ditches (NL: sloten)

sluices

Lake IJssel (?)

Komma invoegen na "supply".

Komma weghalen.

Chapter

Voeg "potential" toe na "irrigation".

flood

delicate (?)

Voeg "the" toe na "of".

flood

flood

flood

dampen

Actual

capacity

e

are

Subscripts "max" en "des" rechtop ipv cursief.

over

months

subject

Subscript "max" rechtop ipv cursief; is er geen netter "kleiner of gelijk"-teken?

Subscript "max" rechtop ipv cursief.

Subscript "max" rechtop ipv cursief; is er geen netter "kleiner of gelijk"-teken?

Welk "voorbeeld"?

Subscript "max" rechtop ipv cursief; ">=" vervangen door ">".

Kan weg.

In deze sectie over reservoirs wordt consequent "I" voor instroom en "O" voor uitstroom gebruikt. Dat moet hier (in deze symbolenlijst) dan ook worden aangepast.

Voeg "required" toe voor "capacity".

probability

such as to

via

over

inflowing

determined

"capacity" kan weg. Niet de "bergingscapaciteit" neemt af, maar de "berging" zelf.

(tangent) lines (NL: raaklijnen)

Subscript "max" rechtop schrijven ipv cursief.

Komma toevoegen na "curve".

In het linkerlid van de vergelijking moeten de d's rechtop geschreven worden ipv cursief; in het rechterlid een subscript "1" toevoegen bij S.

:

In het linkerlid van de vergelijking moeten de d's rechtop geschreven worden ipv cursief.

De term "yield" wordt eerst geintroduceerd, maar vervolgens wordt toch meestal de term "delivery capacity" gebruikt. Waarom?

should only

probability

Volgens mij is de effectieve berging gewoon de hoeveelheid water die geborgen kan worden tussen de dode berging en de noodoverlaat, dus onafhankelijk van de oorspronkelijke berging. Graag even controleren in overleg met Ruud.

Subscript "max" in roman font ipv italic font.

Subscript 1 bij integratielimiet h1 in vergelijking (2.13).

The welfare policies were built to support men and center around men. It seems as if women can never in the eyes of the law be the head of a household without a man, reinforcing the need to marry in order to be economically independent while caring for children.

WHAT THE ACTUAL FUCK

This source talks about a syndrome that I have not heard about. The symptoms are extreme fatigue that lasts at least 6 months. The cause of it is unknown and there is not 1 defining test to determine if you have it. This can be scary because you can brush this off as just being constantly tired from your daily life.

In this article, Alannah Oleson discusses the CIDER technique, which aims to teach inclusive design skills by guiding people through five stages of analytical design evaluations. Going beyond assumptions about "average" users, CIDER encourages the considering of diverse user needs. CIDER stands for Critique, Imagine, Design, Expand, Repeat. The article delves into each type of evaluation in depth and encourages practicing the CIDER activity. This practice can have positive impacts on people's knowledge of inclusive design and improve critical thinking about inclusive technology.

This video explains what is Executive Function and why do ADHD people need it. Executive Function is a process that helps self-regulate so we can effectively plan, organize, sustain our long term goal.

The article discusses Tetrachromatopsia, a rare genetic disorder that enables some people, primarily women, to see multiple colors that most people do not. Concetta Antico, an artist with this disorder, experiences and represents this vivid spectrum in her paintings, offering a unique view of the world that highlights the differences in human color perception. Concetta Antico, who has tetrachromatic iridism, sees richer colors in everyday objects, such as on a cobblestone trail, where she feels the cobblestones shine like jewels displayed by a jeweler in vivid oranges, yellows, greens, blues, and pinks-colors normally invisible to normal eyes.

This is a good point that let people who share the same feeling to use the same social media. In general social media, most of us who haven't experienced such pains may not be able to compassion those people in the same way as those who experienced can. Thus such communication approch would be good.

installed it on Peergos

This reminds me of an analogy that I've heard about the role of UX designers and software engineers. UX designers are the architects and software engineers are the construction workers that build and make that vision come to life. Both are essential for a user's experience, especially in the lens of accessibility and making sure underrepresented groups are heard.

Yes. I really agree with this. If there is no standard rule of making new technologies. Most new technologies will start to innovate and target on large groups of people, and when they are aware of making their technologies, they give engineer a lot of pressure and work.

Persons with disabilities often “mask” their challenges to fit into environments that do not provide accommodations, a practice that can lead to stress and mental health issues. They may also seek roles that match their abilities, highlighting the need for more inclusive environments that recognize and support diverse ways of working.

I've heard about this in designing multiple times. However, I don't think it is possible to create a platform or environment that have options for everyone. There must be someone that we could not empathize on. In Utilitarian approach, as long as our design maximize its potential of usage in groups of people, it should work.

One way design can be more inclusive and diverse is through making it more accessible for those who are color blind. Individuals with color blindness face challenges.

I find learning about this topic quite interesting. As I aspire to be a UX Researcher, I believe it's essential to understand the ethical and moral obligations that come with this role. While UX Research may not directly involve designing programs, this text does raise questions about the ethical considerations involved. For instance, how could designers justify creating a soap dispenser that only operates when it detects light skin? As an aspiring UX Researcher, I'm eager to explore how we can ensure that our designs prioritize inclusivity and accessibility. How can we ensure that users with disabilities are not marginalized but instead empowered by our designs?

After scanning through the article, I am looking back at the title to begin my active reading stage. I think that Ann's intended audience might be an academic audience. Perhaps it is one specifically interested in the study of communication. Or, maybe the audience is broader than this and it is she that has a specific interest in Communication through a medium of text and how it can be explored through podcasts as a medium.

Possible Problem with Cleavers Analysis of Frankfurt: Marcuse apparently evolved from the position he held in One dimensional man. https://marxandphilosophy.org.uk/reviews/8113_the-frankfurt-school-postmodernism-and-the-politics-of-the-pseudo-left-review-by-javier-sethness/ Eric commits the common fallacy of reducing “the Frankfurt School” to One-Dimensional Man. Please reread my words: ‘by the end of the same decade, he had jettisoned such pessimism. In An Essay on Liberation (1969), Marcuse clarifies his belief that the proletariat retains its revolutionary role, amidst the “historical power of the general strike and the factory occupation, of the red flag and the International” (Marcuse 1969, 51-3, 69).’

Cleaver backs up the view of Marcuse as being pessimist on page 56 using Marcuse's later work Counter Revolution and revolt which came out in 1972. One dimensional man came out in 1964 so it does not seem like he evolved.

But in the comment section from above I found this: I am reading the 1974 Paris Vincennes Lectures. These do indeed show a very different side to Marcuse. “Which are today the social and historical agents of radical change in the United States ? First among the working class… I never said that the working class can be replaced by any other class in the transition from capitalism to socialism.”...In fact the Vincennes Lectures make it seem that class struggle is more than just one current but is THE absolutely essential current (into which the other currents must flow in order to be historically effective). So this is very interesting indeed. It’s very important that these Vincennes Lectures become better known.

Wiki makes it seem like an interesting read.

Solicitar, ajuizar, requerer

A cláusula compromissória é a convenção através da qual as partes em um contrato comprometem-se a submeter à arbitragem os litígios que possam vir a surgir, relativamente a tal contrato.

in transactions for which override was requested.

don't need 'details" (it just tracks the transaction identifier not any of its details)

Is "Detail" needed? There are no master detail objects to differentiate.

why deviate from data set

Add Individualized Success: before, like this:

Individualized Success: Zoni American High School offers diverse course options, including electives, credit recovery, AP, and honors courses, providing tailored learning experiences for students.

With design I believe that inclusivity and diversity are the two things that should always be prioritized. Having considered diverse perspectives and needs will underscore the broader issue.

Apple supposedly slashed production for their 3k5 USD ski goggles, a sign they may soon be joining the long list at https://en.wikipedia.org/wiki/List_of_virtual_reality_headsets of attempts that all don't solve the key thing: adding 1kg of ski goggles to your head and shove it between yourself and the world is only acceptable in a very limited set of contexts and uses, and not a mainstream thing.

No hay centro inspiratorio y espiratorio separados

Thomas Clap, An Essay on the Nature and Foundation of Moral Virtue and Obligation: Being a Short Introduction to the Study of Ethics: For the Use of the Students of Yale-College (New Haven, CT: B. Mecom, 1765), 1.

https://globe.engineer/

Found by JM

Github https://github.com/Globe-Engineer -> Ivan Yevenko https://www.linkedin.com/in/iyevenko/

?

reply to u/EquivalentHead3589 at https://www.reddit.com/r/typewriters/comments/1cbzx1n/how_do_you_price_typewriters/

The primary difference is that listing prices don't indicate actual value. That is only determined by actual sales price. Things are worse for the listings which don't indicate much about condition as you're probably more likely to need to have the machine serviced and/or replace or recondition parts. This can often add a few hundred dollars (or significant research and time, tools, and elbow grease) to the bottom line to be able to use a machine.

I do recall a burgundy Olympia SM3 which sold in the last 4 months for right at $300 which was regularly used (loved) and serviced and in excellent condition with some fantastic photos. If you compare it to this Burgundy/Gray machine (https://www.ebay.com/itm/404901285037) for $299, but which has a missing key cap, and a damaged case, and may likely have other hiding issues. If you consider that you'll likely need to put a minimum of another $100 into this to get it up to the fighting shape that the first was in and it's still got damage, you'll start seeing the stark difference. The people with listings at $550-800 know they're not selling and they're just sitting there, so why not email them and ask more specific questions about condition and get a typed typeface sample of all the keys. Then make an offer for $200 +/- with some wiggle room for service costs once you've gotten it to see if they'll sell?

