidence.

(9) The Methods describe P21-P26 animals, whereas the Results include older cohorts (e.g., P90-P100) and additional regions (e.g., mPFC). The Methods should be updated so that all cohorts and regions analyzed in the Results are fully described.

Author response:

We will extend and clarify the text of the paper according to the suggestions of the reviewers. In particular we will extend the description and discussion of the calcium chelator approach, re-patching and multiple probability fluctuation analysis. We will also include in the Results section that volume-averaged calcium signals were measured and extend the description about measurement of the resting calcium and variability between boutons. Literature will be included and discussed as suggested.

In order to avoid any misunderstandings, we will also make it clearer that recordings from

L5PN – L5PN synapses in S1 were published in our preceding papers (Bornschein et al., 2019a, b), but that these data were partially reanalyzed for the comparison with recordings from L5PN – L5PN synapses in PFC (this paper). We will also emphasize that the recordings from L2/3 to L5PN synapses in S1 and PFC were made directly in the present study. We will include a supplementary table, which explicitly shows for each figure which data are from Bornschein et al. (2019a, b) and which data were obtained in the present study. 

We will consider all points of the reviewers and the recommendations of the editors in detail in the revised manuscript and/or our pointwise response.

We recognized one factual error in the public reviews:

Reviewer 2, point 7: “Methods: The authors use Student's t-test for data comparison. The authors should verify that the data distribution was indeed normal, e.g. by using a Shapiro-Wilk test. If this is not the case, non-parametric tests should be used.”

A detailed description of the statistics, including test for normality, is given in the Methods section. In particular we wrote in the Methods: “Normality was tested using the Shapiro-Wilk Test. (…) To compare pre- and post-treatment data the paired t-test or the Wilcoxon signed rank test (WSR) was used, depending on the distribution of the data. (…)”

To further emphasize that the data was tested for normal distribution, we have also extended the description of the statistical tests in the figure legends.

Bornschein G, Brachtendorf S, Schmidt H (2019a) Developmental increase of neocortical  presynaptic efficacy via maturation of vesicle replenishment. Front Synaptic Neurosci 11:36.

Bornschein G, Eilers J, Schmidt H (2019b) Neocortical high probability release sites are formed by distinct Ca2+ channel-to-release sensor topographies during development. Cell Rep 28:1410-1418 e1414.

eLife Assessment

The findings of this study are important since they cover the repurposing of small molecules as metalloprotease and phospholipase inhibitors for early intervention in the treatment of bothropic envenoming in the Neotropics, and thus provide a strong rationale for the progression of these inhibitors into future preclinical and clinical evaluation for snakebite indications across various ecological zones, albeit the current evidence casts doubts on the viability of repurposing nafamostat. The strength of the evidence is solid; however, there are some weaknesses, such as a lack of translatability of the in vivo model and insufficient venom characterization. Thus, the strength of the evidence can be enhanced by using a mouse model in future studies. The paper remains of interest to ophiologists, biochemists and medicinal chemists.

Reviewer #1 (Public review):

Very nice and coherent body of work with appropriate in vitro to in vivo transition in methods.

Lovely and easy to follow figures that can be understood even without the manuscript.

My recommendation is that a sentence or two be added clearly stating the authors think nafamostat is off the table and suggest other approaches/drugs that might be considered instead of just making a general statement. I think all this can be done in a few sentences.

Gabexate was administered to a snakebite victim in this case report from about 20 years ago and also a good example of the now better recognized threat to pregnancy.

Nasu K, Ueda T, Miyakawa I. Intrauterine fetal death caused by pit viper venom poisoning in early pregnancy. Gynecol Obstet Invest. 2004;57(2):114-6. doi: 10.1159/000075676. Epub 2003 Dec 19. PMID: 14691344

Reviewer #2 (Public review):

Summary:

The authors set out to test whether a defined set of small molecules can lessen damaging effects caused by venoms from several Bothrops species, and whether these effects are consistent enough to suggest a broadly applicable approach. They present a cross-venom dataset spanning in-vitro activity readouts and blood-based functional outcomes, and include a chicken embryo model to explore whether venom inhibition can translate into improved survival. The central message is that certain small molecules can reduce specific venom-driven effects across multiple samples, providing a comparative resource for the field and a basis for prioritizing future validation.

Strengths:

The main value of this work is the breadth and structure of the dataset, which places multiple venoms and multiple readouts into a single, comparable framework that should be useful for readers evaluating patterns across samples. The experimental flow is generally coherent, moving from activity measurements to functional outcomes and then to an in-vivo test, which helps the reader understand how the authors link mechanism-oriented assays to more integrated endpoints. The manuscript also provides practical information for the community by highlighting which readouts appear most consistently affected across venoms, which can help guide hypothesis generation and study design in follow-up work.

Comments on revisions:

I would like to thank the authors for answering my questions. The manuscript has gained in quality, knowing the limitations that are now better stated in the manuscript.

Author response:

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

We thank the editors and reviewers for their assessment of this manuscript, and for the positive words highlighting the value of undertaking evaluation of small molecule drugs for snakebite in the neotropics, inclusive of the quality of this work and the value of the validated screening pipeline. We completely agree that the next steps for this work will be to evaluate the preclinical efficacy of the identified drugs in mouse models, though this considerable undertaking will form the basis of future work. Critically, the pipeline that we describe herein facilitates the selection of the most appropriate candidates to progress into such mouse studies, aligning with the 3Rs principles for minimising the need for animal research. The comment around insufficient venom characterisation seems somewhat misplaced – the objective of this project was not to characterise the venoms used, but to evaluate the in vitro inhibition of venom toxin family activities and identify the potential utility of specific repurposed drugs as therapeutics for snakebite in the neotropics. Venom characterisation of the diverse samples used in this project would represent an entire project and manuscript in its own right. We are pleased that the reviewers highlight the gap in research on serine protease inhibitors and the value this paper has in highlighting that more research is required in this area to identify a candidate that is more suitable for future clinical use than nafamostat.

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

Small molecule therapeutics for snakebite have received a lot of attention for their potential to close the gap between bite and treatment, where antivenom is not immediately available.

Strengths:

There has been a lot of focus on Africa, Asia, and India, but very little work related to neotropical regions. The authors seek to begin filling this gap in the preclinical literature. The authors use well-developed methods for preclinical assessment.

Weaknesses:

A clearer and more focused discussion of the limitations of the overall present work would be desirable (e.g. protection vs. rescue, why marimastat over prinomastat for in vivo assays when both have been through clinical trials for other indications; real-world feasibility of nafamostat, which has a half-life of 1-2 minutes compared to camostat, which has a half-life of hours). All of this could be improved in a revision.

We thank the reviewer for their shared opinion of the potential value of small molecules as snakebite envenoming therapeutics and their insight on the gap in focus in the neotropics, which this manuscript aims to address.

Our work in this manuscript included standard practice of pre-incubation between drug and venom for all in vitro studies, and sequential (i.e. not co-incubation) administration in the egg model. In our revised manuscript we will make these distinctions clearer. Use of a ‘rescue’ approach in the in vitro assays is not feasible due to the rapid destruction of the substrates used for assay readouts. The clearest rationale for the use of rescue models relates to their power within in vivo preclinical models (i.e. murine envenoming models) which, following the in vitro characterisations presented in this paper, are the logical next step for evaluating small molecule drugs for inhibiting neotropical snake venoms.

Although both marimastat and prinomastat are repurposed drugs that have undergone clinical evaluation for other indications, marimastat has been more extensively characterised preclinically than prinomastat for snakebite, and will soon enter Phase II clinical trial evaluation for this indication (https://www.ddw-online.com/ophirex-to-produce-snake-venom-inhibitor-for-lstm-study-40669-202602/). Marimastat also has a longer half-life in humans of 8-10 hours (Millar et al. 1998), compared to prinomastat (2-5h, Hande et al. 2004). We will more clearly highlight the rationale for selecting marimastat in the revised manuscript.

Although we appreciate the reviewer’s point regarding the short half-life of nafamostat (which is typically given by continuous iv infusion due to its short half-life), in the manuscript we have already stated that we do not recommend the progression of nafamostat as a snake venom serine protease (SVSP) inhibitor candidate due its low efficacy and off target effects. We highlight the need for the community to identify other serine protease inhibitors that might have utility for snakebite.

Reviewer #2 (Public review):

Summary:

The authors set out to test whether a defined set of small molecules can lessen damaging effects caused by venoms from several Bothrops species, and whether these effects are consistent enough to suggest a broadly applicable approach. They present a cross-venom dataset spanning in-vitro activity readouts and blood-based functional outcomes, and include a chicken embryo model to explore whether venom inhibition can translate into improved survival. The central message is that certain small molecules can reduce specific venom-driven effects across multiple samples, providing a comparative resource for the field and a basis for prioritizing future validation.

Strengths:

The main value of this work is the breadth and structure of the dataset, which places multiple venoms and multiple readouts into a single, comparable framework that should be useful for readers evaluating patterns across samples. The experimental flow is generally coherent, moving from activity measurements to functional outcomes and then to an in-vivo test, which helps the reader understand how the authors link mechanism-oriented assays to more integrated endpoints. The manuscript also provides practical information for the community by highlighting which readouts appear most consistently affected across venoms, which can help guide hypothesis generation and study design in follow-up work.

Weaknesses:

Several aspects of the study design and framing reduce the confidence with which readers can translate the findings beyond the specific experimental context presented. The evidence base is strongest in controlled in-vitro settings, while the bridge to real-world effectiveness remains limited, particularly for understanding performance under conditions that better reflect delayed treatment and systemic exposure. As a result, the manuscript is best interpreted as a well-organized comparative screening study with promising signals, rather than a definitive demonstration of a broadly effective, deployable intervention.

We appreciate the reviewer’s opinion on the thorough and logical workflow we present in this manuscript and the value this pipeline providers the field for future and parallel work. We agree with the reviewer that this provides a well-organized comparative screening study applicable to different snake species or therapeutics. In relation to the comment on this manuscript being a definitive demonstration of a broadly effective, deployable intervention we agree with their opinion and are happy to clarify that while the evidence presented in this manuscript is promising, there is much work still to do before such molecules are ready for deployment for treating snakebite. Ultimately, this manuscript supports the growing evidence of the promising utility of marimastat and varespladib, and extends this evidence to neotropical snake venoms in a comparative manner. The next step will be to evaluate the efficacy of these molecules within in vivo murine preclinical models, which will be crucial for further supporting the evidence base for onward translation.

Reviewer #3 (Public review):

In this work, the authors wanted to evaluate repurposed small molecule inhibitors for the treatment of envenomation by snakes of the Bothrops genus; one of the most medically relevant in the Americas. I believe the objectives of the research were clearly achieved, and compelling evidence for the ability of these molecules to neutralize enzymatic and toxic activities of metalloproteinases and phospholipases in all the tested venoms is provided. Furthermore, the work highlights the limited efficacy of the tested serine protease inhibitor, suggesting a need for drug discovery campaigns to address toxicity caused by this protein family. The methods are well designed and performed, and the use of both in vitro and in vivo methodologies makes this a thorough and robust work.

These results are extremely relevant, since they take us one step further to a potential orally administered snakebite treatment. The existence of such a treatment could improve the outcomes for thousands of snakebite victims worldwide. I have a few comments and questions that I hope will be useful to the authors:

We thank the author for their high regard for the purpose and execution of this work. Their insight in relation to questions are supportive for an improved manuscript and discussion points for the field.

During the introduction, the authors mention that small-molecule inhibitors can neutralize the localized tissue damage via cytotoxicity of some venoms, and cite PLA2s, SVMPs and/or cytotoxic 3FTxs as the main causing agents of this pathology. I am not aware of any direct effect described by small molecule inhibitors on cytotoxic 3FTxs alone. Has this been observed at all? Or is it more likely that the small molecule inhibitors act on the enzymatic toxins only, preventing synergistic effects with 3FTxs?

We apologise for this error on our behalf. While inhibitory molecules have been described for cytotoxic 3FTxs, these are not small molecules as alluded to in the previous version of the manuscript. We have amended this text in our revised manuscript.

I think it would be relevant to address the effects of non-enzymatic PLA2s, such as myotoxin II, which have been described in detail within Bothrops venoms. I believe there is some evidence of Varespladib also having a neutralizing effect on the myotoxicity caused by these non-enzymatic PLA2s. I suggest adding a comment about the contribution of these toxins in the discussion or in the section where PLA2 activity of the venoms is compared. In my opinion, right now it seems like these were overlooked.

We thank the reviewer for highlighting this point. We agree that this is highly relevant and would benefit from discussion in the revised manuscript given the nature of our assays and the non-enzymatic mechanism of action of certain Bothrops PLA₂s. We have added this to the discussion.

Regarding Marimastat and the other MP inhibitors, are there any studies showing that they don't have an effect on endogenous MPs? I understand they have been approved for human use before, but is there any indication that they would not have an effect at the doses that would be required to treat envenomation?

Most matrix metalloproteinases inhibitors will act on endogenous MPs to at least some extent (variable potency on different MMPs). Marimastat has demonstrated activity against endogenous metalloproteinases, including MMP1, which was hypothesised to cause severe joint pain when used chronically (i.e. frequent dosing over many weeks) for indications such as cancer, though this effect was reversible within 8 weeks of cessation of drug administration (Wojtowicz-Praga, 1998). Thus long-term use of matrix metalloproteinases inhibitors can cause safety concerns. However, the anticipated duration of dosing for snakebite, which is an acute life-threatening condition, is a few days. It is therefore unlikely that prior safety concerns observed following chronic dosing in cancer studies would apply to its potential use as a snakebite field therapy.

Regarding the quenched fluorescence substrate used for enzymatic activity. Is there a possibility that some of the SVMPs would not act on this substrate, and therefore their activity or neutralization is not observed? Would it be relevant to test other substrates, such as gelatin, collagen, or even specific clotting factors?

It has been observed that certain SVMPs (specifically several PI SVMPs) are not active against this ES010 substrate in vitro. The substrate used in the in vitro SVMP assay is reported by the manufacturer as a substrate for a wide range of MMPs which target the extracellular matrix components mentioned by the reviewer, i.e. collagenases and gelatinases as well as matrilysins, stromelysins and elastate. This in vitro assay combined with the coagulation assays are complementary in covering the main targets of SVMPs (ECM and clotting cascade), prior to haemorrhagic assessment in the egg model. Thus, we are confident that activity for the broad range of SVMP isoforms will be captured through the screening pipeline we have developed.

Finally, could the authors comment or provide some bibliography regarding the translatability of the chicken embryo model in the context of envenomation?

Our current model is based on an earlier egg embryo model (Sells et al. 1997, Sells et al. 1998 and Sells et al. 2000) which described good correlations (p<0.01) with the standard WHO murine preclinical envenoming model. These studies have assessed correlations for minimal haemorrhagic doses (MHDs), LD50s and ED50s in both models for a selection of viper venoms. As chicken embryos at day 6 of development have incomplete neural arcs, the model is not well suited for assessing neurotoxic effects, but can be effectively used for addressing venom-induced haemorrhage and lethality and for testing therapeutics. In addition, a more recent study (Yusuf et al. 2023) reported almost identical LD50s for the venom of Bitis arietans between the two in vivo approaches. The model is also being pursued as a preclinical testing model by an antivenom manufacturer with the focus of reducing the use of rodents in batch release testing (Verity et al. 2021). We will provide further clarification on the rationale for using the egg model, including the supportive references outlined above, in the revised manuscript.

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

The manuscript provides a useful comparative dataset across multiple Bothrops venoms and supports SVMP inhibition as a broadly effective lever in the authors in-vitro work. However, the strength of the 'pan-Bothrops' and translational claims is currently limited by insufficient characterization of the exact venom samples tested and by experimental designs that fall in clinically realistic rescue.

Major comments:

(1) The venoms used in this study are historical batches and are not formally characterized beyond SDS-PAGE and literature summaries, despite well-known intra- and inter-population venom variability; this weakens the generalization of the conclusions.

To address this comment, we have increased clarity on our venom sources being historic, Due to the historic source locality is not available beyond country of origin, with the exception of B. lanceolatus which is endemic to Martinique. Figure 1 also makes clear that we agree with the reviewer that the variation is high within Bothrops species. We discuss this variation on the limitations in our sampling for making broad conclusions throughout the first paragraph of the discussion, with the final sentence stating Future proteomic characterisations of the specific venom samples used in this study, which were all sourced from a historical collection (except for B. lanceolatus), would be informative in this regard. Although venom composition of our samples has not been characterised, the focus of the manuscript is the characterisation of the whole venom functional activity through a wide ranging screening pipeline, and the generalisation of our findings is supported by the diversity of the venom samples (i.e. several species) despite them not being characterised (which is not critical for the focus of the study).

(2) On a technical comment, the venom inhibition assays appear to rely on drug-first or preincubation conditions, which can easily overestimate efficacy compared with real snakebite envenomation, where toxins distribute and engage targets rapidly. Here, a translational gap is the clinical feasibility of the 'repurposed' inhibitors, as it is unclear whether the drugs central to the conclusions (especially marimastat, prinomastat and varespladib) are realistically available or stocked in hospitals or could be deployed in regions where Bothrops envenoming occurs. I think that the manuscript should clearly distinguish this from candidates with a plausible access and delivery pathway.

Our work in this manuscript includes standard practice of pre-incubation between drug and venom for all in vitro studies, and sequential (i.e. not co-incubation) administration in the egg model. None of our methods administer drug-first. Throughout the methods and figure legends we have made these distinctions clearer. Use of a ‘rescue’ approach in the in vitro assays is not feasible due to the rapid destruction of the substrates used for assay readouts. The clearest rationale for the use of rescue models relates to their power within in vivo preclinical models (i.e. murine envenoming models), which would be the next step for this research programme.

While the evidence presented in this manuscript is promising, there is much work still to do before such molecules are ready for deployment for treating snakebite, inclusive of the requirement to complete clinical trials, cost-benefit analysis and policy change and manufacturing/distribution feasibility assessments. Ultimately, this manuscript supports the growing evidence of the promising utility of marimastat and varespladib, and extends this evidence to neotropical snake venoms in a comparative manner. The next step will be to evaluate the efficacy of these molecules within rescue in vivo murine preclinical models, which will be crucial for further supporting the evidence base for onward translation. To further support this point we have included an additional section to the manuscript discussing the current preclinical and clinical progression of prinomastat and marimastat, which also incorporates the public comment on selection of marimastat over prinomastat.

(3) In my opinion, the Nafamostat results and discussion need reframing, given weak SVSP inhibition and intrinsic anticoagulant behavior at 5 µM. Excluding it from certain analyses undermines interpretability, and it may be more appropriate to include it throughout as an explicit negative control condition (showing its baseline anticoagulant effect) rather than omitting it.

Although we understand the reviewers opinion here, we disagree and believe that including nafomastat as a ‘negative control’ may present a negative reflection on the benefit that an efficacious serine protease inhibitor could provide. Furthermore, as the intrinsic anticoagulant effect of nafamostat cannot be de-coupled from direct SVSP toxin inhibition we were unable to interpret the activity which undermines the results. This can be seen in Figure 3b, which demonstrates that a false positive result would occur. For the serine protease assay, we do clearly discuss the lack of efficacy and justification of why EC₅₀ testing wasn’t appropriate within the guidance of our screening protocols.

In the manuscript we have now further justified our approach in relation to the limitations of nafamostat as a snake venom serine protease (SVSP) inhibitor candidate due its low efficacy and off target effects. We highlight the need for the community to identify other serine protease inhibitors that might have utility for snakebite.

(4) The data presentation needs consistent statistical analyses (currently absent for multiple key figures, including Figures 2, 3, 4, 6 and 7) and a clearer explanation for the dose of venom and drugs you choose. For example, Figure 3 relies on a fixed 5 µM drug concentration and very different venom amounts (50-100-250 ng), but it is not discussed whether such exposures are achievable in vivo, or how these concentrations map onto expected pharmacokinetics in patients. Likewise, Bothrops venoms can contain both pro- and anticoagulant activities, so the authors should justify how their framework accounts for anticoagulant components and why the observed plasma phenotypes are interpreted as they are

In relation to the reviewers comment on the need for consistent analysis we thank the reviewer for flagging this and have now included these in figures 3, 4, 6 and 7. However, Figure 2 is presented to display the variation between all the venoms and ultimately used to select the most relevant doses for the latter inhibition experiments, therefore statistical analysis is not relevant for this figure. The updated statistical analysis now includes the following, which has been included in the relevant figure legends and results sections;

Figure 3 - Bars indicate significant results (p = <0.05) identified through one-way ANOVA with Dunnett’s multiple comparisons test to the DMSO control

Figure 4 - two-way ANOVA with Šídák's multiple comparisons test of each venom control compared to the matched venom treated with inhibitor

Figure 6 – the CT and MCF data were analysed independently using one-way ANOVA with Tukey’s multiple comparisons test

Figure 7 - Log-rank test (Mantel-Cox) with Holm- Šídák's multiple comparisons test against treatment vs venom-only control

We have ensured that all figure legends clearly indicate the venom and drug dose to aid the clarity which the reviewer requested.

The comment Figure 3 relies on a fixed 5 µM drug concentration and very different venom amounts (50-100-250 ng), but it is not discussed whether such exposures are achievable in vivo, or how these concentrations map onto expected pharmacokinetics in patients. is an understandable query however, in vitro assessment such as those carried out in this manuscript are not designed to directly inform pharmacokinetic/pharmacodynmanic interpretations, largely because they do not replicate real world envenoming (i.e. preincubation would not occur between a venom and treatment). This is why, as stated, follow on preclinical and clinical assessments are needed for onward progression of these inhibitors to inform dosing regimens that might achieve the necessary exposures required for in vivo venom neutralisation. That being said, PK/PD work has been initiated within Phase I trials, for example with DMPS Abouyannis et al. 2025 demonstrated a plasma exposure of >10 µg/mL for single doses of 1,200 mg and higher. This is equivalent to 80 µM, which although is lower than the EC₅₀ for some venoms in the clotting assay (Figure 3J), the venom dose (50 to 250 ng/ 50 µL, i.e. 1,000 to 5,000 ng/µL) is estimated to be >1000 times higher than a natural envenoming by Bothrops atrox at less than 1 ng/mL in serum (https://doi.org/10.1016/j.toxicon.2022.09.010). These extrapolations therefore indicate that the doses selected in our studies would have human clinical relevance.

Finally, in terms of anticoagulant venom effects - these would be observed in our experimental approach either as reduced kinetic responses in the plasma clotting assay (as observed with nafamostat in Figure 3B) or as a prolonged clotting time in the thromboelastography assay (Figure 6). As stated in the results section Comparison of coagulation profiles, all of the venoms tested presented with a procoagulant effect. If underlying anticoagulant activity from PLA₂ toxins was to arise after inhibition of the procoagulant toxins (i.e. SVMPs by marimastat), as has been seen for certain other snake venoms previously, this would result in a percentage inhibition far greater than 100% in the plasma assay (Figure 3C to I) or as a prolonged clotting time in the thromboelastography assay. These described anticoagulant profiles were not observed with any venom tested in this study.

(5) Finally, the in vivo evidence is limited to a chicken embryo model. To support your hypothesis, a conventional mouse model with delayed post-envenomation dosing (24-36 h monitoring) is needed to address both safety/toxicity and post-exposure efficacy, and to define a realistic therapeutic window, especially because venom toxins act very quickly and the timing of administration is central to the clinical utility of any small-molecule approach.

We agree with the reviewer that the next important step for this research activity is utilising murine preclinical models to validate the in vitro and preliminary in vivo findings described in this manuscript. However, as stated above, this study provides the initial evidence base that the promising utility of marimastat, DMPS and varespladib as repurposed snakebite drugs extends to a range of neotropical viper venoms. Evaluating the safety, efficacy (both precincubation and rescue approaches) and PK/PD relationships to inform optimal dosing strategies of these molecules will be crucial next steps for the field. However, these activities are far from trivial and will take several years of additional research, and therefore fall outside the scope of this initial manuscript.

To address the concern related to the evidence is limited to a chicken embryo model, we have included additional sentences to discuss the wider use of the egg model within snakebite research and related translation to murine studies.

Minor comments:

(1) Figure 2D: How do you discuss the fact that "no venom" has SVSP activity?

The data for all in vitro assays in Figure 2 is presented as AUC from the raw data (absorbance or fluorescence), for consistency across assay. Therefore, all assays (B to D) have background signal in the absence of venom. The SVSP assay has a greater background signal.

(2) For better understanding, I would suggest adding a dedicated column in Figure 4A with Nafamostat SVSP data reported as "N/D" where applicable.

As stated in the results, due to the weak inhibitory activity EC₅₀ assessment was not justified, therefore adding this column would be redundant.

(3) The introduction is too long relative to the experimental content and would benefit from tightening to sharpen the motivation and unmet need.

We thank the reviewer for their opinion and we have reviewed the introductory section again. While we made minor edits throughout, we decided not to make substantial modifications to it.

Reviewer #3 (Recommendations for the authors):

I only have some minor comments:

(1) In line 100, the word "that" is repeated.

We thank the reviewer for spotting this error, which we have corrected.

(2) Line 433. I believe the word "compromising" should be substituted by "comprising" here.

We thank the reviewer for spotting this.

(3) Figure 1 and supplementary: Bothrops asper venom has been very thoroughly studied, and using only one study from Costa Rica might underestimate the venom variation within the species. I suggest looking at the following study: https://doi.org/10.1016/j.toxicon.2022.106983. Maybe it is not necessary to change anything, but worth looking into.

We appreciate the reviewer flagging this paper, it has been added to the manuscript (reference 48) and has provided additional data for Figure 1 and Supplementary table 1.

(4) Methods: Given the intraspecies variation described for some of these species, I believe it is relevant to add the locality of origin of the venoms, and not only the country. I, of course, understand this is often unknown for historical samples.

We have included the following sentence in the methods. Due to the historic nature of the venom samples, the source locality is not available beyond country of origin, with the exception of B. lanceolatus which is endemic to Martinique.

(5) Figure 3: It is not very accurate to show an SD when the sample number is 2. I suggest, when possible, showing the mean and the two data points in the plots. This also applies to other figures where n=2. Also, in Figure 3D, does Marimastat seem to have an anticoagulant effect, or is this just within normal variation?

We have removed the statement in the statistics paragraph of the methods Standard deviation (SD) for all kinetic reads and standard error for AUC is reported based on Prism v10 but kept the sentence. The sample sizes for HTS assays including the SVMP, PLA₂ and coagulation experiment are the average of the means from independent assays (n >2 within each independent assay). We understand the reviewer’s opinion on limited meaning of SD as well as SE for Fig 3 A to I, therefore we have changed the error bars to range, as we think that displaying the individual points would result in a lack of visual and analytic clarity.

In relation to the query about marimastat anticoagulant effect in Fig 4D, as shown in 4B marimastat has no direct anticoagulant effect. The >100% inhibition for marimastat is likely to be normal variation as this is a biological assay which has high variability. However, it could also be that the strong inhibition of the SVMPs in B. asper along with limited SVSP activity has unmasked an anticoagulant effect of the remaining PLA₂ toxin which has high activity in this venom. That being said, as B. asper has a similar profile, we would have expected to see a similar profile in B. atrox in both the plasma and TEG assays. Therefore, assay variation seems the most likely reason for this observation.

eLife Assessment

This study introduces an important method to estimate the probability of malaria importation with a new Bayesian approach that integrates epidemiological, travel reports, and genetic data. The authors provide convincing evidence for the value of this model in identifying the main sources of malaria transmission and risk factors. This work will be of interest to the area of genomic epidemiology and public health strategies aiming to eliminate malaria.

Reviewer #1 (Public review):

Summary:

This study presents a new Bayesian approach to estimate importation probabilities of malaria combining epidemiological data, travel history, and genetic data through pairwise IBD estimates. Importation is an important factor challenging malaria elimination, especially in low transmission settings. This paper focus on Magude and Matutuine, two districts in south Mozambique with very low malaria transmission. The results show isolation-by-distance in Mozambique, with genetic relatedness decreasing with distances larger than 100 km, and no spatial correlation for distances between 10 and 100 km. But again strong spatial correlation in distances smaller than 10 km. They report high genetic relatedness between Matutuine and Inhambane, higher than between Matutuine and Magude. Inhambane is the main source of importation in Matutuine, accounting for 63.5% of imported cases. Magude, on the other hand, shows smaller importation and travel rates than Matutuine, as it is a rural area with less mobility. Additionally, they report higher levels of importation and travel in the dry season, when transmission is lower. Also, no association with importation was found for occupation, sex and other factors. These data have practical implications for public health strategies aiming malaria elimination, for example, testing and treating travelers from Matutuine in the dry season.

Strengths:

The strength of this study relies in the combination of different sources of data - epidemiological, travel and genetic data - to estimate importation probabilities, the statistical analyses.

Weaknesses:

The authors recognize the limitations related to sample size and the biases of travel reports.

Reviewer #2 (Public review):

Summary:

Based on a detailed dataset, the authors present a novel Bayesian approach to classify malaria cases as either imported or locally acquired.

Strengths:

The proposed Bayesian approach for case classification is simple, well justified, and allows the integration of parasite genomics, travel history, and epidemiological data.

Weakness:

While the authors aim to classify cases as imported or locally acquired, the method does not quantify the contribution of each case type to overall transmission, which the authors leave for future study.

Reviewer #3 (Public review):

This work provides a novel statistical model to identify imported malaria cases, which are an important challenge for elimination, particularly in low-transmission areas. This tool was applied in Plasmodium falciparum populations in Mozambique and determined differences in importation rates in two low-transmission districts in the South.

Strengths:

The study has several strengths, particularly the development of a novel Bayesian model integrating genomic, epidemiological, and travel data to estimate importation probabilities. The findings provided important insights into malaria transmission dynamics, including the identification of importation sources and regional differences in importation rates across Mozambique. These results highlight the potential value of targeted interventions among traveler populations to support malaria elimination efforts. Moreover, this approach could be adapted to other epidemiological settings.