As an example, look at https://www.ebay.com/itm/226016437104 which is a Gray SM3 originally listed for $549 and now on sale for $428. The seller knows it's not moving. They state that they got it at an estate sale (probably for around $25) and they definitely did no work other than quick check of the keys. If you demonstrate that you've savvy enough to know the specific machine (what shape are the rubber washers on the frame next to the feet to prevent the carriage from rubbing against the frame? how what is the durometer measurement on (how hard is) the platen?), the market (in top shape maybe $300), and what servicing/repair costs are, they'd probably accept an offer of $150-200 and you're off to the races and they've made a solid profit.

The biggest issue in the typewriter market at present is the broad lack of information and knowledge about them on both the buyer and seller side. If you can demonstrate you've got more knowledge than the other side, you'll be in a far better position to negotiate, otherwise a seller can sit and wait an undetermined amount of time waiting for a sucker who will likely never show up.

my name was supposed to be josh hutcherson

Can you blow my whistle baby, whistle baby Let me know Girl I'm gonna show you how to do it And we start real slow You just put your lips together And you come real close Can you blow my whistle baby, whistle baby Here we go Look, I'm bettin' you like people And I'm bettin' you love freak mode And I'm bettin' you like girls That give love to girls and stroke your little ego I bet you I'm guilty, your honor That's just how we live in my genre Who in the hell done paved the road wider? There's only one Flo and one Rida I'm a damn shame, order more champagne Pull a damn hamstring tryna put it on ya Let your lips spin back around corner Slow it down, baby, take a little longer Can you blow my whistle baby, whistle baby Let me know Girl I'm gonna show you how to do it And we start real slow You just put your lips together And you come real close Can you blow my whistle baby, whistle baby Here we go (hey) Whistle baby, whistle baby (hey) Whistle baby, whistle baby (hey) Whistle baby, whistle baby (hey) Whistle baby, whistle baby It's like everywhere I go, my whistle ready to blow Shawty don't even know, she can get any for the low Told me she's not a pro, it's okay, it's under control Show me soprano 'cause, girl, you can handle Baby, we start slow then you come up and park close Girl, I'm the whistle man, my Bugatti the same notes Show me your perfect pitch, you got it, my banjo Talented with your lips like you blew out a candle So amusing (amusing) Now you can make a whistle with the music (music) Hope you ain't got no issues, you can do it (do it) Even if it's no picture, never lose it (lose it) Can you blow my whistle baby, whistle baby Let me know Girl I'm gonna show you how to do it And we start real slow You just put your lips together And you come real close Can you blow my whistle baby, whistle baby Here we go (hey) Whistle baby, whistle baby (hey) Whistle baby, whistle baby Whistle baby, whistle baby Whistle baby, whistle baby Go on, girl, you can twerk it Let me see you whistle while you work it I'ma lay back, don't stop it 'Cause I love it how you drop it, drop it, drop it on me Now, shorty, let that whistle blow, oh-oh-oh Yeah, baby, make that whistle blow, oh-oh Can you blow my whistle baby, whistle baby Let me know Girl I'm gonna show you how to do it And we start real slow You just put your lips together And you come real close Can you blow my whistle baby, whistle baby Here we go You blow my whistle baby Whistle baby, whistle baby You blow my whistle baby Whistle baby, whistle baby You blow my whistle baby Whistle baby, whistle baby You blow my whistle baby Whistle baby, whistle baby

https://www.kleinezeitung.at/steiermark/18401298/tu-graz-wirft-letzter-generation-besitzstoerung-und-sachbeschaedigung

.

adjacency - between - Ernest Becker - book - The Birth and Death of Meaning - Eastern meditation to interrogate sense of self - adjacency statement - Becker writes and speculates about the anthropology and cultural history of the origin of the self construct - It is a fascinating question to compare Becker's ideas with Eastern ideas of dissolving the constructed psychological self

Interesting minimalistic alternative to zoxide

TikTok has become a source of income for many Americans, but also isn't the only social media platform that has security issues

Implies that Democrats don't believe, even if voters aren't happy about the bill, they will still support the party because of other "bigger" issues

Going to the other side and examining alternative argument - no one is innocent in this issue

Goes against other statements in the article, potentially utilized to create controversy/debate

Interesting argument without a lot of data to back it up

Way statements are phrased seem to imply that Democrats wanted to extend the timeline in order to keep control of office

Statement from Democratic Senator - interesting that he doesn't mention anything about Tik Tok's global markets, since it is an international platform.

This is also interesting because not only did Biden give more time for himself to be able to use the platform, but he also gave himself a caveat to potentially extend it/get rid of it if necessary.

In another article I annotated, it discussed how the Biden administration was using Tik Tok as a campaign resource, so this is an interesting choice to make to not only appeal to the youth, but also to continue his own campaigning on the platform.

The headline uses punctuation to emphasize a certain part of the message. In this case, the writer seems to want to emphasize that Biden has not only passed a bill that could ban Tik Tok, but that it would take effect either after he is reelected or not in office anymore.

test 1

Or [Shift] for consecutive machines or groups.

This observation is not specific when choosing an MbA job mode, as it is common to every mode. Perhaps this remark should be left on the page where user decide between MbA and MbVBR.

I did not know or even think about doing this. Does this really work or does it cause more problems in the long run?

I have always made the joke that our phone and other devises are listening to us, but come to find out this is true. we can look something up on google and now we are getting adds for it on Facebook. Honestly, this is kind of scary if we really think about it. our phones are keeping tabs on us and figuring out what we like and consuming our "phone life" around it.

Yes I do know that when we download an app we are giving them permission to almost everything if not everything as soon as we download it. I also know that in our phone settings we have the option to deny the app access to somethings, like our location, contacts, and someother things..

4/24/2024

Uniform the title with the recipe

Summary of "Towards a Unified Theory of Reactive UI" by Raph Levien (November 22, 2019)

• Overview and Motivation:

  • Levien explores various reactive UI frameworks to develop a cohesive understanding and communicate effective strategies for the Druid UI system.
  • Reactive UI allows UI construction and updates to be more intuitive and less redundant compared to traditional object-oriented approaches.
  • "My own main motivation for exploring reactive UI is primarily because the object-oriented idioms don’t translate well to Rust" – This highlights the shift towards reactive UI due to language and paradigm limitations in Rust concerning ownership and callbacks.

• Theoretical Framework and Tree Transformations:

  • Reactive UIs fundamentally involve transforming UI component trees across various stages: user data → widget tree → render object tree → draw tree.
  • Levien discusses tree representation either as a data structure or as an execution trace, both impacting how UI transformations are managed.
  • "The main theoretical construct is that reactive UI is at heart a pipeline of tree transformations." – This signifies the central role of managing UI component hierarchies through transformations.

• Diversity in Framework Implementations:

  • Despite common goals, implementation details in reactive UI frameworks vary significantly, influencing how application state is managed and updated.
  • Differences are often seen in how these frameworks handle state, incremental updates, and dependency tracking.
  • "The details of implementation still seem wildly divergent." – Points to a lack of standardization in implementing reactive principles across different systems.

• Incremental Transformations and Diffing:

  • Emphasizes the importance of incremental updates in UI frameworks to minimize the performance costs of updating UI states.
  • Discusses strategies like diffing, which involves comparing new and old UI trees to determine necessary updates.
  • "One of the fundamental goals of a UI framework is to keep the deltas flowing down the pipeline small." – Highlights the challenge of efficiently propagating changes through the UI system.

• Push vs. Pull Interfaces in UI Frameworks:

  • The distinction between push and pull interfaces in managing data flow through UI systems is crucial, with many systems using hybrid approaches.
  • "Pulling from a tree basically means calling a function to access that part of the tree." – Describes how data is retrieved or updated in UI components.

• Case Studies and Examples:

  • Provides insights into specific UI frameworks like Druid, Imgui, Flutter, Jetpack Compose, and React, each illustrating unique approaches to tree transformations and state management.
  • These examples highlight the practical applications of theoretical concepts discussed throughout the blog.

• Future Directions and Academic Gaps:

  • Levien seeks further discussion and academic input to refine these theories and potentially translate them into systematic, robust UI development practices.
  • "I’m also curious if there’s good academic literature I’m missing." – Shows an openness to expanding the theoretical underpinnings of reactive UI based on community and academic feedback.

Concluding Thoughts:

• Raph Levien’s exploration into reactive UI frameworks is a deep dive into understanding and characterizing the diverse implementation strategies that exist, aiming to refine and communicate effective methods for building reactive user interfaces, particularly in the context of systems like Druid that utilize Rust’s programming paradigm.

Let's rephrase it. You cannot map a Veeam Agent backup job configured in VBR to a VA backup chain created in any other mode.

Remove

Author response:

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

Public Review:

Summary:

In this manuscript, the authors set out to understand how different TLR4 agonists trigger Myddosome assembly and seek to examine how the potent LPS agonist induces a heightened TLR4 response. A strength of the study is that the authors employ a novel light sheet imaging modality coupled to nanopipette delivery of TLR4 ligands. The authors use this technological innovation to resolve the dynamics of Myddosome formation within the whole cell volume of macrophage cell lines expressing MyD88-YFP. The main finding is that the kinetics of Myddosome formation is slower for the weaker agonist Abeta than LPS. However, Abeta amyloids resulted in the formation of larger MyD88-YFP puncta that persisted for longer. The authors suggest the slower kinetics of formation and larger puncta size reflect how Abeta amyloids are a less efficient TLR4 agonist. Many Toll-like receptors are now known to recognize endogenous produced danger signals and microbially derived molecules. This work is the first to compare the signaling kinetics of endogenous versus microbially derived TLR agonists.

Strengths:

A key strength of this work is the technological achievement of imaging Myddosomes within the entire cell volume and using a nanopipette to administer ligands directly to single cells. The authors also combine this light sheet microscopy with STORM imaging to gain a super-resolved view of the assembly of Myddosomes. These findings suggest that Myddosomes formed in response to Abeta have a more irregular morphology. We conclude that these technological achievements are significant in improving our understanding of the dynamics of TLR4 signaling in response to diverse agonists. Given the limited literature on the molecular dynamics of innate immune signal transduction, this study is an important addition to the field.

Weaknesses:

One limitation of the paper is that a suitable explanation for how larger Myddosomes would contribute to an attenuated downstream signaling response. Do the larger clusters of nucleated MyD88 polymers reflect inefficiency in assembling fully formed Myddosomes that contain IRAK4/2? Could the MyD88-GFP puncta be stained with antibodies against IRAK4 (or IRAK2) to determine the frequency and probably of the two ligands to stimulate signal transduction beyond MyD88 assembly?

A second weakness is the discussion. The authors should explore other explanations for the observed differences in Myddosome formation between TLR4 agonists. For example, could the observed delay in Myddosome assembly in response to Abeta be due to different binding affinity or kinetics to TLR4? Can this be ruled out?

We thank the reviewer for these comments.

To address the first comment we have added a section on the limitations of the current study and suggested that future work could use IRAK4 or 2 staining to identify Myddosomes that are functional as well as working with cells where the Myddosome expression levels is at physiological levels, which may reduce the formation of larger Myddosomes.

The reviewer is correct that the difference in delay time for Myddosome formation could be due slow formation of a TLR4 dimer or binding to the TLR4 dimer, rather than the time take to assemble the Myddosome after TLR4 dimerisation and binding since we have only measured the delay time for Myddosome formation when triggered by LPS or Aβ aggregates. This delay times involves dimerization of TLR4, binding of LPS or Aβ aggregates to the TLR4 dimer followed by Myddosome formation. These other processes might contribute to the difference in delay time that we observed between LPS or Aβ aggregates. It is worth noting that in our experiments we deliver the LPS or Aβ aggregates directly onto the surface for 5 seconds and that we previously showed the presence of the preformed TLR4 dimers on the cell surface (Latty et al., 2018). The affinity of Aβ aggregates for TLR4 is not known but LPS has a high affinity for TLR4, estimated to ∼3 nM for lipid A–TLR4-MD-2 (Akashi et al., 2003). However, even with this high affinity which implies fast binding, direct delivery directly onto the surface and the presence of preformed TLR4 dimers on the cell surface we observed that it took 80 s to observe Myddosome formation. This indicates that Myddosome formation is the slow step for LPS triggering. This is likely to be the case Aβ aggregates, since pM concentrations of aggregates can trigger TLR4 signalling (Hughes et al., 2020) indicating high affinity. However, it is not possible to rule out a contribution of a difference in affinity to observed difference in delay time without measuring the affinity directly.

We have added both these points to a new paragraph on the limitations of the study in the Discussion.

eLife assessment

This important study uses a novel light sheet imaging technique to investigate how different TLR4 agonists regulate Myddosome formation. The data showing that LPS and A-beta can control the kinetics and size of Myddosome assembly are compelling. This paper should be of substantial interest to the innate immunity field.

Reviewer #1 (Public Review):

Recognition of bacterial lipopolysaccharide by Toll-like Receptor 4 is an essential molecular event triggering inflammation and overcoming Recognition of bacterial lipopolysaccharide by Toll-like Receptor 4 is an essential molecular event in triggering inflammation and overcoming infection by gram-negative bacteria. However, TLR4 has recently been found to respond to other endogenously derived ligands. This has implicated TLR4 signaling in the development of disease pathology, for example, Alzheimer's disease, through the recognition of amyloid-beta. Intriguingly, the signaling response to these non-bacterial-derived ligands differs from that of bacterial-derived LPS, suggesting mechanistic differences between endogenous and bacterial-derived agonists. In this work, the authors set out to characterize these mechanistic differences. TLR4 signals through two large macromolecular complexes that assemble at activated receptors: the Myddosome and Triffosome. One hypothesis the authors aimed to test was that different ligands alter these signaling complexes' kinetics and nano-scale features. The authors focused on testing this hypothesis by examining the formation of the Myddosome in live cells. A significant strength of the paper is that the authors developed technological innovations to address this problem. Using a nanopipette delivery mechanism combined with light sheet microscopy, the authors could observe Myddosome signaling in the whole cell volume of live macrophages. This allowed them to accurately quantify the Myddosome number, size, and kinetics of complex formation and compare cells stimulated with amyloid-beta and LPS. The authors discovered differences in Myddosomes formed under LPS versus amyloid-beta stimulation. In general, amyloid-beta TLR4 stimulation resulted in slower Myddosome formation with altered morphology. One limitation of the work, which the authors point out in the discussion, is that they could not distinguish signaling-competent Myddosomes. Future work will be needed to understand whether these amyloid beta induced Myddosomes assembly have a similar or altered complement of downstream signaling proteins (such as the IRAK4/1 and TRAF6). Secondly, the structural basis for how TLR4 would distinguish between different radically agonists remains speculative, and will need further investigation. Nonetheless, this paper is important for the technological innovation to look at the molecular dynamics of signal transduction, a technology that could be adapted to study other receptor signaling pathways.

It is already known that the subcellular location of intracellular TLRs is important for limiting the recognition of self-derived ligands and maintaining tolerance. This work hints at another possible layer of regulation: that a cell surface TLR (TLR4) generates diverse signaling outcomes to extrinsic or intrinsically derived agonists by changing the dynamic behavior of signaling proteins. If correct (and much further work is required to understand endogenous TLR ligands better), it might suggest that the innate immune system employs the same molecular hardware but with altered kinetics to distinguish between exogenous and endogenous inflammatory signals. Thus, pathological aggregates or markers of sterile inflammation might be recognized and responded to by a specific signaling program that is defined kinetically. It will be an interesting direction for future studies to investigate whether and how diverse pathogen and endogenous inflammatory signals modulate the dynamics of signaling complexes.

Why we don't use the Domain layer

Author response:

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

We are grateful for these balanced, nuanced evaluations of our work concerning the observed epistatic trends and our interpretations of their mechanistic origins. Overall, we think the reviewers have done an excellent job at recognizing the novel aspects of our findings while also discussing the caveats associated with our interpretations of the biophysical effects of these mutations. We believe it is important to consider both of these aspects of our work in order to appreciate these advances and what sorts of pertinent questions remain.

Notably, both reviewers are concerned that our lack of experimental approaches to compare the conformational properties of GnRHR variants weakens our claims. We would first humbly suggest that this constitutes a more general caveat that applies to nearly all investigations of the cellular misfolding of α-helical membrane proteins. Whether or not any current in vitro folding measurements report on conformational transitions that are relevant to cellular protein misfolding reactions remains an active area of debate (discussed further below). Nevertheless, while we concede that our structural and/ or computational evaluations of various mutagenic effects remain speculative, prevailing knowledge on the mechanisms of membrane protein folding suggest our mutations of interest (V276T and W107A) are highly unlikely to promote misfolding in precisely the same way. Thus, regardless of whether or not we were able experimentally compare the relevant folding energetics of GnRHR variants, we are confident that the distinct epistatic interactions formed by these mutations reflect variations in the misfolding mechanism and that they are distinct from the interactions that are observed in the context of stable proteins. In the following, we provide detailed considerations concerning these caveats in relation to the reviewers’ specific comments.

Reviewer #1 (Public Review):

The paper carries out an impressive and exhaustive non-sense mutagenesis using deep mutational scanning (DMS) of the gonadotropin-releasing hormone receptor for the WT protein and two single point mutations that I) influence TM insertion (V267T) and ii) influence protein stability (W107A), and then measures the effect of these mutants on correct plasma membrane expression (PME).

Overall, most mutations decreased mGnRHR PME levels in all three backgrounds, indicating poor mutational tolerance under these conditions. The W107A variant wasn't really recoverable with low levels of plasma membrane localisation. For the V267T variant, most additional mutations were more deleterious than WT based on correct trafficking, indicating a synergistic effect. As one might expect, there was a higher degree of positive correlation between V267T/W107A mutants and other mutants located in TM regions, confirming that improper trafficking was a likely consequence of membrane protein co-translational folding. Nevertheless, context is important, as positive synergistic mutants in the V27T could be negative in the W107A background and vice versa. Taken together, this important study highlights the complexity of membrane protein folding in dissecting the mechanism-dependent impact of disease-causing mutations related to improper trafficking.

Strengths

This is a novel and exhaustive approach to dissecting how receptor mutations under different mutational backgrounds related to co-translational folding, could influence membrane protein trafficking.

Weaknesses

The premise for the study requires an in-depth understanding of how the single-point mutations analysed affect membrane protein folding, but the single-point mutants used seem to lack proper validation.