Weaknesses:

The study has some limitations, including uneven sample representation across provinces, incomplete metadata for risk factor analysis and a proxy for transmission intensity. Future work will include a new sample collection effort and the incorporation of monthly malaria incidence estimates.

Author response:

The following is the authors’ response to the previous reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study presents a new Bayesian approach to estimate importation probabilities of malaria combining epidemiological data, travel history, and genetic data through pairwise IBD estimates. Importation is an important factor challenging malaria elimination, especially in low transmission settings. This paper focus on Magude and Matutuine, two districts in south Mozambique with very low malaria transmission. The results show isolation-by-distance in Mozambique, with genetic relatedness decreasing with distances larger than 100 km, and no spatial correlation for distances between 10 and 100 km. But again strong spatial correlation in distances smaller than 10 km. They report high genetic relatedness between Matutuine and Inhambane, higher than between Matutuine and Magude. Inhambane is the main source of importation in Matutuine, accounting for 63.5% of imported cases. Magude, on the other hand, shows smaller importation and travel rates than Matutuine, as it is a rural area with less mobility. Additionally, they report higher levels of importation and travel in the dry season, when transmission is lower. Also, no association with importation was found for occupation, sex and other factors. These data have practical implications for public health strategies aiming malaria elimination, for example, testing and treating travelers from Matutuine in the dry season.

Strengths:

The strength of this study relies in the combination of different sources of data - epidemiological, travel and genetic data - to estimate importation probabilities, the statistical analyses.

Weaknesses:

The authors recognize the limitations related to sample size and the biases of travel reports.

We appreciate the review and comment about the manuscript.

Reviewer #2 (Public review):

Summary:

Based on a detailed dataset, the authors present a novel Bayesian approach to classify malaria cases as either imported or locally acquired.

Strengths:

The proposed Bayesian approach for case classification is simple, well justified, and allows the integration of parasite genomics, travel history, and epidemiological data.

Weakness:

While the authors aim to classify cases as imported or locally acquired, the work lacks a quantification of the contribution of each case type to overall transmission.

Comments on revisions:

All my questions and concerns were satisfactorily addressed.

We appreciate the review and comment about the manuscript. In fact, the approach does not pretend to quantify the contribution of each case to overall transmission. In the discussion we state it and refer to future work with this scope.

Reviewer #3 (Public review):

This work provides a novel statistical model to identify imported malaria cases, which are an important challenge for elimination, particularly in low-transmission areas. This tool was applied in Plasmodium falciparum populations in Mozambique and determined differences in importation rates in 2 low-transmission districts in the South.

Strengths:

The study has several strengths, mainly the development of a novel Bayesian model that integrates genomic, epidemiological, and travel data to estimate importation probabilities. The results showed insights into malaria transmission dynamics, particularly identifying importation sources and differences in importation rates in Mozambique. Finally, the relevance of the findings is to suggest interventions focusing on the traveler population to support efforts for malaria elimination.

Weaknesses:

The study also has some limitations, although the authors have plans to address them. The sample collection was not representative of some provinces, and not all samples had sufficient metadata for the risk factor analysis. Additionally, the authors used a proxy for transmission intensity and assumed some other conditions to calculate the importation probability for specific scenarios. They plan to conduct a new sample collection and include monthly malaria incidence estimates in the future.

Comments on revisions:

Delete "We added this text to the discussion" in line 302 (Discussion)

I recommend adding the plans to address limitations indicated in the Response to Reviewers document in the Discussion. This would really strengthen the limitation section.

Thank you for pointing to these aspects. We deleted the sentence mentioned. In the discussion section, we now finish the paragraph on limitations with the proposed future work to address them.

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

Learn more at Review Commons

Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)): __ In this manuscript, the authors describe the discovery of a molecular regulator of the immune transcriptional program, which is activated by intestinal distension upon bacterial colonization of the C. elegans intestine. Taking advantage of the fact that inhibition of aex-5 is known to cause intestinal distension and a C-type lectin gene clec-60 as a marker for the immune response to intestinal distension (clec-60p::gfp), the authors performed a forward genetic screen for suppressors of the immune response activation. Of the two mutants isolated, they focused on the stronger suppressor, which corresponded to a cysteine-type DUB, the Ubiquitin Specific Peptidase-14 (usp-14). Through rescue experiments, phenocopy analyses, and quantitative RT-PCR, they validated usp-14 as the causal gene and initiated characterization of its role in immune response activation. To this end, the authors investigated the tissue of action, identifying the intestine as the tissue in which usp-14 mediates the regulation of the immune response. Through transcriptomic analyses, they found that the signalling pathway likely regulated by usp-14 in response to intestinal distension is the Wnt pathway, as they have observed reduction in the transcriptional level of some of the Wnt pathway components in usp-4(tm1481), in response to infection with S. aureus. Additionally, transcriptomic data indicate that usp-14 plays a role in immunity regulation even in the absence of infection. Based on these findings, the authors propose that usp-14 has a dual role in immune regulation: one in surveillance immunity, preventing overactivation of immune responses, and another as a mediator of pathogen-induced responses, such as those triggered by P. aeruginosa or S. aureus. The experiments are rigorous and the results robust; however, some points would benefit from further investigation or clarification. __Response: We thank the reviewer for an excellent summary of our work and for the valuable feedback.

Comment: The expression domain of usp-14 appears to be quite expanded based on single cell RNAseq data (e.g. PMID: 28818938) therefore it is likely that the transgenes used for expression analysis are lacking key regulatory information. Alternative methods like smFISH would be more appropriate to characterise the spatiotemporal pattern of usp-14 expression in more detail. Response: We thank the reviewer for this valuable suggestion. In the original version of the manuscript, we used a 714 bp region upstream of the usp-14 start codon to generate the transcriptional reporter. In the revised manuscript, we reconstructed the reporter using a longer 1924 bp upstream promoter region together with a portion of exon 1. Using this updated reporter, we observed substantially broader expression of usp-14, particularly during the early larval stages. These results are described on page 6, lines 147-152: “We next examined the spatiotemporal expression pattern of usp-14 in C. elegans. To this end, we generated transgenic worms expressing GFP under the control of the usp-14 promoter (usp-14p::gfp). During early larval development, usp-14 was broadly expressed across multiple tissues (Figure 3A). However, in L4 larvae and adult animals, expression became more restricted and was predominantly observed in the intestine and a subset of neuronal cells. Notably, both intestinal and neuronal expression persisted throughout development (Figure 3A).”

Comment: __The mutation mapped in usp-14(jsn19) is a missense mutation (E122K) that suppresses the immune response to a degree comparable to the usp-14(tm1481) deletion allele. However, the authors do not show the functional domains in Fig. 1E potentially affected by this missense mutation. __Response: We have now updated Figure 1E to include the functional domains of USP-14 and mapped both the usp-14(jsn19) missense allele and the usp-14(tm1481) deletion allele onto the protein schematic.

Comment: __How USP-14 regulates Wnt and how Wnt signalling relates to activation of immune responses is not fully supported. Are the Wnt components mentioned in the study induced specifically in the intestine upon infection and does USP-14 act in the intestine in the context of this regulation? How do the authors interpret that both Wnt ligands and receptors are induced ? Does Wnt signalling appear as a GO term in the transcriptomic analysis? The authors can include Wnt signalling components in the analysis of the transcriptomic results. __Response: We thank the reviewer for these insightful comments. Previous studies have shown that the Wnt pathway components examined in our study are induced in the intestine upon infection and function within the intestine to regulate host defense against bacterial pathogens (PMID: 29768179; PMID: 36323254).

We did not observe significant enrichment of Wnt signaling terms in the GO analysis of our transcriptomic dataset. We believe this is likely due to the stringent thresholds used for differential expression analysis (fold change > 2 and p At present, the precise mechanism by which USP-14 regulates Wnt pathway components remains unclear. One possibility is that USP-14 influences Wnt signaling indirectly through additional substrates or interacting proteins that regulate transcriptional outputs. We have now clarified this point in the Discussion (page 12, lines 340–345): “These observations raise the possibility that additional USP-14 substrates or interacting proteins modulate transcriptional outputs downstream of intestinal distension. Future studies aimed at identifying the direct substrates of USP-14 and defining how USP-14 interfaces with neuronal ACC-4 signaling and other distension-responsive pathways will provide important mechanistic insight into how intestinal distension is coupled to innate immune activation.”

Regarding the simultaneous induction of Wnt ligands and receptors, we interpret this as a potential amplification or reinforcement mechanism that enhances Wnt/β-catenin signaling during infection-induced intestinal distension. However, further studies will be required to determine the mechanistic significance of this coordinated transcriptional regulation.

Comment: __Overall, in most of the figures, the micrographs are in general quite dark and exhibit poor contrast between signal and background, particularly in Fig. 1, panels B and J, and Fig. 2, panels B and F (upper rows). Even though these panels are intended to show absence of response, the outlines of the worms are difficult to discern. __Response: We thank the reviewer for the feedback. We have now improved the image presentation throughout the manuscript by either increasing the intensity or adding dotted outlines to more clearly indicate worm positions.

Comment: __In Figure S3, panels A and B, the pmk-1(km25); usp-14(tm1481) animals subjected to aex-5 RNAi show some level of fluorescence/response induction comparable to pmk-1(km25) alone. This observation is not discussed in the text. __Response: We have now discussed this observation in the text. These results are described on page 9, lines 240-244: “Although pmk-1(km25);usp-14(tm1481) worms displayed relatively higher GFP levels than usp-14(tm1481) single mutants upon aex-5 RNAi treatment, this effect likely reflects the elevated basal GFP expression observed in pmk-1(km25) mutants (Figure S4B). Importantly, pmk-1(km25);usp-14(tm1481) animals still exhibited significantly lower GFP levels than pmk-1(km25) single mutants.”

Reviewer #1 (Significance (Required)): __ __Comment: __The work is interesting because it expands some previous work in the field demonstrating immune response induction as a consequence of intestinal distension even in the absence of bacterial infection. This is known to be mediated by the neuronal acetylcholine receptor ACC-4, which signals to the intestine where it regulates immune genes via the Wnt pathway. However, how USP-14 relates to ACC-4 is currently unclear and whether USP-14 function is really required in the intestine to control Wnt signalling is not demonstrated. The authors should include a model to describe how their findings relate to the previous literature and how USP-14 may link mechanistically to Wnt signalling pathway activation. __Response: We thank the reviewer for this insightful comment. We agree that the relationship between USP-14, ACC-4, and Wnt signaling requires further clarification. As suggested by the reviewer, we have now included a model summarizing the current understanding of intestinal distension-induced immune activation and integrating our findings with previous literature (Figure 6H).

Comment: __It remains also unclear whether usp-14 is the only deubiquitinase involved in intestinal distension-induced signalling via the Wnt pathway, or whether other paralog usp genes might also contribute to regulation of immune-responsive transcription. Notably, several mammalian deubiquitinases have established roles in cancer suppression and inflammatory response and innate immunity in other systems so this would increase the potential significance of the work. __Response: We thank the reviewer for this valuable suggestion. To systematically examine whether additional DUBs contribute to intestinal distension-induced immune activation, we performed an RNAi screen targeting all DUBs available in the Ahringer RNAi library using the aex-5(sa23);clec-60p::gfp reporter strain. Among the DUBs tested, knockdown of usp-14 produced the strongest suppression of clec-60p::gfp expression. Although knockdown of usp-5 also partially suppressed GFP induction, usp-5 RNAi did not affect survival during P. aeruginosa infection, suggesting that usp-5 is not required for host defense under these conditions. Together, these findings identify USP-14 as the major DUB required for intestinal distension-induced immune activation in our experimental system. These results are now included in Figure 1G, H, and Figure S2.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): __ Summary C. elegans are soil-dwelling nematodes that feed on bacteria and fungi and thus must be able to distinguish between innocuous and pathogenic species of microbes to survive. Though they lack adaptive immunity, these animals have an ancient version of an innate immune system that has no circulating sentinel or phagocytic cells yet can still mount a response to pathogen exposure. A consequence of the mode of infection of some ingested bacterial pathogens is intestinal distension which by itself, even in the absence of pathogens, is sufficient to trigger the expression of genes encoding immune effectors, including proteins that are bactericidal. The complete mechanistic scheme connecting intestinal distension to the expression of immunity genes has not been resolved, motivating the authors to perform a forward genetic screen for additional components of this pathway. One mutant that the authors isolated was usp-14, encoding an evolutionarily conserved deubiqutinating enzyme. Functional analysis revealed that usp-14 confers protection from microbial pathogens and that the intestine is its primary site of action for its role in host defense. The authors' data indicate that while USP-14 regulates the expression of innate immunity genes that are induced by intestinal distension, surprisingly it functions independently of several canonical innate immune signaling pathways, including the pmk-1/p38 MAPK pathway. Instead, USP-14 appears to act through Wnt signaling to regulate immune effectors by upregulating the expression of several components of that pathway, including the C. elegans ß-catenin ortholog bar-1. This places usp-14 within a gut-brain axis previously shown to control the C. elegans innate immune response through acetylcholine-mediated activation of Wnt signaling. The authors' findings provide new mechanistic insight to this pathway and add to the understanding of ubiqutination as an immune regulatory module. __Response: We thank the reviewer for providing an excellent summary of our work.

Major comments __1. There are three types of experiments in which the authors use the same set of controls across several different figure panels, as stated in the legend to Figure 2. First, when quantifying GFP levels of clec-60::gfp in RNAi-treated animals, the authors use the same clec-60p::gfp and usp-14(jsn19);clec-60p::gfp controls for Fig. 1K, 2C, and 2G. For infection assays with S. aureus NCTC8325, the survival plots for the clec-60p::gfp and usp-14(jsn19);clec-60p::gfp controls shown in Fig. 2E are the same as the ones used in Fig. 1M. Similarly, for infection assays with P. aeruginosa PA14, the survival plots for the clec-60p::gfp and usp-14(jsn19);clec-60p::gfp controls shown in Fig. 2I is the same as was used for Fig 1I. In each case, if the authors in fact collected all of the data for each strain that they studied at the same time but then chose to parse larger datasets into separate figure panels to make it more clear to the reader, then this approach is valid but the authors need to explicitly state that this is what they did. However, if the data pertaining to the control strains were collected at a different time or if it comes from a separate biological replicate, then re-using data from the controls is not appropriate because it would not accurately reflect the specific conditions of the experiment to which the data are being compared. If this is indeed the scenario, then the authors will need to repeat these experiments and include the appropriate control in each iteration. __Response: While preparing the manuscript, these experiments were performed simultaneously. Therefore, all panels that share controls have results from experiments performed simultaneously and represent the same biological replicate. We have added this additional information in the relevant figure legends.

Comment: __2. From the legends describing figure panels that include data pertaining to clec-60p::gfp expression levels as assessed by fluorescence microscopy it seems that, in general, the authors measured GFP fluorescence in about 30 animals to produce quantitative data. How many biological replicates of these types of experiments were carried out? This is not explicitly stated in the section describing fluorescence imaging in the Methods section. Following the description of their methodology regarding statistical analysis of survival curves from microbial infection assays, however, the authors state that, "[a]ll experiments were performed independently at least three times unless otherwise noted." Does this statement apply to microscopy or only to experiments involving infection assays? If the data reporting quantitation of GFP signal is based on only 30 animals, then additional biological replicates are necessary, along with appropriate statistical analyses. __Response: The quantified GFP fluorescence data are derived from three independent biological replicates. In each experiment, we typically imaged and quantified approximately 10 worms per condition, yielding a total of ~30 worms analyzed per genotype or treatment across all replicates (except Figure S1B, where we had two independent replicates). We have added the number of experiments in the figure legends for these data.