Given our limited understanding of the structural properties of misfolded membrane proteins, it is unclear whether the relevant conformational effects of these mutations can be unambiguously validated using current biochemical and/ or biophysical folding assays. X-ray crystallography, cryo-EM, and NMR spectroscopy measurements have demonstrated that many purified GPCRs retain native-like structural ensembles within certain detergent micelles, bicelles, and/ or nanodiscs. However, helical membrane protein folding measurements typically require titration with denaturing detergents to promote the formation of a denatured state ensemble (DSE), which will invariably retain considerable secondary structure. Given that the solvation provided by mixed micelles is clearly distinct from that of native membranes, it remains unclear whether these DSEs represent a reasonable proxy for the misfolded conformations recognized by cellular quality control (QC, see https://doi.org/10.1021/acs.chemrev.8b00532). Thus, the use and interpretation of these systems for such purposes remains contentious in the membrane protein folding community. In addition to this theoretical issue, we are unaware of any instances in which GPCRs have been found to undergo reversible denaturation in vitro- a practical requirement for equilibrium folding measurements (https://doi.org/10.1146/annurev-biophys-051013-022926). We note that, while the resistance of GPCRs to aggregation, proteolysis, and/ or mechanical unfolding have also been probed in micelles, it is again unclear whether the associated thermal, kinetic, and/ or mechanical stability should necessarily correspond to their resistance to cotranslational and/ or posttranslational misfolding. Thus, even if we had attempted to validate the computational folding predictions employed herein, we suspect that any resulting correlations with cellular expression may have justifiably been viewed by many as circumstantial. Simply put, we know very little about the non-native conformations are generally involved in the cellular misfolding of α-helical membrane proteins, much less how to measure their relative abundance. From a philosophical standpoint, we prefer to let cells tell us what sorts of broken protein variants are degraded by their QC systems, then do our best to surmise what this tells us about the relevant properties of cellular DSEs.

Despite this fundamental caveat, we believe that the chosen mutations and our interpretation of their relevant conformational effects are reasonably well-informed by current modeling tools and by prevailing knowledge on the physicochemical drivers of membrane protein folding and misfolding. Specifically, the mechanistic constraints of translocon-mediated membrane integration provide an understanding of the types of mutations that are likely to disrupt cotranslational folding. Though we are still learning about the protein complexes that mediate membrane translocation (https://doi.org/10.1038/s41586-022-05336-2), it is known that this underlying process is fundamentally driven by the membrane depth-dependent amino acid transfer free energies (https://doi.org/10.1146/annurev.biophys.37.032807.125904). This energetic consideration suggests introducing polar side chains near the center of a nascent TMDs should almost invariably reduce the efficiency of topogenesis. To confirm this in the context of TMD6 specifically, we utilized a well-established biochemical reporter system to confirm that V276T attenuates its translocon-mediated membrane integration (Fig. S1)- at least in the context of a chimeric protein. We also constructed a glycosylation-based topology reporter for full-length GnRHR, but ultimately found its’ in vitro expression to be insufficient to detect changes in the nascent topological ensemble.

In contrast to V276T, the W107A mutation is predicted to preserve the native topological energetics of GnRHR due to its position within a soluble loop region. W107A is also unlike V276T in that it clearly disrupts tertiary interactions that stabilize the native structure. This mutation should preclude the formation of a structurally conserved hydrogen bonding network that has been observed in the context of at least 25 native GPCR structures (https://doi.org/10.7554/eLife.5489). However, without a relevant folding assay, the extent to which this network stabilizes the native GnRHR fold in cellular membranes remains unclear. Overall, we admit that these limitations have prevented us from measuring how much V276T alters the efficiency of GnRHR topogenesis, how much the W107A destabilizes the native fold, or vice versa. Nevertheless, given these design principles and the fact that both reduce the plasma membrane expression of GnRHR, as expected, we are highly confident that the structural defects generated by these mutations do, in fact, promote misfolding in their own ways. We also concede that the degree to which these mutagenic perturbations are indeed selective for specific folding processes is somewhat uncertain. However, it seems exceedingly unlikely that these mutations should disrupt topogenesis and/ or the folding of the native topomer to the exact same extent. From our perspective, this is the most important consideration with respect to the validity of the conclusions we have made in this manuscript.

Furthermore, plasma membrane expression has been used as a proxy for incorrect membrane protein folding, but this not necessarily be the case, as even correctly folded membrane proteins may not be trafficked correctly, at least, under heterologous expression conditions. In addition, mutations can affect trafficking and potential post-translational modifications, like glycosylation.

While the reviewer is correct that the sorting of folded proteins within the secretory pathway is generally inefficient, it is also true that the maturation of nascent proteins within the ER generally bottlenecks the plasma membrane expression of most α-helical membrane proteins. Our group and several others have demonstrated that the efficiency of ER export generally appears to scale with the propensity of membrane proteins to achieve their correct topology and/ or to achieve their native fold (see https://doi.org/10.1021/jacs.5b03743 and https://doi.org/10.1021/jacs.8b08243). Notably, these investigations all involved proteins that contain native glycosylation and various other post-translational modification sites. While we cannot rule out that certain specific combinations of mutations may alter expression through their perturbation of post-translational GnRHR modifications, we feel confident that the general trends we have observed across hundreds of variants predominantly reflect changes in folding and cellular QC. This interpretation is supported by the relationship between observed trends in variant expression and Rosetta-based stability calculations, which we identified using unbiased unsupervised machine learning approaches (compare Figs. 6B & 6D).

Reviewer #2 (Public Review):

Summary:

In this paper, Chamness and colleagues make a pioneering effort to map epistatic interactions among mutations in a membrane protein. They introduce thousands of mutations to the mouse GnRH Receptor (GnRHR), either under wild-type background or two mutant backgrounds, representing mutations that destabilize GnRHR by distinct mechanisms. The first mutant background is W107A, destabilizing the tertiary fold, and the second, V276T, perturbing the efficiency of cotranslational insertion of TM6 to the membrane, which is essential for proper folding. They then measure the surface expression of these three mutant libraries, using it as a proxy for protein stability, since misfolded proteins do not typically make it to the plasma membrane. The resulting dataset is then used to shed light on how diverse mutations interact epistatically with the two genetic background mutations. Their main conclusion is that epistatic interactions vary depending on the degree of destabilization and the mechanism through which they perturb the protein. The mutation V276T forms primarily negative (aggravating) epistatic interactions with many mutations, as is common to destabilizing mutations in soluble proteins. Surprisingly, W107A forms many positive (alleviating) epistatic interactions with other mutations. They further show that the locations of secondary mutations correlate with the types of epistatic interactions they form with the above two mutants.

Strengths:

Such a high throughput study for epistasis in membrane proteins is pioneering, and the results are indeed illuminating. Examples of interesting findings are that: (1) No single mutation can dramatically rescue the destabilization introduced by W107A. (2) Epistasis with a secondary mutation is strongly influenced by the degree of destabilization introduced by the primary mutation. (3) Misfolding caused by mis-insertion tends to be aggravated by further mutations. The discussion of how protein folding energetics affects epistasis (Fig. 7) makes a lot of sense and lays out an interesting biophysical framework for the findings.

Weaknesses:

The major weakness comes from the potential limitations in the measurements of surface expression of severely misfolded mutants. This point is discussed quite fairly in the paper, in statements like "the W107A variant already exhibits marginal surface immunostaining" and many others. It seems that only about 5% of the W107A makes it to the plasma membrane compared to wild-type (Figures 2 and 3). This might be a low starting point from which to accurately measure the effects of secondary mutations.

The reviewer raises an excellent point that we considered at length during the analysis of these data and the preparation of the manuscript. Though we remain confident in the integrity of these measurements and the corresponding analyses, we now realize this aspect of the data required further discussion and documentation which we have provided in the revised version of the manuscript as is described in the following.

Still, the authors claim that measurements of W107A double mutants "still contain cellular subpopulations with surface immunostaining intensities that are well above or below that of the W107A single mutant, which suggests that this fluorescence signal is sensitive enough to detect subtle differences in the PME of these variants". I was not entirely convinced that this was true.

We made this statement based on the simple observation that the surface immunostaining intensities across the population of recombinant cells expressing the library of W107A double mutants was consistently broader than that of recombinant cells expressing W107A GnRHR alone (see Author response image 1 for reference). Given that the recombinant cellular library represents a mix of cells expressing ~1600 individual variants that are each present at low abundance, the pronounced tails within this distribution presumably represent the composite staining of many small cellular subpopulations that express collections of variants that deviate from the expression of W107A to an extent that is significant enough to be visible on a log intensity plot.

Author response image 1.

Firstly, I think it would be important to test how much noise these measurements have and how much surface immunostaining the W107A mutant displays above the background of cells that do not express the protein at all.

For reference, the average surface immunostaining intensity of HEK293T cells transiently expressing W107A GnRHR was 2.2-fold higher than that of the IRES-eGFP negative, untransfected cells within the same sample- the WT immunostaining intensity was 9.5-fold over background by comparison. Similarly, recombinant HEK293T cells expressing the W107A double mutant library had an average surface immunostaining intensity that was 2.6-fold over background across the two DMS trials. Thus, while the surface immunostaining of this variant is certainly diminished, we were still able to reliably detect W107A at the plasma membrane even under distinct expression regimes. We have included these and other signal-to-noise metrics for each experiment in the Results section of the revised manuscript.

Beyond considerations related to intensity, we also previously noticed the relative intensity values for W107A double mutants exhibited considerable precision across our two biological replicates. If signal were too poor to detect changes in variant expression, we would have expected a plot of the intensity values across these two replicates to form a scatter. Instead, we found DMS intensity values for individual variants to be highly correlated from one replicate to the next (Pearson’s R2 = 0.95, see Author response image 2 for reference). This observation empirically demonstrates that this assay consistently differentiated between variants that exhibit slightly enhanced immunostaining from those that have even lower immunostaining than W107A GnRHR. We have included these discussion points in the Results section as well as scatter plots for replicate variant intensities within all three genetic backgrounds in Figure S3 of the revised manuscript.

Author response image 2.

But more importantly, it is not clear if under this regimen surface expression still reports on stability/protein fitness. It is unknown if the W107A retains any function or folding at all. For example, it is possible that the low amount of surface protein represents misfolded receptors that escaped the ER quality control.

While we believe that such questions are outside the scope of this work, we certainly agree that it is entirely possible that some of these variants bypass QC without achieving their native fold. This topic is quite interesting to us but is quite challenging to assess in the context of GPCRs, which have complex fitness landscapes that involve their propensity to distinguish between different ligands, engage specific components associated with divergent downstream signaling pathways, and navigate between endocytic recycling/ degradation pathways following activation. In light of the inherent complexity of GPCR function, we humbly suggest our choice of a relatively simple property of an otherwise complex protein may be viewed as a virtue rather than a shortcoming. Protein fitness is typically cast as the product of abundance and activity. Rather than measuring an oversimplified, composite fitness metric, we focused on one variable (plasma membrane expression) and its dominant effector (folding). We believe restraining the scope in this manner was key for the elucidation of clear mechanistic insights.

The differential clustering of epistatic mutations (Fig. 6) provides some interesting insights as to the rules that dictate epistasis, but these too are dominated by the magnitude of destabilization caused by one of the mutations. In this case, the secondary mutations that had the most interesting epistasis were exceedingly destabilizing. With this in mind, it is hard to interpret the results that emerge regarding the epistatic interactions of W107A. Furthermore, the most significant positive epistasis is observed when W107A is combined with additional mutations that almost completely abolish surface expression. It is likely that either mutation destabilizes the protein beyond repair. Therefore, what we can learn from the fact that such mutations have positive epistasis is not clear to me. Based on this, I am not sure that another mutation that disrupts the tertiary folding more mildly would not yield different results. With that said, I believe that the results regarding the epistasis of V276T with other mutations are strong and very interesting on their own.

We agree with the reviewer. In light of our results we believe it is virtually certain that the secondary mutations characterized herein would be likely to form distinct epistatic interactions with mutations that are only mildly destabilizing. Indeed, this insight reflects one of the key takeaway messages from this work- stability-mediated epistasis is difficult to generalize because it should depend on the extent to which each mutation changes the stability (ΔΔG) as well as initial stability of the WT/ reference sequence (ΔG, see Figure 7). Frankly, we are not so sure we would have pieced this together as clearly had we not had the fortune (or misfortune?) of including such a destructive mutation like W107A as a point of reference.

Additionally, the study draws general conclusions from the characterization of only two mutations, W107A and V276T. At this point, it is hard to know if other mutations that perturb insertion or tertiary folding would behave similarly. This should be emphasized in the text.

We agree. Our findings suggest different mutations may not behave similarly, which we believe is a key finding of this work. We have emphasized this point in the Discussion section of the revised manuscript as follows:

“These findings suggest the folding-mediated epistasis is likely to vary among different classes of destabilizing mutations in a manner that should also depend on folding efficiency and/ or the mechanism(s) of misfolding in the cell.”

Some statistical aspects of the study could be improved:

(1) It would be nice to see the level of reproducibility of the biological replicates in a plot, such as scatter or similar, with correlation values that give a sense of the noise level of the measurements. This should be done before filtering out the inconsistent data.

We thank the reviewer for this suggestion and will include scatters for each genetic background like the one shown above in Figure S3 of the revised version of the manuscript.

(2) The statements "Variants bearing mutations within the C- terminal region (ICL3-TMD6-ECL3-TMD7) fare consistently worse in the V276T background relative to WT (Fig. 4 B & E)." and "In contrast, mutations that are 210 better tolerated in the context of W107A mGnRHR are located 211 throughout the structure but are particularly abundant among residues 212 in the middle of the primary structure that form TMD4, ICL2, and ECL2 213 (Fig. 4 C & F)." are both hard to judge. Inspecting Figures 4B and C does not immediately show these trends, and importantly, a solid statistical test is missing here. In Figures 4E and F the locations of the different loops and TMs are not indicated on the structure, making these statements hard to judge.

We apologize for this oversight and thank the reviewer for pointing this out. We utilized paired Wilcoxon-Signed Rank Tests to evaluate the statistical significance of these observations and modified the description of these findings in the revised version of the results section as follows:

“Variants bearing mutations within the C-terminal regions including ICL3, TMD6, and TMD7 fare consistently worse in the V276T background relative to WT (paired Wilcoxon-Signed Rank Test p-values of 0.0001, 0.02, and 0.005, respectively) (Fig. 4 B & E). Given that V276T perturbs the cotranslational membrane integration of TMD6 (Fig. S1, Table S1), this directional bias potentially suggests that the apparent interactions between these mutations manifest during the late stages of cotranslational folding. In contrast, mutations that are better tolerated in the context of W107A mGnRHR are located throughout the structure but are particularly abundant among residues in the middle of the primary structure that form ICL2, TMD4, and ECL2 (paired Wilcoxon-Signed Rank Test p-values of 0.0005, 0.0001, and 0.004, respectively) (Fig. 4 C & F).”

(3) The following statement lacks a statistical test: "Notably, these 98 variants are enriched with TMD variants (65% TMD) relative to the overall set of 251 variants (45% TMD)." Is this enrichment significant? Further in the same paragraph, the claim that "In contrast to the sparse epistasis that is generally observed between mutations within soluble proteins, these findings suggest a relatively large proportion of random mutations form epistatic interactions in the context of unstable mGnRHR variants". Needs to be backed by relevant data and statistics, or at least a reference.

We thank the reviewer for this reasonable suggestion. In the revised manuscript, we included the results of a paired Wilcoxon-Signed Rank Test that confirms the statistical significance of this observation and modified the Results section to reflect this as follows:

“Notably, these 98 variants are enriched with TMD variants (65% TMD) relative to the overall set of 251 variants (45% TMD, Fisher’s Exact Test p = 0.0019). These findings suggest random mutations form epistatic interactions in the context of unstable mGnRHR variants in a manner that depends on the specific folding defect (V276T vs. W107A) and topological context.”

Reviewer #1 (Recommendations for the Authors):

As far as this reviewer is aware, the effect of the V267T variant on MP insertion has not been measured directly; its position corresponds to T277 in TMD6 of human GnRHR that has been measured for TM insertion, but given the clear lack of conservation (threonine vs valine) the mutation in TM6 could potentially have a different impact on the mouse homologue. Please clarify what the predicted delta TM for insertion is between human and mouse GnRHR is? Moreover, I would argue that single TM insertion by tethering to Lep is insufficient to understand MP insertion/folding, as neighbouring TM helices could help to drive TM6 insertion. Has ER microsome experiments for mouse GnRHR also been carried out in the context of neighbouring helices?

We included measurements (and predictions) of the impact of the V276T substitution on the translocon-mediated membrane integration of the mouse TMD6 in the context of a chimeric Lep protein (see Fig. S1 & Table S1). Our results reveal that this substitution decreases the efficiency of TMD6 membrane integration by ~10%. Though imperfect, this prevailing biochemical assay remains popular for a variety of theoretical and technical reasons. Importantly, extensive experimental testing of this system has shown that these measurements report apparent equilibrium constants that are well-described by two-state equilibrium partitioning models (see DOIs 10.1038/nature03216 and 10.1038/nature06387). This observation provides a reasonable rationale to interpret these measurements using energetic models as we have in this work (see Table S1). From a technical perspective, the Lep system is also advantageous due to the fact that this protein is generally well expressed in the context of in vitro translation systems containing native membranes, which generally ensures a consistent signal to noise and dynamic range for membrane integration measurements. Nevertheless, the reviewers are correct that membrane integration efficiencies are likely distinct in the context of the native mGnRHR protein. For these reasons, we attempted to develop a glycosylation-based topology reporter prior to the posting and submission of this manuscript. However, all GnRHR reporters we tested were poorly expressed in vitro and the resulting 35S-labeled proteins only generated faint smears on our phosphorimaging screens that could not be interpreted. For these reasons, we chose to rely the Lep measurements for these investigations.

The lack of a more relevant topological reporter is one of many challenges we faced in our investigations of this unstable, poorly behaved protein. We share the reviewer’s frustrations concerning the speculative aspects of this work. Nevertheless, there is increasing appreciation for the fact that our perspectives on protein biophysics have been skewed by our continuing choice to focus on the relatively small set of model proteins that are compatible with our favored methodologies (doi: 10.1016/j.tibs.2013.05.001). We humbly suggest this work represents an example of how we can gain a deeper understanding of the limits of biochemical systems when we instead choose to study the unsavory bits of cellular proteomes. But this choice requires a willingness to make some reasonable assumptions and to lean on energetic/ structural modeling from time to time. Despite this limitation, we believe there is still tremendous value in this compromise.