Comment: __3. The authors have made all of the RNASeq data publicly available on the Sequence Read Archive, and they include data from several pairwise comparisons for differential gene expression analysis in their supplemental files. One of the most important facts to come out of the authors' Gene Ontology analyses of their RNASeq data is that the genes that are upregulated in a usp-14-dependent manner upon intestinal distension are enriched for those whose products play a role in innate immunity/host defense. The authors should say more about these genes. Are there any commonalities between them with regard to function? Are any of them targets of transcription factors that are known to function in C. elegans innate immunity? If so, this could provide clues as to what the substrates of USP-14 might be. Importantly, the specific identity of the genes assigned in the GO analyses to biological processes pertaining to innate immunity and host defense should be revealed in a supplemental file, and designated as being dependent on or independent of usp-14 for their expression during intestinal distension. __Response: We thank the reviewer for this insightful suggestion. We have now expanded the Results section to describe the functional categories enriched among the USP-14-dependent intestinal distension-induced immune genes, including C-type lectins, ShK toxin domain-containing proteins, and lysozymes (page 7, lines 193-195).

In addition, we compared our transcriptomic dataset with previously published transcription factor-regulated gene sets using WormExp analysis and identified a substantial overlap with genes regulated by the GATA transcription factor ELT-2. These new analyses are described on page 7, lines 196-206: “To identify transcription factors potentially involved in intestinal distension-induced immune activation, we performed transcription factor enrichment analysis using WormExp on genes upregulated in N2 worms following aex-5 RNAi treatment. This analysis revealed a substantial overlap between aex-5 RNAi-induced genes and genes regulated by the GATA transcription factor ELT-2 (Figure S3D). We next examined whether USP-14-dependent immune genes overlapped with ELT-2-dependent immunity genes induced by intestinal distension. To this end, we identified innate immune genes common to both ELT-2-regulated gene sets and aex-5 RNAi-induced genes. Strikingly, these ELT-2-dependent intestinal distension-induced immune genes showed substantial overlap with USP-14-dependent immune genes (Figure S3E and Table S5), suggesting that USP-14 may regulate distension-induced immunity, at least in part, through ELT-2-dependent transcriptional programs.”

Finally, we have created a new table (Table S5) that specifies the identity of the genes assigned in the GO analyses to biological processes pertaining to innate immunity and host defense, for USP-14-dependent and independent genes.

Comment: __4. The authors' data suggest that in response to bacterial infection USP-14 upregulates the expression of bar-1, along with other components of the Wnt signaling pathway, which in turn upregulates innate immunity genes. This could be further substantiated by directly demonstrating that there are USP-14-regulated innate immunity genes whose induced expression in the presence of microbial pathogens also requires bar-1. Along those lines, an initial test would be to assess clec-60p::gfp expression in bar-1 animals versus bar-1;usp-14 double mutants, similar to the experiment whose results are reported in Fig. S4. If generating the bar-1;usp-14 double mutant is not feasible, then RNAi could be used to knockdown bar-1 expression in clec-60p::gfp;usp-14(tm1481) animals. To expand this analysis, the expression of the six innate immunity genes shown to be regulated upon intestinal distension in usp-14-dependent manner could be measured in the presence and absence of intestinal distension or microbial infection in bar-1 and bar-1;usp-14 animals by qRT-PCR. At a minimum, the authors should conduct a bioinformatics analysis to compare the USP-14-regulated innate immunity genes identified in their RNAseq studies to lists of known BAR-1 transcriptional targets to look for potential overlap. __Response: We agree that extending these analyses to qRT-PCR experiments examining additional immune genes would be informative. However, both bar-1 mutants and bar-1 RNAi-treated worms exhibited severe developmental and physiological defects, including sick and dead animals during development, likely reflecting the pleiotropic developmental roles of BAR-1. Although fluorescence imaging and survival assays could be performed by selectively transferring surviving adults, we were concerned that bulk collection of worms for qRT-PCR analyses would introduce confounding effects arising from developmental defects and reduced viability.

To further address the reviewer’s suggestion, we carried out a comparative analysis between USP-14-dependent intestinal distension-induced immune genes and previously identified BAR-1-dependent immune genes. Although transcriptome-wide datasets for BAR-1-dependent pathogen-induced immune genes are not currently available, an earlier study identified seven immune response genes regulated by BAR-1 during infection (PMID: 18981407). We found that six of these genes overlap with the USP-14-dependent intestinal distension-induced immune genes identified in our study. These analyses have now been added to the Results section and included in Table S5.

Comment: __5. While in their Discussion section the authors mention evolutionarily conserved roles for protein ubiquitination as means of immunomodulation, there are few if any comments regarding ubiqutination as a regulatory scheme in C. elegans innate immunity or how their findings enhance our understanding of this phenomenon. Ubiquitination affects C. elegans immunity at multiple levels, from avoidance behavior to gene regulation, and it seems appropriate for the authors to address this in order to more fully contextualize their findings. __Response: We thank the reviewer for the suggestion. We have now added a new paragraph to the Discussion that places our findings in the context of the existing literature on ubiquitination, deubiquitination, and innate immunity in C. elegans. The discussion is added on pages 10-11, lines 295-308: “Although ubiquitin-mediated signaling has emerged as a central regulator of innate immunity across metazoans (Jiang & Chen, 2011; Mello-Vieira & Dikic, 2026), the contribution of DUBs to host defense in C. elegans remains poorly understood. Previous studies in C. elegans have shown that ubiquitin-dependent processes regulate diverse aspects of immunity, including immune surveillance, xenophagy, and pathogen tolerance (Garcia-Sanchez et al, 2021). Perturbations in proteasome function have also been shown to activate surveillance immunity (Ghosh & Singh, 2026; Troemel et al, 2026), highlighting the importance of ubiquitin-associated pathways in sensing pathogen-induced cellular damage. However, most prior studies have focused on ubiquitin ligases, proteasome-associated pathways, or global ubiquitin signaling rather than on specific DUBs directly regulating antibacterial immune responses. To our knowledge, our study provides the first direct evidence that a specific DUB regulates antibacterial innate immunity in C. elegans. Thus, our findings establish USP-14 as a previously unrecognized regulator of host defense and identify deubiquitination as an important regulatory layer in intestinal distension-mediated immunity.”

__Minor comments __1. In the Results section, the authors state that "[k]nockdown of cec-10 led to only a marginal decrease in survival during P. aeruginosa infection" (lines 92 and 93) and that cec-10 "has minimal impact on C. elegans survival during infection" (lines 93 and 94). However, as reported in Supplemental Table 5 the magnitude of the calculated difference in mean survival time between animals treated with RNAi targeting cec-10 and untreated control animals (-20% to -24% and statistically significant in 3/3 replicates) closely approximates the difference in mean survival between usp-14 mutants and controls (-19% to -28% and statistically significant in 3/3 replicates), which the authors clearly find to be significant. If by this metric usp-14 is important for host defense, then so too is cec-10. In light of this, the authors should use different language to describe the impact of cec-10 knockdown on the susceptibility of C. elegans to microbial infection and the potential role of cec-10 in immunity.

Response: We chose not to pursue cec-10 further primarily because it lacks a clear human homolog and because the mutant exhibited reduced expression of the co-injection marker, raising the possibility of broader transgene-related effects. We have modified the text on page 4, lines 92-96: “Knockdown of cec-10 resulted in a significant reduction in survival during P. aeruginosa infection (Figure S1C). However, we did not pursue cec-10 further for two reasons: (i) cec-10(jsn20) mutants exhibited a modest but significant reduction in the myo-2p::mCherry co-injection marker (Figure 1D), raising the possibility of broader transgene-related defects, and (ii) cec-10 lacks a clear human homolog.”

Comment: __2. All of the micrographs in Fig. 1B appear very dark. The GFP expression in the control animals appears dim, making it difficult for the reader to compare the signal in those animals to the GFP expression levels in the mutants. I recommend adjusting the brightness level in an equivalent manner across all of the micrographs to account for this. __Response: We have increased the brightness of all the images, as suggested by the reviewer.

__Comment: __3. Fig. 1E depicts a gene structure diagram for usp-14 with the position of the point mutation in the jsn19 allele isolated in the authors' forward genetic screen indicated by the amino acid substitution symbol drawn over the second exon. Instead of mixing gene- and protein-level information about the jsn19 allele, I recommend replacing the gene structure diagram with a domain structure diagram of the USP-14 protein that depicts the conserved C19 peptidase and ubiquitin-like domains. The relative position of the E122K substitution should still be noted. __Response: __We have now updated Figure 1E to include the functional domains of USP-14 and mapped both the usp-14(jsn19) missense allele and the usp-14(tm1481) deletion allele onto the protein schematic.

Comment: __4. Since all of the information in Fig. 1F appears elsewhere in the text, I recommend eliminating this panel. __Response: We have removed it.

Comment: __5. Regarding the RNAseq analysis, the authors state that 1241 genes are upregulated upon aex-5 knockdown (line 162). The authors then ask which of these genes are regulated by usp-14 in the context of intestinal distension and find that 633 are upregulated a usp-14-dependent manner when aex-5 is targeted by RNAi and that 595 are upregulated even in the absence of usp-14 (Fig. 3D). This accounts for 1228 genes in total, not 1241. Can the authors explain this discrepancy? __Response: We thank the reviewer for carefully noting this discrepancy. The difference arises from the criteria used to classify genes into the categories shown in Figure 5D (previously Figure 3D). Specifically, genes uniquely upregulated in usp-14(tm1481) worms were defined as genes that were either exclusively induced in usp-14(tm1481) worms or expressed at levels more than 2-fold higher in usp-14(tm1481) worms compared to N2 worms. During this classification, 13 genes that were initially identified as upregulated in N2 worms following aex-5 RNAi were found to be expressed at levels more than 2-fold higher in usp-14(tm1481) worms than in N2 worms (Table S4). These genes were therefore reassigned to the “usp-14(tm1481)-specific” category in the Venn diagram. Consequently, the total number of genes represented in the Venn diagram becomes 1228 instead of 1241. To clarify this point, we have now added an explanation to the figure legend.

Comment: __6. For the sake of clarity, in the legend to Fig. 3D I recommend expanding the description of the categories of genes depicted in the Venn diagram by using the same language as in the first worksheet of Supplemental Table 4. __Response: We thank the reviewer for the suggestion. We have now added these details to the legend of Figure 5D (previously Figure 3D). The legend reads: “(D) Venn diagram showing the overlap between genes upregulated upon aex-5 RNAi in N2 and usp-14(tm1481) worms. The GO analyses for the biological processes of unique and common genes are shown. USP-14-dependent genes were defined as genes that were either exclusively upregulated in N2 worms or expressed at levels greater than 2-fold higher in N2 worms than in usp-14(tm1481) worms. USP-14-independent genes were defined as genes upregulated in both N2 and usp-14(tm1481) worms with expression differences of less than 2-fold between the two strains. Genes uniquely upregulated in usp-14(tm1481) worms were defined as genes that were either exclusively induced in usp-14(tm1481) worms or expressed at levels greater than 2-fold higher in usp-14(tm1481) worms than in N2 worms. Thirteen genes classified as upregulated in N2 worms were more than 2-fold higher in usp-14(tm1481) worms than in N2 worms (Table S4) and were therefore included in the usp-14(tm1481)-specific category.”

Comment: __7. In Fig. 4B, the authors' annotation indicates that there is a statistically significant difference (**, p __Comment: __8. In Fig. S5, the shade of blue used to represent the data from the nhr-49(nr2041);usp-14(tm1481);clec60p::gfp animals in panel E is different from that used to represent data from the same animals in panel B. This breaks the pattern of all of the other panels of this figure in which the data pertaining to a given phenotype are depicted in the same color. Also, in the symbol key in panel E there is an extra semi-colon before clec-60p::gfp that should be eliminated in the second genotype notation. __Response: We thank the reviewer for carefully examining the figure and for bringing these issues to our attention. We have made the changes.

Comment: __9. The authors' data show that USP-14 regulates bar-1 expression, and in the Discussion section they mention that in mammals beta-catenin is a substrate of USP14. Can the authors comment on the possibility of/evidence for BAR-1 autoregulation in C. elegans and the prospect of it being facilitated by USP-14? This could be a minor point to add to the Discussion. __Response: In both contexts, USP-14 appears to stabilize BAR-1 by regulating it at either the transcriptional or post-translational level. However, it is currently unknown whether BAR-1 regulates USP-14 expression and thereby participates in an autoregulatory mechanism. Nevertheless, we have added to the Discussion that USP14 may regulate the Wnt pathway through both transcriptional and post-translational mechanisms, depending on the biological context. __Reviewer #2 (Significance (Required)): __ The study described in this manuscript ties in to the findings from two prior genetic screens carried out in C. elegans that aimed to identify immune regulators (Ren et al., Cell Reports, 2022 and Labed et al., Immunity, 2018). Though their strategies differed, both of these previous studies uncovered a role for acetylcholine receptors in modulating the response to ingested microbial pathogens, especially when infection is associated with intestinal distension, indicating that a neuron-to-gut axis controls innate immunity in C. elegans. Labed and colleagues were the first to show that activation of this pathway results in the upregulation of genes encoding Wnt signaling pathway components, including the worm ortholog of beta-catenin called bar-1, which are necessary for the expression of immune effectors in the intestine. The Labed study also revealed that protein ubiquitination could contribute to regulating host defense gene induction because knockdown of lin-23, the substate binding subunit of a ubiquitin ligase complex that mediates BAR-1 degradation, results in constitutive expression of clec-60p::gfp, the same transcription reporter used by Ghosh and Singh as a readout for the expression of innate immunity genes. In their screen that revisits the Ren et al. approach, Ghosh and Singh find that another protein implicated in regulating protein stability via ubiquitination status, USP-14, also controls the expression of innate immunity genes in response to intestinal distension. Interestingly, their data indicate that it does so by upregulating bar-1. This discovery therefore adds an element of mechanistic detail regarding the regulation of Wnt signaling in immunity. While the Labed data suggest that ubiquitination may regulate BAR-1 at the post-translational level, Ghosh and Singhs' results indicate a second layer of regulation of bar-1 at the transcriptional level that also appears to involve ubiquitination. In this case, USP-14 is predicted to modulate the ubiquitination status of a yet-to-be-identified substrate that directly or indirectly governs bar-1 expression. The authors' findings thus bring the field closer to having a complete picture of the Ach-Wnt pathway in C. elegans. As they point out in the Discussion section of their manuscript, ubiquitination is an evolutionarily conserved yet complex means of tuning the immune system. The work described here helps to shed light on this important immune regulatory mode and could have implications for aspects of epithelial immunity that are in common to both invertebrates and vertebrates.