What is the experimental evidence the W107A variant affects the protein structure? Has its melting temperature with and without inverse agonist binding for WT vs the W107A variant been measured, for example? Even heat-FSEC of detergent-solubilised membranes would be informative to know how unstable the W107A variant is. If is very unstable in detergent, then it could be that recovery mutants are going to be unlikely as you are already starting with a poor construct showing poor folding/localisation.

We again understand the rationale for this concern, but do not believe that thermal melting measurements are likely to report the same sorts of conformational transitions involved in cellular misfolding. Heating up a protein to the point in which membranes (or micelles) are disrupted and the proteins begin to form insoluble aggregates is a distinct physical process from those that occur during co- and post-translational folding within intact ER membranes at physiological temperatures (discussed further in the Response to the Reviews). Indeed, as the reviewer points out below, there seems to be little evidence that secretion is linked to thermal stability or various other metrics that others have attempted to optimize for the sake of purification and/ or structural characterization. Thus, we believe it would be just as speculative to suggest thermal aggregation represents a relevant metric for the propensity of membrane proteins to fold in the cell. The physical interpretation of membrane protein misfolding reaction remains contentious in our field due to the key fact that the denatured states of helical membrane proteins remain highly structured in a manner that is hard to generalize beyond the fact that the denatured states retain α-helical secondary structure (doi: 10.1146/annurev-biophys-051013-022926). This is in stark contrast to soluble proteins, where random coil reference states have proven to be generally useful for energetic interpretations of protein stability. For reference, our lab is currently working to leverage epistatic measurements like this to map the prevailing physiological denatured states of an integral membrane protein. Our current findings suggest that non-native electrostatic interactions form in the context of misfolded states. We hope that more information on the structural aspects of these states will help us to develop and interpret meaningful folding measurements within the membrane.

For reference, even in cases when quantitative folding measurements can be achieved, their relevance remains actively debated. As a point of reference, the corresponding author of this work previously worked on the stability and misfolding of another human α-helical membrane protein (PMP22). Like GnRHR, PMP22 is prone to misfolding in the secretory pathway and is associated with dozens of pathogenic mutations that cause protein misfolding. To understand how the thermodynamic stability of this protein is linked to secretion, the corresponding author purified PMP22, reconstituted it into n-Dodecyl-phosphocholine (DPC) micelles, and measured its resistance to denaturation by an anionic denaturing detergent (Lauryl Sarcosine, LS). The results were initially perplexing due to the fact that equilibrium unfolding curves manifested as an exponential decay (rather than a sigmoid) and relaxation kinetics appeared to be dominated by the rate constant for unfolding (doi: 10.1021/bi301635f). Unfortunately, these data could not be fit with existing folding models due to the lack of a folded protein baseline and the absence of a folding arm in the chevron plot. We eventually found that a full sigmoidal unfolding transition and refolding kinetics could be measured upon addition of 15% (v/v) glycerol. Our measurements revealed that the free energy of unfolding in DPC micelles was 0 kcal/ mol (without glycerol). This shocking lack of WT stability made it impossible to directly measure the effects of destabilizing mutations that enhance misfolding- you can’t measure the unfolding of a protein that is already unfolded. We ultimately had to instead infer the energetic effects of such mutations from the thermodynamic coupling between cofactor binding and folding (doi: 10.1021/jacs.5b03743). Finally, after demonstrating the resulting ΔΔGs correlated with both cellular trafficking and disease phenotype, we still faced justified scrutiny about the relevance of these measurements due to the fact that they were carried out in micelles. For these reasons, we do not feel that additional biophysical measurements will add much to this work until more is understood about the nature of misfolding reactions in the membrane and how to effectively recapitulate it in vitro. We also note that PMP22 is secreted with 20% efficiency in mammalian cell lines, which is 20-fold more efficient than human GnRHR under similar conditions (doi: 10.1016/j.celrep.2021.110046). Thus, we suspect equilibrium unfolding measurements are likely out of reach using previously described measurements.

Our greatest evidence suggesting W107A destabilizes the protein has to do with the fact that it deletes a highly conserved structural contact and that this structural modification kills its secretion. The fact that this mutation clearly reduces the escape of GnRHR from ER quality control is a classic indicator of misfolding that represents the cell’s way of telling us that the mutation compromises the folding of the nascent protein in some way or another. Precisely how this mutation remodels the nascent conformational ensemble of nascent GnRHR and how this relates to the free energy difference between the native and non-native portions of its conformational ensemble under cellular conditions is a much more challenging question that lies beyond the scope of this investigation (and likely beyond the scope of what’s currently possible). Indeed, there is an entire field dedicated to understanding such. Nevertheless, the difference in the epistatic interactions formed by W107A and V276T is at the very least consistent with our speculative interpretation that these two mutations vary in their misfolding mechanism and/ or in the extent to which they destabilize the protein. For these reasons, we feel the main conclusions of this manuscript are well-justified.

Please clarify if the protein is glycosylated or not and, if it is, how would this requirement affect the conclusions of your analysis?

As we noted in the Response to the Reviewers, which also constitutes a published portion of the final manuscript, this protein is indeed glycosylated. We were well aware of this aspect of the protein since inception of this project and do not think this changes our interpretation at all. Most membrane proteins are glycosylated, and several groups have demonstrated in various ways that the secretion efficiency of glycoproteins is proportional to certain stability metrics for secreted soluble proteins and membrane proteins alike. Generally, mutations that enhance misfolding do not change the propensity of the nascent chain to undergo N-linked glycosylation, which occurs during translation before protein synthesis and/ or folding is complete. Misfolded proteins typically carry lower weight glycans, which reflects their failure to advance from the ER to the Golgi, where N-linked glycans are modified and O-linked glycans are added. From our perspective, glycosyl modifications just ensure that nascent proteins are engaged by calnexin and other lectin chaperones involved in QC. It does not decouple folding from secretion efficiency. In the case of PMP22 (described above), we found that removal of its glycosylation site allows the nascent protein to bypass the lectin chaperones in a manner that enhances its plasma membrane expression eight-fold (doi: 10.1016/j.jbc.2021.100719). Similar to WT, the expression of several misfolded PMP22 variants also significantly increases upon removal of the glycosylation site. Nevertheless, their expression is still significantly lower than the un-glycosylated WT protein, and the expression patterns of the mutants relative to WT was quite similar across this panel of un-glycosylated proteins. Thus, while glycosylation certainly impacts secretion, it does not change its dependence on folding efficiency within the ER. There are many layers of partially redundant QC within the ER, and it seems that folding imposes a key bottleneck to secretion regardless of which QC proteins are involved. For these reasons, we do not think glycosylation (or other PTMs) should factor into our interpretation of these results.

One caveat with the study is that there is a poor understanding of the factors that decide if the protein should be trafficked to the PM or not. Even secretory proteins not going through the calnexin/reticulum cycle (as they have no N-linked glycans), might still get stuck in the ER, despite the fact they are functional. Could this be a technical issue of heterologous expression overloading the Sec system?

While we agree that there is much to be learned about this topic, we disagree with the notion that our understanding of folding and secretion is insufficient to generally interpret the molecular basis of the observed trends. In collaboration with various other groups, the corresponding author of this paper has shown for several other proteins that the stability of the native topology and the native tertiary structure can constrain secretion efficiency (see dois: 10.1021/jacs.8b08243, 10.1021/jacs.5b03743, and 10.1016/j.jbc.2021.100423). Moreover, the Balch and Kelly groups demonstrated many years ago that relatively simple models for the coupling between folding and chaperone binding can recapitulate the observed effects of mutations on the secretion efficiency of various proteins (doi: 10.1016/j.cell.2007.10.025). Given a wide body of prevailing knowledge in this area, we believe it is entirely reasonable to assume that the conformational effects of these mutation have a dominant effect on plasma membrane expression.

Whether or not some of the proteins retained in the ER are folded and/ or functional is an interesting question, but is outside the scope of this work. Various lines of evidence concerning approaches to rescue misfolded membrane proteins suggest many of these variants are likely to retain residual function once they escape the ER, which may suggest there are pockets of foldable/ folded proteins within the ER. But it seems generally clear that the efficiency of folding in the ER bottlenecks secretion regardless of whether or not the ER contains some fraction of folded/ functional protein. We note that it is certainly possible, if not likely, that secretion efficiency is likely to be higher at lower expression levels (doi: 10.1074/jbc.AC120.014940). However, the mutational scanning platform used in this work was designed such that all variants are expressed from an identical promoter at the same location within the genome. Thus, for the purposes of these investigations, we believe it is entirely fair to draw “apples-to-apples” comparisons of their relative effects on plasma membrane expression.

Please see Francis Arnold's paper on this point and their mutagenesis library of the channelrhodopsin (https://www.pnas.org/doi/10.1073/pnas.1700269114), which further found that 20% of mutations improved WT trafficking. Some general comparisons to this paper might be informative.

We agree that it may be interesting to compare the results from this paper to those in our own. Indeed, we find that 20% of the point mutations characterized herein also enhance the expression of WT mGnRHR, as mentioned in the Results section. However, we think it might be a bit premature to suggest this is a more general trend in light of the fact that the channelrhodopsins engineered in those studies were not of eukaryotic origin and have likely resulted from distinct evolutionary constraints. We ultimately decided against adding more on this to our already lengthy discussion in order to maintain focus on the mechanisms of epistasis.