Response: We thank the reviewer for providing such a thoughtful overview of the field and for placing our findings in the context of previous studies on intestinal distension-induced immunity in C. elegans. We also sincerely appreciate the reviewer’s constructive feedback and insightful comments, which have helped us improve the quality and clarity of the manuscript.

My research interest and specific area of expertise pertains to evolutionarily conserved genetic pathways that control healthspan through affecting cellular resilience later in life. Using C. elegans as a surrogate for aging humans, my group studies age-dependent changes in the activity of regulatory modules that protect older animals from the molecular damage associated with intrinsic and extrinsic sources of cellular stress, with a particular emphasis on microbial infection and oxidative stress.

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Referee #2

Evidence, reproducibility and clarity

Summary

C. elegans are soil-dwelling nematodes that feed on bacteria and fungi and thus must be able to distinguish between innocuous and pathogenic species of microbes to survive. Though they lack adaptive immunity, these animals have an ancient version of an innate immune system that has no circulating sentinel or phagocytic cells yet can still mount a response to pathogen exposure. A consequence of the mode of infection of some ingested bacterial pathogens is intestinal distension which by itself, even in the absence of pathogens, is sufficient to trigger the expression of genes encoding immune effectors, including proteins that are bactericidal. The complete mechanistic scheme connecting intestinal distension to the expression of immunity genes has not been resolved, motivating the authors to perform a forward genetic screen for additional components of this pathway. One mutant that the authors isolated was usp-14, encoding an evolutionarily conserved deubiqutinating enzyme. Functional analysis revealed that usp-14 confers protection from microbial pathogens and that the intestine is its primary site of action for its role in host defense. The authors' data indicate that while USP-14 regulates the expression of innate immunity genes that are induced by intestinal distension, surprisingly it functions independently of several canonical innate immune signaling pathways, including the pmk-1/p38 MAPK pathway. Instead, USP-14 appears to act through Wnt signaling to regulate immune effectors by upregulating the expression of several components of that pathway, including the C. elegans ß-catenin ortholog bar-1. This places usp-14 within a gut-brain axis previously shown to control the C. elegans innate immune response through acetylcholine-mediated activation of Wnt signaling. The authors' findings provide new mechanistic insight to this pathway and add to the understanding of ubiqutination as an immune regulatory module.

Major comments

1. There are three types of experiments in which the authors use the same set of controls across several different figure panels, as stated in the legend to Figure 2. First, when quantifying GFP levels of clec-60::gfp in RNAi-treated animals, the authors use the same clec-60p::gfp and usp-14(jsn19);clec-60p::gfp controls for Fig. 1K, 2C, and 2G. For infection assays with S. aureus NCTC8325, the survival plots for the clec-60p::gfp and usp-14(jsn19);clec-60p::gfp controls shown in Fig. 2E are the same as the ones used in Fig. 1M. Similarly, for infection assays with P. aeruginosa PA14, the survival plots for the clec-60p::gfp and usp-14(jsn19);clec-60p::gfp controls shown in Fig. 2I is the same as was used for Fig 1I. In each case, if the authors in fact collected all of the data for each strain that they studied at the same time but then chose to parse larger datasets into separate figure panels to make it more clear to the reader, then this approach is valid but the authors need to explicitly state that this is what they did. However, if the data pertaining to the control strains were collected at a different time or if it comes from a separate biological replicate, then re-using data from the controls is not appropriate because it would not accurately reflect the specific conditions of the experiment to which the data are being compared. If this is indeed the scenario, then the authors will need to repeat these experiments and include the appropriate control in each iteration.
2. From the legends describing figure panels that include data pertaining to clec-60p::gfp expression levels as assessed by fluorescence microscopy it seems that, in general, the authors measured GFP fluorescence in about 30 animals to produce quantitative data. How many biological replicates of these types of experiments were carried out? This is not explicitly stated in the section describing fluorescence imaging in the Methods section. Following the description of their methodology regarding statistical analysis of survival curves from microbial infection assays, however, the authors state that, "[a]ll experiments were performed independently at least three times unless otherwise noted." Does this statement apply to microscopy or only to experiments involving infection assays? If the data reporting quantitation of GFP signal is based on only 30 animals, then additional biological replicates are necessary, along with appropriate statistical analyses.
3. The authors have made all of the RNASeq data publicly available on the Sequence Read Archive, and they include data from several pairwise comparisons for differential gene expression analysis in their supplemental files. One of the most important facts to come out of the authors' Gene Ontology analyses of their RNASeq data is that the genes that are upregulated in a usp-14-dependent manner upon intestinal distension are enriched for those whose products play a role in innate immunity/host defense. The authors should say more about these genes. Are there any commonalities between them with regard to function? Are any of them targets of transcription factors that are known to function in C. elegans innate immunity? If so, this could provide clues as to what the substrates of USP-14 might be. Importantly, the specific identity of the genes assigned in the GO analyses to biological processes pertaining to innate immunity and host defense should be revealed in a supplemental file, and designated as being dependent on or independent of usp-14 for their expression during intestinal distension.
4. The authors' data suggest that in response to bacterial infection USP-14 upregulates the expression of bar-1, along with other components of the Wnt signaling pathway, which in turn upregulates innate immunity genes. This could be further substantiated by directly demonstrating that there are USP-14-regulated innate immunity genes whose induced expression in the presence of microbial pathogens also requires bar-1. Along those lines, an initial test would be to assess clec-60p::gfp expression in bar-1 animals versus bar-1;usp-14 double mutants, similar to the experiment whose results are reported in Fig. S4. If generating the bar-1;usp-14 double mutant is not feasible, then RNAi could be used to knockdown bar-1 expression in clec-60p::gfp;usp-14(tm1481) animals. To expand this analysis, the expression of the six innate immunity genes shown to be regulated upon intestinal distension in usp-14-dependent manner could be measured in the presence and absence of intestinal distension or microbial infection in bar-1 and bar-1;usp-14 animals by qRT-PCR. At a minimum, the authors should conduct a bioinformatics analysis to compare the USP-14-regulated innate immunity genes identified in their RNAseq studies to lists of known BAR-1 transcriptional targets to look for potential overlap.
5. While in their Discussion section the authors mention evolutionarily conserved roles for protein ubiquitination as means of immunomodulation, there are few if any comments regarding ubiqutination as a regulatory scheme in C. elegans innate immunity or how their findings enhance our understanding of this phenomenon. Ubiquitination affects C. elegans immunity at multiple levels, from avoidance behavior to gene regulation, and it seems appropriate for the authors to address this in order to more fully contextualize their findings.

Minor comments

1. In the Results section, the authors state that "[k]nockdown of cec-10 led to only a marginal decrease in survival during P. aeruginosa infection" (lines 92 and 93) and that cec-10 "has minimal impact on C. elegans survival during infection" (lines 93 and 94). However, as reported in Supplemental Table 5 the magnitude of the calculated difference in mean survival time between animals treated with RNAi targeting cec-10 and untreated control animals (-20% to -24% and statistically significant in 3/3 replicates) closely approximates the difference in mean survival between usp-14 mutants and controls (-19% to -28% and statistically significant in 3/3 replicates), which the authors clearly find to be significant. If by this metric usp-14 is important for host defense, then so too is cec-10. In light of this, the authors should use different language to describe the impact of cec-10 knockdown on the susceptibility of C. elegans to microbial infection and the potential role of cec-10 in immunity.
2. All of the micrographs in Fig. 1B appear very dark. The GFP expression in the control animals appears dim, making it difficult for the reader to compare the signal in those animals to the GFP expression levels in the mutants. I recommend adjusting the brightness level in an equivalent manner across all of the micrographs to account for this.
3. Fig. 1E depicts a gene structure diagram for usp-14 with the position of the point mutation in the jsn19 allele isolated in the authors' forward genetic screen indicated by the amino acid substitution symbol drawn over the second exon. Instead of mixing gene- and protein-level information about the jsn19 allele, I recommend replacing the gene structure diagram with a domain structure diagram of the USP-14 protein that depicts the conserved C19 peptidase and ubiquitin-like domains. The relative position of the E122K substitution should still be noted.
4. Since all of the information in Fig. 1F appears elsewhere in the text, I recommend eliminating this panel.
5. Regarding the RNAseq analysis, the authors state that 1241 genes are upregulated upon aex-5 knockdown (line 162). The authors then ask which of these genes are regulated by usp-14 in the context of intestinal distension and find that 633 are upregulated a usp-14-dependent manner when aex-5 is targeted by RNAi and that 595 are upregulated even in the absence of usp-14 (Fig. 3D). This accounts for 1228 genes in total, not 1241. Can the authors explain this discrepancy?
6. For the sake of clarity, in the legend to Fig. 3D I recommend expanding the description of the categories of genes depicted in the Venn diagram by using the same language as in the first worksheet of Supplemental Table 4.
7. In Fig. 4B, the authors' annotation indicates that there is a statistically significant difference (**, p<0.01) in the fluorescence signal from clec-60p::gfp in usp-14(jsn19);aex-5(sa23);clec-60p::gfp_EV versus usp-14(jsn19);aex-5(sa23);clec-60p::gfp_bar-1 animals. This is likely a typographical error that should be changed to "ns" to indicate no significant difference in the fluorescence signal between these two groups, which is consistent with what the data show and with the authors' description of these data in the text (lines 211-214).
8. In Fig. S5, the shade of blue used to represent the data from the nhr-49(nr2041);usp-14(tm1481);clec60p::gfp animals in panel E is different from that used to represent data from the same animals in panel B. This breaks the pattern of all of the other panels of this figure in which the data pertaining to a given phenotype are depicted in the same color. Also, in the symbol key in panel E there is an extra semi-colon before clec-60p::gfp that should be eliminated in the second genotype notation.
9. The authors' data show that USP-14 regulates bar-1 expression, and in the Discussion section they mention that in mammals beta-catenin is a substrate of USP14. Can the authors comment on the possibility of/evidence for BAR-1 autoregulation in C. elegans and the prospect of it being facilitated by USP-14? This could be a minor point to add to the Discussion.

Significance

The study described in this manuscript ties in to the findings from two prior genetic screens carried out in C. elegans that aimed to identify immune regulators (Ren et al., Cell Reports, 2022 and Labed et al., Immunity, 2018). Though their strategies differed, both of these previous studies uncovered a role for acetylcholine receptors in modulating the response to ingested microbial pathogens, especially when infection is associated with intestinal distension, indicating that a neuron-to-gut axis controls innate immunity in C. elegans. Labed and colleagues were the first to show that activation of this pathway results in the upregulation of genes encoding Wnt signaling pathway components, including the worm ortholog of beta-catenin called bar-1, which are necessary for the expression of immune effectors in the intestine. The Labed study also revealed that protein ubiquitination could contribute to regulating host defense gene induction because knockdown of lin-23, the substate binding subunit of a ubiquitin ligase complex that mediates BAR-1 degradation, results in constitutive expression of clec-60p::gfp, the same transcription reporter used by Ghosh and Singh as a readout for the expression of innate immunity genes. In their screen that revisits the Ren et al. approach, Ghosh and Singh find that another protein implicated in regulating protein stability via ubiquitination status, USP-14, also controls the expression of innate immunity genes in response to intestinal distension. Interestingly, their data indicate that it does so by upregulating bar-1. This discovery therefore adds an element of mechanistic detail regarding the regulation of Wnt signaling in immunity. While the Labed data suggest that ubiquitination may regulate BAR-1 at the post-translational level, Ghosh and Singhs' results indicate a second layer of regulation of bar-1 at the transcriptional level that also appears to involve ubiquitination. In this case, USP-14 is predicted to modulate the ubiquitination status of a yet-to-be-identified substrate that directly or indirectly governs bar-1 expression. The authors' findings thus bring the field closer to having a complete picture of the Ach-Wnt pathway in C. elegans. As they point out in the Discussion section of their manuscript, ubiquitination is an evolutionarily conserved yet complex means of tuning the immune system. The work described here helps to shed light on this important immune regulatory mode and could have implications for aspects of epithelial immunity that are in common to both invertebrates and vertebrates.

My research interest and specific area of expertise pertains to evolutionarily conserved genetic pathways that control healthspan through affecting cellular resilience later in life. Using C. elegans as a surrogate for aging humans, my group studies age-dependent changes in the activity of regulatory modules that protect older animals from the molecular damage associated with intrinsic and extrinsic sources of cellular stress, with a particular emphasis on microbial infection and oxidative stress.

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

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Referee #1

Evidence, reproducibility and clarity

In this manuscript, the authors describe the discovery of a molecular regulator of the immune transcriptional program, which is activated by intestinal distension upon bacterial colonization of the C. elegans intestine. Taking advantage of the fact that inhibition of aex-5 is known to cause intestinal distension and a C-type lectin gene clec-60 as a marker for the immune response to intestinal distension (clec-60p::gfp), the authors performed a forward genetic screen for suppressors of the immune response activation. Of the two mutants isolated, they focused on the stronger suppressor, which corresponded to a cysteine-type DUB, the Ubiquitin Specific Peptidase-14 (usp-14). Through rescue experiments, phenocopy analyses, and quantitative RT-PCR, they validated usp-14 as the causal gene and initiated characterization of its role in immune response activation. To this end, the authors investigated the tissue of action, identifying the intestine as the tissue in which usp-14 mediates the regulation of the immune response. Through transcriptomic analyses, they found that the signalling pathway likely regulated by usp-14 in response to intestinal distension is the Wnt pathway, as they have observed reduction in the transcriptional level of some of the Wnt pathway components in usp-4(tm1481), in response to infection with S. aureus. Additionally, transcriptomic data indicate that usp-14 plays a role in immunity regulation even in the absence of infection. Based on these findings, the authors propose that usp-14 has a dual role in immune regulation: one in surveillance immunity, preventing overactivation of immune responses, and another as a mediator of pathogen-induced responses, such as those triggered by P. aeruginosa or S. aureus. The experiments are rigorous and the results robust; however, some points would benefit from further investigation or clarification.

The expression domain of usp-14 appears to be quite expanded based on single cell RNAseq data (e.g. PMID: 28818938) therefore it is likely that the transgenes used for expression analysis are lacking key regulatory information. Alternative methods like smFISH would be more appropriate to characterise the spatiotemporal pattern of usp-14 expression in more detail.

The mutation mapped in usp-14(jsn19) is a missense mutation (E122K) that suppresses the immune response to a degree comparable to the usp-14(tm1481) deletion allele. However, the authors do not show the functional domains in Fig. 1E potentially affected by this missense mutation.

How USP-14 regulates Wnt and how Wnt signalling relates to activation of immune responses is not fully supported. Are the Wnt components mentioned in the study induced specifically in the intestine upon infection and does USP-14 act in the intestine in the context of this regulation? How do the authors interpret that both Wnt ligands and receptors are induced ? Does Wnt signalling appear as a GO term in the transcriptomic analysis? The authors can include Wnt signalling components in the analysis of the transcriptomic results.

Overall, in most of the figures, the micrographs are in general quite dark and exhibit poor contrast between signal and background, particularly in Fig. 1, panels B and J, and Fig. 2, panels B and F (upper rows). Even though these panels are intended to show absence of response, the outlines of the worms are difficult to discern.