Chris Tate and others have shown that there is a high frequency of finding stabilising point mutations in GPCRs and this is the premise of the StAR technology used to thermostabilise GPCRs in the presence of different ligands, i.e. agonist vs inverse agonists. As far as I am aware, there is a poor correlation between expression levels and thermostability (measured by ligand binding to detergent-solubilised membranes). As such, it is possible that some of the mutants might be more stable than WT even though they have lower levels of PME.

We believe the disconnect between thermostability and expression precisely speaks to our main point about the suitability of current membrane protein folding assays for the questions we address herein. The degradative activity of ER quality control has not necessarily selected for proteins that are resistant to thermal degradation and/ or are suitable for macromolecular crystallography. For this reason, it is often not so difficult to engineer proteins with enhanced thermal stability. We do not believe this disconnect signals that quality control is insensitive to protein folding and stability, but rather that it is more likely to recognize conformational defects that are distinct from those involved in thermal degradation and/ or aggregation. Indeed, recent work from the Fluman group, which builds on a wider body of previous observations, has shown that the exposure of polar groups within the membrane is a key factor that recruits degradation machinery (doi: 0.1101/2023.12.12.571171). It is hard to imagine that these sorts of conformational defects are the same as those involved in thermal aggregation.

Reviewer #2 (Recommendations For The Authors):

(1) I believe that by focusing more on the epistasis with V276T, and less on W107A, the paper could be strengthened significantly.

We appreciate this sentiment. But we believe the comparison of these two mutants really drive home the point that destabilizing mutations are not equivalent with respect to the epistatic interactions they form.

(2) In the abstract - please define the term epistasis in a simple way, to make it accessible to a general audience. For example - negative epistasis means that... this should be explicitly explained.

We thank the reviewer for this suggestion. To meet eLife formatting, we had to cut down the abstract significantly. We simplified this as best we could in the following statement:

“Though protein stability is known to shape evolution, it is unclear how cotranslational folding constraints modulate the synergistic, epistatic interactions between mutations.”

We also define positive and negative epistasis in the results section as follows:

“Positive Ɛ values denote double mutants that have greater PME than would be expected based on the effects of single mutants. Negative Ɛ values denote double mutants that have lower PME than would be expected based on the effects of single mutants. Pairs of mutations with Ɛ values near zero have additive effects on PME.”

(3) The title is quite complex and might deter readers from outside the protein evolution field. Consider simplifying it.

We thank the reviewer for this suggestion. We have simplified the title to the following:

“Divergent Folding-Mediated Epistasis Among Unstable Membrane Protein Variants”

(4) The paper could benefit from a simple figure explaining the different stages of membrane protein folding (stages 1+2) to make it more accessible to readers from outside the membrane protein field.

This is a great suggestion. We incorporated a new schematic in the revised manuscript that outlines the nature of these processes (see Fig. 1A in the revised manuscript).

(5) For the FACS-Seq experiment - it was not clear to me if and when all cells are pulled together. For example - are the 3 libraries mixed together already at the point of transfection, or are the transfected cells pulled together at any point before sorting? This could have some implications on batch effects and should, therefore, be explicitly mentioned in the main text.

We thank the reviewer for this suggestion. We modified the description of the DNA library assembly to emphasize that the mutations were generated in the context of three mixed plasmid pools, which were then transfected into the cells and sorted independently:

“We then generated a mixed array of mutagenic oligonucleotides that collectively encode this series of substitutions (Table S3) and used nicking mutagenesis to introduce these mutations into the V276T, W107A, and WT mGnRHR cDNAs (Medina-Cucurella et al., 2019), which produced three mixed plasmid pools.”

(6) The following description in the text is quite confusing. It would be better to simplify it considerably or remove it: "scores (Ɛ) were then determined by taking the log of the double mutant fitness value divided by the difference between the single mutant fitness values (see Methods)."

We thank the reviewer for this valuable feedback and have simplified the text as follows:

“To compare epistatic trends in these libraries, we calculated epistasis scores (Ɛ) for the interactions that these 251 mutations form with V276T and W107A by comparing their relative effects on PME of the WT, V276T, and W107A variants using a previously described epistasis model (product model, see Methods) (Olson et al. 2014).”

eLife assessment

This important study describes exhaustive deep mutational scanning (DMS) of the gonadotropin-releasing hormone wild-type receptor and for two single point mutations that impact its folding and structure, monitoring how plasma membrane expression levels are influenced by the introduced mutations. With solid evidence, the authors have pioneered an exploration of the interaction between mutations (epistasis) in a membrane protein, with a potential for explaining membrane protein evolution and genetic diseases.

Reviewer #1 (Public Review):

Summary:

The paper carries out an impressive and exhaustive non-sense mutagenesis using deep mutational scanning (DMS) of the gonadotropin-releasing hormone receptor for the WT protein and two single point mutations that I) influences TM insertion (V267T) and ii) influences protein stability (W107A) and then measures the effect of these mutants on correct plasma membrane expression (PME).

Overall, most mutations decreased mGnRHR PME levels in all three backgrounds, indicating poor mutational tolerance under these conditions. The W107A variant wasn't really recoverable with low levels of plasma membrane localisation. For the V267T variant, most additional mutations were more deleterious than WT based on correct trafficking, indicating a synergistic effect. As one might expect, there was a higher degree of positive correlation between V267T/W107A mutants and other mutants located in TM regions, confirming that improper trafficking was a likely consequence of membrane protein co-translational folding. Nevertheless, context is important, as positive synergistic mutants in the V27T could be negative in the W107A background and vice versa. Taken together, this important study highlights the complexity of membrane protein folding in dissecting the mechanism-dependent impact of disease-causing mutations related to improper trafficking.

Strengths:

This is a novel and exhaustive approach to dissect how receptor mutations under different mutational backgrounds related to co-translational folding, could influence membrane protein trafficking.

Weaknesses:

The premise for the study requires an in-depth understanding of how the single point mutations analysed effect membrane protein folding in context of DMS, but the single point mutants used could do with further validation. The V267T mutant only reduced MP insertion by 10% and the effect of W107A on protein stability was not assessed. Furthermore, plasma membrane expression has been used as a proxy for incorrect membrane protein folding, but this not necessarily be the case, as even correctly folded membrane proteins may not be trafficked correctly, at least, under heterologous expression conditions. In addition, mutations can effect trafficking and potential post-translational modifications, like glycosylation.

Reviewer #2 (Public Review):

Summary:

In this paper, Chamness and colleagues make a pioneering effort to map epistatic interactions among mutations in a membrane protein. They introduce thousands of mutations to the mouse GnRH Receptor (GnRHR), either under wild-type background or two mutant backgrounds, representing mutations that destabilize GnRHR by distinct mechanisms. The first mutant background is W107A, destabilizing the tertiary fold, and the second, V276T, perturbing the efficiency of cotranslational insertion of TM6 to the membrane, which is essential for proper folding. They then measure surface expression of these three mutant libraries, using it as a proxy for protein stability, since misfolded proteins do not typically make it to the plasma membrane. The resulting dataset is then used to shed light on how diverse mutations interact epistatically with the two genetic background mutations. Their main conclusion is that epistatic interactions vary depending on the degree of destabilization and the mechanism through which they perturb the protein. The mutation V276T forms primarily negative (aggravating) epistatic interactions with many mutations, as is common to destabilizing mutations in soluble proteins. Surprisingly, W107A forms many positive (alleviating) epistatic interactions with other mutations. They further show that the locations of secondary mutations correlate with the types of epistatic interactions they form with the above two mutants.

Strengths:

Such a high throughput study for epistasis in membrane proteins is pioneering, and the results are indeed illuminating. Examples of interesting findings are that: (1) No single mutation can dramatically rescue the destabilization introduced by W107A. (2) Epistasis with a secondary mutation is strongly influenced by the degree of destabilization introduced by the primary mutation. (3) Misfolding caused by mis-insertion tends to be aggravated by further mutations. The discussion of how protein folding energetics affects epistasis (Fig. 7) makes a lot of sense and lays out an interesting biophysical framework for the findings.

Weaknesses:

The major weakness comes from the potential limitations in the measurements of surface expression of severely misfolded mutants. It seems that only about 5% of the W107A makes it to the plasma membrane compared to wild-type. This point is discussed quite fairly in the paper. (Figures 2 and 3). This might be a low starting point from which to accurately measure the effects of secondary mutations. I am concerned about the extent to which surface expression can report on protein stability, especially when it comes to double mutants where each mutation alone severely decreases surface expression. It is possible that in these cases, both the single and double mutants are completely misfolded, beyond repair. The surface-expressed proteins in such mutants may not be stable, folded or active at all, and the authors do not provide any indication that the combined effects of the mutations are derived from effects on folding stability or misfolding. Therefore, the reason for the epistatic effects of these mutations is hard to interpret, leaving a notable gap in our understanding. However, I find that this point is discussed much more fairly in the current manuscript.

With that said, I believe that the results regarding the epistasis of V276T with other mutations are strong and very interesting on their own.

Another concern relates to the measurements of the epistatic effects of mutations in the background of the V107A mutation. I am concerned about their measurement accuracy. Firstly, the authors note that the surface immunostaining measurements of these mutants are on average only 2-fold above background, which is quite a low signal-to-noise regimen. Secondly, I believe that the authors still haven't demonstrated the reproducibility of their surface expression measurements. To showcase the reproducibility, the authors show the correlation of two biological replicates in Figure S3. However, these are shown only for the 251 mutations that passed a reproducibility filter, after the authors "discarded variant scores for which the difference in percentile rank across the two replicates was greater than 25%. " . this means that all mutations that showed irreproducible results were filtered out before the analysis in Figure S3. It is, therefore, no surprise that the remaining mutations are highly reproducible, and such an analysis cannot serve as an indication of the reproducibility. It remains possible that a large fraction of the surface immunostaining scores of the V107A variants are dominated by noise and that their correlation in these two replicates might be random and may not necessarily be reproduced in a third replicate, for example.