In Figure S3, panels A and B, the pmk-1(km25); usp-14(tm1481) animals subjected to aex-5 RNAi show some level of fluorescence/response induction comparable to pmk-1(km25) alone. This observation is not discussed in the text.

Significance

The work is interesting because it expands some previous work in the field demonstrating immune response induction as a consequence of intestinal distension even in the absence of bacterial infection. This is known to be mediated by the neuronal acetylcholine receptor ACC-4, which signals to the intestine where it regulates immune genes via the Wnt pathway. However, how USP-14 relates to ACC-4 is currently unclear and whether USP-14 function is really required in the intestine to control Wnt signalling is not demonstrated. The authors should include a model to describe how their findings relate to the previous literature and how USP-14 may link mechanistically to Wnt signalling pathway activation.

It remains also unclear whether usp-14 is the only deubiquitinase involved in intestinal distension-induced signalling via the Wnt pathway, or whether other paralog usp genes might also contribute to regulation of immune-responsive transcription. Notably, several mammalian deubiquitinases have established roles in cancer suppression and inflammatory response and innate immunity in other systems so this would increase the potential significance of the work.

Please remove SaaS from everywhere, see migration.mdx for more detials

investigate, might be AI slop from current docs

Dari segi legal risks, penggunaan sistem “mass balance” ini boleh menimbulkan risiko misleading or deceptive environmental claims kerana label “Better Cotton” mungkin memberi gambaran bahawa produk sepenuhnya mesra alam sedangkan ia sebenarnya boleh bercampur dengan cotton biasa yang tidak dapat dikesan secara fizikal. Ini boleh melanggar prinsip asas undang-undang perlindungan pengguna yang mewajibkan semua maklumat pemasaran adalah tepat, jelas dan tidak mengelirukan. Jika terbukti pengguna dipengaruhi oleh maklumat yang tidak lengkap atau kurang telus, syarikat boleh berdepan tindakan daripada badan pengawal selia, aduan pengguna, atau isu reputasi undang-undang.

I would like to offer the following comments. While Asia is socially important due to its large population, research on thermophilization in this region remains strikingly insufficient. In this context, I believe that studies detecting thermophilization based on reliable plot‑based data, such as those used here, are extremely valuable. I also agree with the detection that thermophilization is occurring in Japanese forest communities.

eLife Assessment

This important study presents a theoretically grounded framework for dimensionality reduction in single-cell RNA sequencing data, utilizing the principles of Riemannian manifolds. The proposed method addresses a critical challenge in bioinformatics-extracting highly informative latent dimensions without relying on the heuristic assumptions common in existing workflows. The evidence supporting the method's utility in estimating intrinsic dimensionality and identifying cell types is convincing, though the work would benefit from more rigorous validation against established ground truths and a clearer strategy for addressing prevalent batch effects.

Reviewer #1 (Public review):

Summary:

Sidarta-Oliveira et al. present TopOMetry, a novel dimensionality reduction method based on the eigendecomposition of approximated Laplace-Beltrami Operator. Shortly, TopOMetry is an iterative version of the existing spectral methods (e.g., Laplacian Eigenmap or Diffusion map). It approximates the Laplacian operators twice, once in a "phenotypic space" and then once again in the eigenbases space. By doing this the approximated operator will contain more information of the manifold, which allows for more robust and accurate downstream analyses.

Strengths:

- Introduces operator-native fidelity scores and Riemannian diagnostics to single-cell analysis, enabling researchers to evaluate and trust embeddings - functionality absent in prior methods.<br /> - The approach was rigorously tested based on synthetic and real single-cell RNA-seq datasets.<br /> - The package is well-made and easily scalable to millions of cells.<br /> - The comprehensive documentation helps the end-users to run desired analyses.

Weaknesses:

- The method is an extension of the current state-of-art methods, not a fundamentally new one.

Comments on revised version:

The revised manuscript partially addresses the concerns raised in the prior review. The jargon weakness has been substantially mitigated by relocating mathematical derivations to the Methods section and simplifying language in the main text; this weakness has been updated accordingly.

The introduction of operator-native fidelity scores and Riemannian diagnostics represents a meaningful addition and has been added to the Strengths. The benchmarking scope has also been notably expanded.

The core weakness - that the method is an extension of existing spectral methods rather than a fundamentally new contribution - remains unchanged, as the authors' rebuttal did not provide a sufficiently precise mathematical argument to overturn it.

Reviewer #2 (Public review):

Summary:

This work introduces a novel framework to systematically learn the latent dimensions of single-cell data, grounded in the theory of the Riemannian manifold. The authors demonstrate how this framework can be applied to various important tasks, such as estimating intrinsic dimensionalities, annotating cell types, etc. They did a great job of tackling an important but not yet established problem in the field and approaching it with a theoretically sound and novel approach. I think after a more rigorous and comprehensive validation, this work could be impactful.

Strengths:

- Dimensionality reduction is a routine step in analyzing many high-dimensional data, such as molecular data. While the downstream analysis results depend heavily on this step, existing methods rely on strong assumptions and are sometimes heuristic. The authors present a novel, theoretically grounded approach to address this important problem.

- The authors demonstrated its usability in downstream analysis in a comprehensive manner. Especially, they show evidence suggesting novel T-cell subpopulations.

- I commend the authors for releasing and maintaining their software well with comprehensive documentation. This significantly increases the usability and accessibility of the method.

Weaknesses:

- The paper lacks experiments that validate the results. It would be beneficial to see additional evaluation settings with better-established ground truths to more strongly demonstrate the method's effectiveness.

- Batch effects are prevalent in single-cell data. The paper does not adequately address how the proposed method handles this issue.

Author response:

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

Reviewer #1 (Public review):

(1) The method is an extension of the current state-of-art methods, not a fundamentally new one.

We respectfully disagree with this characterization. While TopoMetry is inspired by the theory of spectral geometry, it is not a simple extension of existing dimensionality reduction methods such as Diffusion Maps. Instead, TopoMetry introduces a new framework for single-cell analysis that:

Iteratively approximates manifold geometry by constructing refined diffusion operators on spectral scaffolds (“the geometry of the geometry”), a procedure not present in existing methods.

Provides a unified workflow for dimensionality estimation, clustering, visualization, imputation, lineage inference, and diagnostics, all within the same geometric framework.

Introduces operator-native fidelity scores and Riemannian diagnostics to single-cell analysis, enabling researchers to evaluate and trust embeddings—functionality absent in prior methods.

Thus, TopoMetry represents a new paradigm for geometry-aware single-cell analysis, not merely a reimplementation of existing algorithms.

(2) The paper contains a lot of jargon.

We have thoroughly simplified the text throughout the manuscript. We now introduce geometric concepts in accessible terms, avoiding technical details where they are not essential for biological interpretation. For example, references to the Laplace–Beltrami operator and its eigenfunctions have been reduced and reframed in terms of “geometry,” “diffusion,” and “spectral scaffolds,” which are more intuitive for a general audience.

Reviewer #1 (Recommendations for the authors):

(1) What happens if the LBO is approximated more than twice? As the main idea of the method is an iterative approach to approximate LBO more precisely, then the authors would have already considered this. If so, this could be additionally discussed in the manuscript.

We thank the reviewer for this important point. Indeed, TopoMetry’s design naturally supports iterating the Laplace–Beltrami operator (LBO) approximation beyond two steps. However, additional iterations (three or more) lead to only marginal improvements in final results while significantly increasing computational cost. In some tested cases, additional iterations could even over-smooth the data, reducing the resolution of fine-scale structure. The revised manuscript avoids an excessive focus on iterative LBO approximations and instead centers the narrative around representing and evaluating the underlying geometry of single-cell data.

(2) As the paper describes the method in a very comprehensive way, as a result, it contains a lot of mathematical equations and jargon. This could hinder the visibility of the whole manuscript to biologists who do not have a background in mathematics. Thus, I strongly recommend that the authors consider moving a considerable amount of text to the supplementary material, and the main text should focus on the benchmarking results and the possible applications.

We appreciate this recommendation and have substantially revised the manuscript to make it more accessible to a broad biological audience. In the revised version:

We moved detailed mathematical derivations and operator definitions to the Methods section, keeping only the most essential concepts in the main text.

We reframed technical terms (e.g., Laplace–Beltrami operator, eigenfunctions) in simpler and more intuitive language in the main text. 

The Results section now emphasizes benchmarking outcomes and biological applications.

Reviewer #2 (Public review):

(1) To encourage the single-cell community to adopt this method, the authors should more clearly demonstrate its advantages over existing methods. There are many single cell analysis algorithms that are proposed in each task and some of them are widely used by biologists. However, the comparison in this work is somewhat limited. For example, Even methods mentioned in the relevant work paragraph (2nd paragraph) on page 2 are not all compared, or the reason why they are not included is not discussed. Also, I am curious how PC dimensions are determined. The choice of 300 PCs on page 11 seems arbitrary. Furthermore, the usefulness of dimension-reduced data also depends a lot on the preceding processing steps, such as highly variable gene selection. I understand it is hard to control all those factors, but I think there is room for improvement.

We have substantially expanded the benchmarking and discussion of competing methods. These additions more clearly demonstrate TopoMetry’s advantages and robustness compared to widely adopted alternatives. In the revised manuscript:

We now benchmark TopoMetry against 68 diverse single-cell datasets, far exceeding the scope of the original version.

We explicitly compare TopoMetry with PCA→UMAP, standalone UMAP, and scVI. These workflows represent the de facto current standard in single-cell analysis. While numerous other approaches exist, a comprehensive benchmark of every possible workflow lies beyond the scope of this study and would itself warrant a dedicated report.

We adopt the exact same preprocessing steps for all evaluated workflows to ensure a fair comparison, except for scVI, which requires gene counts data and performs its own internal preprocessing.

We adjust the number of PCs used for each dataset based on the currently adopted “elbow point” ad hoc.

(2) The paper lacks experiments that validate the results. It would be beneficial to see additional evaluation settings with better-established ground truths to more strongly demonstrate the method's effectiveness.

We agree that validation is crucial and have strengthened this aspect:

We introduce new geometry-preservation metrics and validate that TopoMetry outperforms current de facto standards.

We demonstrate that TopoMetry resolves well-established ground-truth structures, such as the cell cycle in pancreas development and T cell proliferation, which PCA→UMAP fails to capture (Suppl. Fig. S3).

We validate the biological relevance of novel T cell subpopulations by linking them to TCR clonotypes and clonal expansion patterns using datasets with paired VDJ information (ECCITE-TCR, TICA).

We show that TopoMetry faithfully recovers expected lineage trajectories in atlas-scale datasets (MOCA).

These analyses demonstrate that TopoMetry not only preserves geometry but also recovers biologically meaningful ground-truth structures. Further experimental investigation of biological insights obtained from the presented examples exceeds the scope of the presented methodological work.

(3) The effect of various parameters, such as those involved in k-nearest neighbors (KNN) or choosing the appropriate Laplacian operator, is not comprehensively explored. How can we ensure the analysis is not overly sensitive to these parameters?

We now explicitly address parameter robustness and show that results are stable across a wide range of k values (30–200) in the neighborhood graph (Suppl. Fig. S1e).

The range of possible Laplacian operators was a design choice aimed at increasing user freedom, but we agree with the reviewer that this option could confuse readers and users. TopoMetry now only uses the appropriate operator (density-normalized graph Laplacian, a.k.a. diffusion operator), reducing variability and improving usability.

(4) Batch effects are prevalent in single-cell data. The paper does not adequately address this issue.

Several of the datasets we analyzed include cells from multiple donors and experimental batches, and TopoMetry successfully recovers consistent biological structure across these.

TopoMetry’s spectral scaffolds can be integrated with data integration methods such as Harmony and Scanorama, which are employed to correct the latent PCA space in current practice.

Reviewer #2 (Recommendations for the authors):

(1) The paper introduces technical jargon without sufficient explanation abruptly many times. This makes it difficult for readers from a biological background to follow. Even I, with a more computational background, struggled to grasp some parts.

We thank the reviewer for this feedback and have streamlined terminology throughout the manuscript, replacing jargon with more intuitive language and providing brief explanations when technical terms are first introduced. This makes the text more accessible to both computational and biological audiences.

(2) There is no comparison of the computational cost of this method with existing approaches, which is an important factor for practical adoption. Including a benchmarking section on this would be useful.

We thank the reviewer for this suggestion and have now included a runtime benchmark against PCA→UMAP, PHATE, and scVI (Suppl. Fig. 1f), showing that while TopoMetry is slightly slower than PCA→UMAP, it scales more favorably than alternative geometry-aware methods (PHATE) and neural networks (scVI).

(3) TopOMetry allows users to obtain and evaluate dozens of possible representations. However, I wonder if this could introduce a user burden, increasing uncertainty and subjectivity, as users should examine them manually. I think this should be clarified.

We appreciate this concern and have streamlined the workflow to minimize user burden. As shown in the original manuscript, representations learned with different TopoMetry kernels and Laplacian variants converge to highly similar results. Based on this, TopoMetry now defaults to the best-performing kernel and the most appropriate Laplacian operator, yielding only two scaffold representations (fixed-time and multiscale) and corresponding visualizations rather than dozens of alternatives. This removes the need for manual selection while retaining flexibility for advanced users. In addition, we introduced a single-line command that runs the entire analysis and generates a comprehensive PDF report, allowing users to evaluate results in a standardized and user-friendly way. Together, these changes eliminate unnecessary subjectivity and ensure consistent outputs across analyses.

(4) Formatting. There are errors in figure numbering within the main text. For instance, it should be Figure 4 instead of Figure 3 on page 11. Some figures are not concise. For example, Figure 2 contains too much text, which detracts from its visibility. I recommend trimming the figures to improve clarity. A color map is missing in Figure 2, which could help better interpret the data.

We have thoroughly adjusted the manuscript and figures for improved visibility and clarity.

Broader Impact and Reception

Since our preprint, TopoMetry has been used by Hale et al. (Science, 2024), where it helped reveal morphological T cell subpopulations, and in a recent preprint by Tedeschi et al. (2025). These independent applications highlight the utility and impact of TopoMetry beyond our group, supporting its relevance to diverse biological contexts. In addition, two independent studies performing multimodal integration of RNA and TCR data (Zhang et al., 2023 and Drost et al., 2024) have identified a diversity of T cell subpopulations that resembles the clusters identified by TopoMetry using only RNA data.

eLife Assessment

This study reports the relative importance of Tie1 and Tie2 signaling for atrial versus ventricular trabeculation. It is an important study and is one of the few works to date that have carefully and simultaneously analyzed these two processes. In line with a previous study in zebrafish, the authors demonstrate key differences between atrial and ventricular trabeculation. While the imaging and quantitative data were conducted with solid and validated methodology throughout the manuscript, the work would benefit from more rigourous approaches where Tie1/2 signaling is disrupted prior to the onset of atrial/ventricular trabeculation, to allow for a more direct comparison.

Reviewer #1 (Public review):

Summary:

In this manuscript, Ding et al. use genetic mouse models to demonstrate that atrial trabeculation is more dependent on Tie1/Tie2 signaling than ventricular trabeculation. With additional experimentation that would support the current claims, the results may hold significant value, as atrial trabeculation remains an understudied phenomenon in cardiac biology with potential implications for atrial cardiomyopathy and atrial fibrillation.

Strengths:

Detailed characterization of atrial versus ventricular trabeculation across different developmental timepoints, and the use of appropriate animal models to address the scientific question at hand.

Weaknesses:

The authors have consistently treated mice with tamoxifen after ventricular, but not atrial, trabeculation has already started. As such, the observed cardiac phenotypes - where predominantly atrial trabeculation is affected - might be a mere consequence of the precise time window in which Tie1/2 signaling was impaired, rather than a direct measurement of its relative importance for atrial versus ventricular trabeculation. The conclusions of the paper may thus be significantly strengthened by depleting Tie1/2 signaling prior to the onset of ventricular trabeculation, as is done for atrial trabeculation.

Reviewer #2 (Public review):

Summary:

Ding et al. examine the role of TIE1 in cardiac chamber morphogenesis using genetic mouse models targeting Tie1, Tek, or both, and analyzing endocardial cell-mediated chamber formation across multiple embryonic developmental and postnatal stages, supported by analysis of published single-cell datasets and new bulk RNA seq analyses of murine cardiac tissue. The authors find that Tie1 and Tek expression is higher in atrial than ventricular endocardial cells. Notably, endothelial Tie1 is required for atrial trabeculation at E12.5, but is less critical in ventricular trabeculation. TIE1 also acts synergistically with TIE2 during atrial trabeculation. While Tie1 deficiency alone does not cause defects at E10.5, combined heterozygous deletion of Tek disrupts both atrial and ventricular development at E10.5. This synergy is further supported by analyses at later embryonic stages and in postnatal hearts.

Strengths:

The study is well-designed, clearly written, and supported by high-quality figures. The performed experiments demonstrate a previously unrecognized role for Tie1 in cardiac development and identify synergistic control of cardiac morphogenesis by Tie1 and Tie2. This synergy is consistent with the previously identified roles of Tie1 and Tek in venous development and with Tie1 involvement in angiopoietin-dependent postnatal vascular and lymphatic remodeling. Together, these findings support a role for Tie1 as a contributor to Ang1-Tie2 signaling during heart development.

Weaknesses:

The manuscript does not include direct mechanistic studies; however, RNA seq analysis of atria and ventricles showed reduced expression of Tek, Dll1, and Notch1 upon Tie1 deficiency in developing hearts. Although previously reported mechanisms, such as TIE1-TIE2 heterodimer formation and effects on endothelial junctions, migration, or survival are discussed, no direct mechanistic experiments are performed. Addressing some of these mechanisms would have clarified the basis of Tie1-Tie2 synergy. As two distinct Tie1 models are used, including one targeting the kinase domain, the authors should state whether phenotypes differed or were similar between models.

Reviewer #3 (Public review):

Summary:

Ding et al. investigate the roles of TIE1 and TEK (Tie2) in mouse cardiac development, with a particular focus on atrial trabeculation. The authors employ multiple genetic models, including Tie1ICDflox/flox (with Cdh5-CreERT2), a knockout-first allele (EUCOMM, Tie1 tm1a/tm1a), and a Tek deletion model.

Based on the dataset from Feng et al. 2022 Nat Commun, the authors report increased expression of Tie1 and Tek transcripts in atrial endocardial cells compared to ventricular cells at embryonic day (E) 14.5. Loss of Tie1 leads to early atrial trabeculation defects detectable at E12.5, whereas ventricular defects appear later and are less pronounced at E14.5. Chamber-specific RNA sequencing reveals stronger transcriptional changes in atrial tissue.

Conditional deletion of Tek results in a similar phenotype, with more pronounced atrial defects. Combined deletion of Tie1 and Tek (Tie1 ΔICD/ΔICD; Tek+/-) leads to earlier and more severe defects in both atrial and ventricular trabeculation and results in embryonic lethality around E12.5, suggesting a synergistic interaction between the two genes.

Conditional endothelial deletion of Tie1 combined with heterozygous global Tek at later embryonic stages allows analysis at later time points and again shows more severe defects in atrial trabeculation. Postnatal analysis of this model reveals reduced heart-to-body weight ratios and potential mild atrial abnormalities.

Strengths:

(1) The authors address chamber-specific signaling mechanisms underlying atrial versus ventricular trabeculation, an area of high developmental and clinical relevance.

(2) The study provides a comprehensive temporal analysis across multiple embryonic stages.

(3) The use of multiple genetic models strengthens the overall conclusions and allows comparative interpretation.

(4) While focusing on trabeculation, the authors also include observations on coronary vessel development, increasing the broader relevance of the work. The findings are therefore of interest to the wider cardiovascular research community.

Weaknesses:

(1) Timing of recombination vs. trabeculation onset

Ventricular trabeculation begins earlier than atrial trabeculation. Since tamoxifen (in contrast to 4-hydroxytamoxifen) requires metabolic activation, Cre-mediated recombination will occur with a delay. This suggests that atrial trabeculation may be targeted before its onset, whereas ventricular trabeculation may already be underway for 2-3 days at the time of effective gene deletion.

How do the authors account for this discrepancy in their interpretation?

Have earlier induction time points been tested to better capture the onset of ventricular trabeculation? This limitation should be explicitly discussed.

(2) Clarity of genetic models and experimental design

The study employs several genetic constructs. It would improve clarity if, for each experiment, the specific genetic model and tamoxifen regimen were clearly described before presenting the results.

(3) Tie1 tm1a/tm1a phenotype vs. known global knockout

Previous studies (PMID: 8846781, 7596437) show that complete Tie1 loss leads to severe edema, vascular rupture, and embryonic lethality around E13.5-E14.5.

How does the Tie1 tm1a/tm1a allele differ, given that animals appear to survive longer? Is this allele hypomorphic rather than a full knockout?

This point requires clarification.

(4) Limited mechanistic insight

While the authors aim to investigate underlying mechanisms, the current study is largely descriptive and based on mRNA expression and genetic interaction analyses (Tie1/Tek co-deletion). Direct mechanistic insights into signaling pathways remain limited. However, the dataset provides a valuable foundation for future mechanistic studies, which should be more clearly acknowledged in the discussion.

Author response:

eLife Assessment

This study reports the relative importance of Tie1 and Tie2 signaling for atrial versus ventricular trabeculation. It is an important study and is one of the few works to date that have carefully and simultaneously analyzed these two processes. In line with a previous study in zebrafish, the authors demonstrate key differences between atrial and ventricular trabeculation. While the imaging and quantitative data were conducted with solid and validated methodology throughout the manuscript, the work would benefit from more rigourous approaches where Tie1/2 signaling is disrupted prior to the onset of atrial/ventricular trabeculation, to allow for a more direct comparison.

We thank the editors for the eLife assessment. We would like to request that the following statement be modified: “…the work would benefit from more rigourous approaches where Tie1/2 signaling is disrupted prior to the onset of atrial/ventricular trabeculation, to allow for a more direct comparison”. We request this change for the following reasons:

We utilized two distinct genetic mouse models in this study (as summarized in Fig. 7I), comprising conventional knockouts (Tie1^tm1a/tm1a, Tie1^ΔICD/ΔICD and Tie1^ΔICD/ΔICD;Tek^+/-) and inducible gene deletion models (Tek^iECKO, Tie1ICD^iECKO, and Tie1ICD^iECKO;Tek^+/-) [1-3]. The Tie1^tm1a/tm1a line is equivalent to the previously published Tie1<sup>-/-</sup mouse line, as demonstrated in our prior work and by others [1, 2, 4-6]. Therefore, the Tie1 or Tek alleles were inactivated prior to the onset of atrial and ventricular trabeculations, as shown in Fig. 1, Fig. 2, Fig. 3, Fig. 5A-D, and Supplemental Fig. 3. Based on these findings, we propose that TIE1 is differentially required for atria versus ventricle morphogenesis, and acts synergistically with TIE2 during cardiac trabeculation.

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this manuscript, Ding et al. use genetic mouse models to demonstrate that atrial trabeculation is more dependent on Tie1/Tie2 signaling than ventricular trabeculation. With additional experimentation that would support the current claims, the results may hold significant value, as atrial trabeculation remains an understudied phenomenon in cardiac biology with potential implications for atrial cardiomyopathy and atrial fibrillation.

Strengths:

Detailed characterization of atrial versus ventricular trabeculation across different developmental timepoints, and the use of appropriate animal models to address the scientific question at hand.

Weaknesses:

The authors have consistently treated mice with tamoxifen after ventricular, but not atrial, trabeculation has already started. As such, the observed cardiac phenotypes - where predominantly atrial trabeculation is affected - might be a mere consequence of the precise time window in which Tie1/2 signaling was impaired, rather than a direct measurement of its relative importance for atrial versus ventricular trabeculation. The conclusions of the paper may thus be significantly strengthened by depleting Tie1/2 signaling prior to the onset of ventricular trabeculation, as is done for atrial trabeculation.

We thank the reviewer for the comments.

Regarding the timeline of gene deletion and tamoxifen treatment, we would like to provide the following clarification.

Fig. 1-3: As described in the Methods and Materials, Tie1^tm1a/tm1a is a knockout first mouse model established from EUCOMM embryonic stem cells (EPD0735-3B07) targeting Tie1 gene. Therefore, the Tie1^tm1a/tm1a line is equivalent to the previously published Tie1 null mice (Tie1^-/-). The Tie1^Flox/Flox mouse line (with exon 8 floxed) was generated when the lacZ reporter and neo-cassette were excised using the FLPeR mice.

Fig. 5A-D: To investigate the synergy of TIE1 and TIE2 in cardiac trabeculation, we utilized the Tek^+/- and Tie1^ΔICD/+ mouse lines and they were crossbred to generate double mutant mice harboring a homozygous Tie1 mutation and a single null Tek allele (Tie1^ΔICD/ΔICD;Tek^+/-). Although no obvious defects were observed in atrial or ventricular structures following Tie1 deficiency alone at E10.5, both atria and ventricle development were disrupted in Tie1^ΔICD/ΔICD;Tek^+/- mutants at the same stage (Fig. 5A-D).

Supplemental Fig. 3: To verify the role of TIE1 in atrial development, we employed alternative knockout mouse line targeting the Tie1 intracellular domain by floxing exons 15 and exon 16 (Tie1ICD^Flox/Flox). Mutants harboring these null alleles are designated as Tie1^{ΔICD/ ΔICD}. As detailed in the previous publication [2], the line is also equivalent to the previously published Tie1 null mice (Tie1^-/-). The cardiac phenotypes shown in Supplemental Fig. 3 are indeed similar to those of Tie1^tm1a/tm1a mutant mice.

For the inducible knockouts targeting Tie1, Tek and both, the results are shown in Fig. 4, Fig. 5E-H, Fig. 6, Fig. 7.

Fig. 4: As mice homozygous for Tek mutation (Tek^-/-) die before E10.5 [3, 7], we performed studies using the inducible knockout line targeting Tek (Tek^Flox/-;Cdh5-Cre^ERT2 named as Tek^iECKO), as shown in Fig. 4.

Fig. 5-7: To investigate the synergy of TIE1 and TIE2 in the cardiac trabeculation at the later stages of embryogenesis (Fig. 5E-H, Fig. 6) and the postnatal stage (Fig. 7), we used the inducible knockout models targeting Tie1/Tek, including Tie1ICD^iECKO (Tie1ICD^Flox/-;Cdh5-Cre^ERT2) and Tie1ICD^iECKO;Tek^+/- (Tie1ICD^Flox/-;Cdh5-Cre^ERT2;Tek^+/-).

Reviewer #2 (Public review):

Summary:

Ding et al. examine the role of TIE1 in cardiac chamber morphogenesis using genetic mouse models targeting Tie1, Tek, or both, and analyzing endocardial cell-mediated chamber formation across multiple embryonic developmental and postnatal stages, supported by analysis of published single-cell datasets and new bulk RNA seq analyses of murine cardiac tissue. The authors find that Tie1 and Tek expression is higher in atrial than ventricular endocardial cells. Notably, endothelial Tie1 is required for atrial trabeculation at E12.5, but is less critical in ventricular trabeculation. TIE1 also acts synergistically with TIE2 during atrial trabeculation. While Tie1 deficiency alone does not cause defects at E10.5, combined heterozygous deletion of Tek disrupts both atrial and ventricular development at E10.5. This synergy is further supported by analyses at later embryonic stages and in postnatal hearts.

Strengths:

The study is well-designed, clearly written, and supported by high-quality figures. The performed experiments demonstrate a previously unrecognized role for Tie1 in cardiac development and identify synergistic control of cardiac morphogenesis by Tie1 and Tie2. This synergy is consistent with the previously identified roles of Tie1 and Tek in venous development and with Tie1 involvement in angiopoietin-dependent postnatal vascular and lymphatic remodeling. Together, these findings support a role for Tie1 as a contributor to Ang1-Tie2 signaling during heart development.

Weaknesses:

The manuscript does not include direct mechanistic studies; however, RNA seq analysis of atria and ventricles showed reduced expression of Tek, Dll1, and Notch1 upon Tie1 deficiency in developing hearts. Although previously reported mechanisms, such as TIE1-TIE2 heterodimer formation and effects on endothelial junctions, migration, or survival are discussed, no direct mechanistic experiments are performed. Addressing some of these mechanisms would have clarified the basis of Tie1-Tie2 synergy. As two distinct Tie1 models are used, including one targeting the kinase domain, the authors should state whether phenotypes differed or were similar between models.

We thank the reviewer for the comments. In this study, we have provided genetic evidence that TIE1 is differentially required for atrial versus ventricular trabeculation. Although the precise molecular mechanisms underlying TIE1 function require further investigation, we have provided compelling genetic evidence of its synergistic role with TIE2 during this process. The two genetic models targeting Tie1 (Tie1^tm1a/tm1a, Tie1^ΔICD/ΔICD) produced consistent cardiac and vascular phenotypes as shown in this study and our previous work [1, 2].

Reviewer #3 (Public review):

Summary:

Ding et al. investigate the roles of TIE1 and TEK (Tie2) in mouse cardiac development, with a particular focus on atrial trabeculation. The authors employ multiple genetic models, including Tie1ICDflox/flox (with Cdh5-CreERT2), a knockout-first allele (EUCOMM, Tie1 tm1a/tm1a), and a Tek deletion model.

Based on the dataset from Feng et al. 2022 Nat Commun, the authors report increased expression of Tie1 and Tek transcripts in atrial endocardial cells compared to ventricular cells at embryonic day (E) 14.5. Loss of Tie1 leads to early atrial trabeculation defects detectable at E12.5, whereas ventricular defects appear later and are less pronounced at E14.5. Chamber-specific RNA sequencing reveals stronger transcriptional changes in atrial tissue.

Conditional deletion of Tek results in a similar phenotype, with more pronounced atrial defects. Combined deletion of Tie1 and Tek (Tie1 ΔICD/ΔICD; Tek+/-) leads to earlier and more severe defects in both atrial and ventricular trabeculation and results in embryonic lethality around E12.5, suggesting a synergistic interaction between the two genes.

Conditional endothelial deletion of Tie1 combined with heterozygous global Tek at later embryonic stages allows analysis at later time points and again shows more severe defects in atrial trabeculation. Postnatal analysis of this model reveals reduced heart-to-body weight ratios and potential mild atrial abnormalities.

Strengths:

(1) The authors address chamber-specific signaling mechanisms underlying atrial versus ventricular trabeculation, an area of high developmental and clinical relevance.

(2) The study provides a comprehensive temporal analysis across multiple embryonic stages.

(3) The use of multiple genetic models strengthens the overall conclusions and allows comparative interpretation.

(4) While focusing on trabeculation, the authors also include observations on coronary vessel development, increasing the broader relevance of the work. The findings are therefore of interest to the wider cardiovascular research community.

Weaknesses:

(1) Timing of recombination vs. trabeculation onset

Ventricular trabeculation begins earlier than atrial trabeculation. Since tamoxifen (in contrast to 4-hydroxytamoxifen) requires metabolic activation, Cre-mediated recombination will occur with a delay. This suggests that atrial trabeculation may be targeted before its onset, whereas ventricular trabeculation may already be underway for 2-3 days at the time of effective gene deletion.

How do the authors account for this discrepancy in their interpretation?

Have earlier induction time points been tested to better capture the onset of ventricular trabeculation? This limitation should be explicitly discussed.

(2) Clarity of genetic models and experimental design

The study employs several genetic constructs. It would improve clarity if, for each experiment, the specific genetic model and tamoxifen regimen were clearly described before presenting the results.

We thank the reviewer for the detailed and constructive comments. For studies employing the inducible gene deletion mouse models, the genetic models and tamoxifen treatment schemes have been provided in the related figures. For the rest of studies, we used the conventional knockouts targeting Tie1 and Tek (Tie1^tm1a/tm1a, Tie1^ΔICD/ΔICD and Tie1^ΔICD/ΔICD;Tek^+/-), as detailed above.

(3) Tie1 tm1a/tm1a phenotype vs. known global knockout

Previous studies (PMID: 8846781, 7596437) show that complete Tie1 loss leads to severe edema, vascular rupture, and embryonic lethality around E13.5-E14.5.

How does the Tie1 tm1a/tm1a allele differ, given that animals appear to survive longer? Is this allele hypomorphic rather than a full knockout?

This point requires clarification.

Tie1^tm1a/tm1a is equivalent to the full knockout (Tie1^-/-). As demonstrated in our prior work, the Tie1^ΔICD/ΔICD model produced lymphatic and blood vascular phenotypes similar to those of Tie1^-/- mutants [1, 2, 5, 6].

(4) Limited mechanistic insight

While the authors aim to investigate underlying mechanisms, the current study is largely descriptive and based on mRNA expression and genetic interaction analyses (Tie1/Tek co-deletion). Direct mechanistic insights into signaling pathways remain limited. However, the dataset provides a valuable foundation for future mechanistic studies, which should be more clearly acknowledged in the discussion.

We thank the reviewer for the comments. The manuscript will be revised accordingly, and a detailed response will be provided in our final submission.

Reference

(1) Cao, X., et al., Endothelial TIE1 Restricts Angiogenic Sprouting to Coordinate Vein Assembly in Synergy With Its Homologue TIE2. Arterioscler Thromb Vasc Biol, 2023. 43(8): p. e323-e338.

(2) Shen, B., et al., Genetic dissection of tie pathway in mouse lymphatic maturation and valve development. Arterioscler Thromb Vasc Biol, 2014. 34(6): p. 1221-30.

(3) Chu, M., et al., Angiopoietin receptor Tie2 is required for vein specification and maintenance via regulating COUP-TFII. Elife, 2016. 5:e21032.

(4) Rodewald, H.R. and T.N. Sato, Tie1, a receptor tyrosine kinase essential for vascular endothelial cell integrity, is not critical for the development of hematopoietic cells. Oncogene, 1996. 12(2): p. 397-404.

(5) D'Amico, G., et al., Loss of endothelial Tie1 receptor impairs lymphatic vessel development-brief report. Arterioscler Thromb Vasc Biol, 2010. 30(2): p. 207-9.

(6) Qu, X., et al., Abnormal embryonic lymphatic vessel development in Tie1 hypomorphic mice. Development, 2010. 137(8): p. 1285-95.

(7) Dumont, D.J., et al., Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo. Genes Dev, 1994. 8(16): p. 1897-909.

These people will be behind the curve when it comes to GAI and how it can apply in a meaningful way in education.

This pre-print is now published in a peer-reviewed journal: https://doi.org/10.1111/psyp.70271

However, a match between the pre-print and the publication is not visible in bioRxiv.

DOI: 10.1038/s41419-026-08701-5

Resource: The Cancer Genome Atlas (RRID:SCR_003193)

Curator: @scibot

SciCrunch record: RRID:SCR_003193

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Reply to the reviewers

Please see the uploaded "Response to Reviewers" PDF file which contains figures.

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Referee #2

Evidence, reproducibility and clarity

In the manuscript titled "Multiple Molecular Pathways to Longevity: Opposing Gene Expression Programs Define Distinct Aging Strategies", the authors investigated diverse genetic pathways that contribute to lifespan extension in Caenorhabditis elegans and aimed to identify shared and distinct molecular mechanisms among various longevity mutants. Through comprehensive RNA sequencing of different longevity mutants representing seven distinct pathways, the authors showed that these mutants cluster into three primary groups based on their gene expression profiles. This transcriptomic analysis revealed that while some longevity genes are commonly regulated across multiple pathways, others exhibit opposing expression patterns, suggesting that distinct molecular strategies can lead to increased lifespan. Specifically, they identified a set of 196 genes that are consistently upregulated in most longevity mutants, many of which are involved in innate immunity and stress defense. By performing RNAi-based screening, the authors further validated the functional roles of several candidates, including C08F11.7, ugt-62, and K05C4.9, supporting their contributions to longevity and stress resistance. The authors conclude that longevity is mediated through multiple molecular pathways and provide a public online tool to study these complex transcriptomic landscapes.

Major comments

1. While the authors identified a set of 196 upregulated genes, the rationale for narrowing these down to the three final candidates (C08F11.7, ugt-62, and K05C4.9) is not clearly described. The authors show that genetic inhibition of several genes, including DC2.5, C05B5.5, T07C4.5, and W03B1.7, decreases lifespan in both nuo-6 mutants and wild-type animals. However, the authors did not describe why these additional validated candidates, which also showed significant effects on longevity, were not pursued for further characterization. The authors should explicitly state the criteria used to prioritize these three genes over the other validated genes.
2. The authors conclude that longevity can be mediated by multiple molecular pathways. However, it remains unclear whether these distinct strategies can operate simultaneously or are mutually exclusive. The authors need to test whether lifespan extension in a Group 1 mutant is further enhanced or suppressed by the knockdown of a key Group 2-specific genes. These experiments would help determine these pathways act additively, antagonistically, or as partially redundant survival programs.
3. The authors provide interesting data on overexpression of the three candidate genes. However, whereas C08F11.7 clearly demonstrates both necessity and sufficiency for lifespan extension, overexpression of ugt-62 and K05C4.9 does not independently extend lifespan. To strengthen the manuscript, the authors should expand the discussion of these divergent results and clarify possible explanations.
4. Key citations are missing and the authors should add multiple citations including the following ones. Please cite the following paper and discuss the authors' finding with respect to the related work (Lee et al PMID: 40814218). Add citations in the sentence describing changes in the transcriptome of C. elegans associated with age (Lee et al., PMID: 38508494). Furthermore, please cite papers describing the overviews of survival assay using C. elegans (Kwon et al., PMID: 40436148, Hwang et al., PMID: 40436147).

Minor comments

1. To improve readability, please provide the full names for all abbreviations at their first appearance in the manuscript.
2. Please ensure that the labels in the figures match the text exactly. For instance, if different promoters are used for generating overexpression animals, it may be helpful to indicate the specific promoter in the figure panel or legend for clarity.
3. For all lifespan and stress resistance assays, please include the total number of animals (n) and the number of independent biological replicates (N) in the figure legends to confirm statistical reliability.
4. Please clearly specify the exact developmental stage of the animals used for the survival assays in the Materials and Methods section.

Referees cross-commenting

I also agree with reviewer #1's comments and recommend revision to further improve the manuscript.

Significance

This study provides a systematic, side-by-side transcriptomic comparison of nine genetically distinct long-lived C. elegans mutants, revealing that lifespan extension arises from both shared and opposing gene expression programs. By identifying three distinct longevity groups and demonstrating that key pathways can be modulated in opposite directions to achieve long life, the work challenges the notion of a single universal transcriptional signature of aging. Importantly, functional validation shows that select commonly regulated genes can directly modulate lifespan and stress resistance, highlighting actionable molecular targets for promoting healthy aging.

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Referee #1

Evidence, reproducibility and clarity

Summary

This manuscript by Rudich ZD et al. systematically profiled the transcriptomic changes in nine long-lived C. elegans mutants and presented a careful and informative comparative analysis of these aging-related changes. In addition to these valuable datasets and bioinformatics analyses, the authors performed a large-scale RNAi screen to assess the role of the differentially expressed genes (DEGs) in these mutants and identify several potential targets to promote healthy aging. Moreover, the authors have provided a user-friendly website to examine genes of interest in those longevity mutants from their datasets.

Major comments

The conclusions of this manuscript are generally well supported. The study is also technically sound. Yet, I still have a few concerns that should be carefully addressed.

1. Although I myself believe that the datasets in this study should be more consistent and comprehensive, the authors should perform a data mining analysis of previously reported transcriptomic changes of these mutants or similar mutants in the same longevity pathway and compare the reported changes with their findings to highlight the necessity and advances of this study.
2. This manuscript does not perform any regulon or transcription factor (TF) analyses. TFs are the drivers of the transcriptomic changes and multiple conserved TFs (e.g., daf-16) have already been identified in these pathways. Therefore, it is necessary to examine and compare the regulons/TFs in these new datasets by bioinformatics. Such analyses can: a) provide more information of the driving force of these transcriptomic changes; b) show the role of these known longevity TFs; c) propose new TFs driving longevity; d) support the findings of 'longevity strategies' and 'longevity groups' from the perspective of TFs.
3. osm-5 and daf-2 are categorized into two different groups in this study. Since the longevity of cilia (-) mutants is through daf-16, the same master TF driving daf-2 longevity, please perform further analyses or discussion to clarify this issue.
4. This manuscript focused on genes whose RNAi suppressed the mutants longevity. Please also use bioinformatics to analyze the functions of those whose RNAi extends the mutants longevity, because these genes could tell the health price these mutants pay and help improve ageing interventions by reducing side effects.
5. (OPTIONAL) I strongly suggest a comprehensive comparison of these transcriptomic changes in long-lived mutants with published age-related transcriptomic changes in wild type worms. This comparison will significantly All the suggested analyses are pure bioinformatics and should be realistic to finish in several months.

Minor comments

I also have a few minor comments on data presentation: 1. Please further clarify the analysis of DEGs correlated with lifespan extension in Fig. 2 by a depiction. In Fig. 2C and D, please label data dots from different strains with different colors. 2. In Fig. 3 and S20, please label the percentage of overlapping genes on top of each bars.

Referees cross-commenting

I agree with Reviewer #2's comments and would suggest giving the authors enough time to revise their manuscript.

Significance

Compared to previous transcriptomic analyses of these mutants in differen reports, this study minimized the technical variations and benefitted from the advances in RNA-Seq technology and bioinformatics tools. Therefore, it should provide a more consistent and comprehensive view of the molecular mechanisms underlying the longevity of these mutants. The datasets in this manuscript are valuable to other researchers in the biology of aging.

Meanwhile, since these mutants have been extensively studied, the advance of this study in unknown mechanisms remains limited. Therefore, I would recommend its publication as a 'Resource' article after addressing my concerns.

(I am an expert in the biology of ageing, using C. elegans and mouse as major models.)

This article mentions how doxing means publicly sharing someone's personal info without consent and has grown from a niche hacker tactic in the 90s into a widespread form of online harassment. It covers real examples like Gamergate, the Boston Marathon bombing misidentifications, and journalists revealing anonymous public figures. It also touches on how laws around doxing are pretty limited, especially in the US.

I have witnessed harassment on social media, my friend was being harassed by a kid at our school and everytime she would block them, they would make a new account. It got to the point where we had to tell the staff at our school.

Greenwashing happens when an organisation claims to be environmentally friendly but does not provide enough proof or action to support the claim. In UUM cafés, the “no-plastic” campaign is a positive step because it encourages students to reduce single-use plastic. However, the campaign may become greenwashing if cafés still use plastic cups, food containers, or non-recyclable packaging while promoting themselves as environmentally friendly. Therefore, cafés should provide clearer evidence and specific actions to make the campaign more credible and transparent.

Enforced configuration là cấu hình được hệ thống bắt buộc thực thi bất kể AI quyết định gì.

CLAUDE.md chỉ là text, được đưa vào context window như một tin nhắn.(có thể làm theo, quên, hoặc hiểu sai.)

I would ordinarily ask to define "good enough" -- but to be fair "good enough" here is in the context of marketing.

The specification of 'cinematic' video generation implies a deep, model-inherent understanding of professional filmmaking principles like shot composition, pacing, and narrative structure. This goes beyond simple video creation into the realm of professional content production.

The claim to 'design advanced algorithms' for mathematics and computing places this model in a meta-cognitive category. It's not just solving problems but creating new methodologies, positioning it as a potential co-architect for future AI and scientific discovery.

The phrase 'learns with you' is a subtle but powerful deviation from standard AI terminology. It implies a collaborative, co-evolutionary learning process rather than a one-way training dynamic, suggesting a more human-like interactive agent.

The explicit inclusion of 'use tools' alongside core cognitive functions like 'perceive' and 'reason' highlights a significant architectural focus on embodied AI. This suggests the model is being designed with a direct path to physical agency, a non-obvious but critical distinction.

This demonstrates the model's capacity for complex, structured problem-solving. To apply this, frame your prompts around a specific problem, provide all necessary constraints and requirements, and ask the model to design a step-by-step solution or algorithm.

This suggests a focus on complex, multi-step reasoning and tool use. To apply this, structure your prompts as a sequence of tasks or a workflow, where the model must first perceive information, then reason, and finally decide on a tool or action to take.

This points to the model's specialized audio generation. To apply this, provide specific prompts that reference musical genres, instruments, tempo, and mood to guide the creation of high-fidelity audio outputs.

This underscores the importance of detailed language for visual generation. To apply this, use rich, evocative language in your prompts, specifying lighting, composition, style, and subject details to achieve the desired image quality.

This highlights the model's advanced creative capabilities. To apply this, be highly descriptive in your prompts, specifying mood, shot type, pacing, and audio cues to guide the model towards producing a specific cinematic result.

This indicates the model's strength in creating agents with specific roles and behaviors. To apply this, use persona prompting by defining a character, its expertise, its communication style, and its goals before asking it to perform a task.

This tagline suggests a core capability: use diverse inputs to generate diverse outputs. To apply this, pair unexpected modalities in your prompt, such as asking the model to generate a poem based on a data table or a musical score from a photograph.