Author response:

Reviewer #1 (Public Review):

Summary:

For many years, there has been extensive electrophysiological research investigating the relationship between local field potential patterns and individual cell spike patterns in the hippocampus. In this study, using state-of-the-art imaging techniques, they examined spike synchrony of hippocampal cells during locomotion and immobility states. In contrast to conventional understanding of the hippocampus, the authors demonstrated that hippocampal place cells exhibit prominent synchronous spikes locked to theta oscillations.

Strengths:

The voltage imaging used in this study is a highly novel method that allows recording not only suprathreshold-level spikes but also subthreshold-level activity. With its high frame rate, it offers time resolution comparable to electrophysiological recordings. Moreover, it enables the visualization of actual cell locations, allowing for the examination of spatial properties (e.g., Figure 4G).

We thank the reviewer for pointing out the technical novelty of this work.

Weaknesses:

There is a notable deviation from several observations obtained through conventional electrophysiological recordings. Particularly, as mentioned below in detail, the considerable differences in baseline firing rates and no observations of ripple-triggered firing patterns raise some concerns about potential artifacts from imaging and analysis, such as cell toxicity, abnormal excitability, and false detection of spikes. While these findings are intriguing if the validity of these methods is properly proven, accepting the current results as new insights is challenging.

We appreciate the reviewer’s insightful comments regarding the intriguing aspect of our findings. Indeed, the emergence of a novel form of CA1 population synchrony presents exciting implications for hippocampal memory research and beyond.

While we acknowledge the deviations from conventional electrophysiological recordings, we respectfully contend that these differences do not necessarily imply methodological flaws. All experiments and analyses were conducted with meticulous adherence to established standards in the field.

Regarding the observed variations in averaging firing rates, it is important to note the well-documented heterogeneity in CA1 pyramidal neuron firing rates, spanning from 0.01 to 10 Hz, with a skewed distribution toward lower frequencies (Mizuseki et al., 2013). Our exclusion criteria for neurons with low estimated firing rates may have inadvertently biased the selection towards more active neurons. Moreover, prior research has indicated that averaging firing rates tend to increase during exposure to novel environments (Karlsson et al., 2008), and among deep-layer CA1 pyramidal neurons (Mizuseki et al., 2011). Given our recording setup in a highly novel environment and the predominance of deep CA1 pyramidal neurons in our sample, the observed higher averaging firing rates could be influenced by these factors. Considering these points, our mean firing rates (3.2 Hz) are reasonable estimations compared to previously reported values obtained from electrophysiological recordings (2.1 Hz in McHugh et al., 1996 and 2.4-2.6 Hz in Buzsaki et al., 2003).

Regarding concerns about potential cell toxicity, previous studies have shown that Voltron expression and illumination do not significantly alter membrane resistance, membrane capacitance, resting membrane potentials, spike amplitudes, and spike width (see Abdelfattah 2019, Science, Supplementary Figure 11 and 12). In our recordings, imaged neurons exhibit preserved membrane and dendritic morphology during and after experiments (Author response image 1), supporting the absence of significant toxicity.

Author response image 1.

Voltron-expressing neurons exhibit preserved membrane and dendritic morphology. (A) Images of two-photon z-stack maximum intensity projection showing Voltron-expressing neurons taken after voltage image experiments in vivo. (B) Post-hoc histological images of neurons being voltage-imaged.

Regarding spike detection, we use validated algorithms (Abdelfattah et al., 2019 and 2023) to ensure robust and reliable detection of spikes. Spiking activity was first separated from slower subthreshold potentials using high-pass filtering. This way, a slow fluorescence increase will not be detected as a spike, even if its amplitude is large. We benchmarked the detection algorithm in computer simulation. The sensitivity and specificity of the algorithm exceed 98% at the level of signal-to-noise ratio of our recordings. While we acknowledge that a small number of spikes, particularly those occurring later in a burst, might be missed due to their smaller amplitudes (as illustrated in Figure 1 and 2 of the manuscript), we anticipate that any missed spikes would lead to a decrease rather than an increase in synchrony between neurons. Overall, we are confident that spike detection is performed in a rigorous and robust manner.

To further strengthen these points, we will include the following in the revision:

(1) Histological images of recorded neurons during and after experiments.

(2) Further details regarding the validation of spike detection algorithms.

(3) Analysis of publicly available electrophysiological datasets.

(4) Discussion regarding the reasons behind the novelty of some of our findings compared to previous observations.

In conclusion, we assert that our experimental and analysis approach upholds rigorous standards. We remain committed to reconciling our findings with previous observations and welcome further scrutiny and engagement from the scientific community to explore the intriguing implications of our findings.

Reviewer #2 (Public Review):

Summary:

This study employed voltage imaging in the CA1 region of the mouse hippocampus during the exploration of a novel environment. The authors report synchronous activity, involving almost half of the imaged neurons, occurred during periods of immobility. These events did not correlate with SWRs, but instead, occurred during theta oscillations and were phased-locked to the trough of theta. Moreover, pairs of neurons with high synchronization tended to display non-overlapping place fields, leading the authors to suggest these events may play a role in binding a distributed representation of the context.

We thank the reviewer for a thorough and thoughtful review of our paper.

Strengths:

Technically this is an impressive study, using an emerging approach that allows single-cell resolution voltage imaging in animals, that while head-fixed, can move through a real environment. The paper is written clearly and suggests novel observations about population-level activity in CA1.

We thank the reviewer for pointing out the technical strength and the novelty of our observations.

Weaknesses:

The evidence provided is weak, with the authors making surprising population-level claims based on a very sparse data set (5 data sets, each with less than 20 neurons simultaneously recorded) acquired with exciting, but less tested technology. Further, while the authors link these observations to the novelty of the context, both in the title and text, they do not include data from subsequent visits to support this. Detailed comments are below:

We understand the reviewer’s concerns regarding the size of the dataset. Despite this limitation, it is important to note that synchronous ensembles beyond what could be expected from chance (jittering) were detected in all examined data. In the revision, we plan to add more data, including data from subsequent visits, to further strengthen our findings.

(1) My first question for the authors, which is not addressed in the discussion, is why these events have not been observed in the countless extracellular recording experiments conducted in rodent CA1 during the exploration of novel environments. Those data sets often have 10x the neurons simultaneously recording compared to these present data, thus the highly synchronous firing should be very hard to miss. Ideally, the authors could confirm their claims via the analysis of publicly available electrophysiology data sets. Further, the claim of high extra-SWR synchrony is complicated by the observation that their recorded neurons fail to spike during the limited number of SWRs recorded during behavior- again, not agreeing with much of the previous electrophysiological recordings.

We understand the reviewer’s concern. We will examine publicly available electrophysiology datasets to gain further insights into any similarities and differences to our findings. Based on these results, we will discuss why these events have not been previously observed/reported.

(2) The authors posit that these events are linked to the novelty of the context, both in the text, as well as in the title and abstract. However, they do not include any imaging data from subsequent days to demonstrate the failure to see this synchrony in a familiar environment. If these data are available it would strengthen the proposed link to novelty if they were included.

We thank the reviewer’s constructive suggestion. We will acquire more datasets from subsequent visits to gain further insights into these synchronous events.

3) In the discussion the authors begin by speculating the theta present during these synchronous events may be slower type II or attentional theta. This can be supported by demonstrating a frequency shift in the theta recording during these events/immobility versus the theta recording during movement.

We thank the reviewer’s constructive suggestion. We did demonstrate a frequency shift to a lower frequency in the synchrony-associated theta during immobility than during locomotion (see Fig. 4B, the red vs. blue curves). We will enlarge this panel and specifically refer to it in the corresponding discussion paragraph.

(4) The authors mention in the discussion that they image deep-layer PCs in CA1, however, this is not mentioned in the text or methods. They should include data, such as imaging of a slice of a brain post-recording with immunohistochemistry for a layer-specific gene to support this.

We thank the reviewer’s constructive suggestion. We do have images of brain slices post-recordings (Author response image 2). Imaged neurons are clearly located in the deep CA1 pyramidal layer. We will add these images and quantification in the revised manuscript.

Author response image 2.

Imaged neurons are located in the deep pyramidal layer of the dorsal hippocampal CA1 region.

Reviewer #3 (Public Review):

Summary:

In the present manuscript, the authors use a few minutes of voltage imaging of CA1 pyramidal cells in head-fixed mice running on a track while local field potentials (LFPs) are recorded. The authors suggest that synchronous ensembles of neurons are differentially associated with different types of LFP patterns, theta and ripples. The experiments are flawed in that the LFP is not "local" but rather collected in the other side of the brain, and the investigation is flawed due to multiple problems with the point process analyses. The synchrony terminology refers to dozens of milliseconds as opposed to the millisecond timescale referred to in prior work, and the interpretations do not take into account theta phase locking as a simple alternative explanation.

We genuinely appreciate the reviewer’s feedback and acknowledge the concerns raised. However, we believe these concerns can be effectively addressed without undermining the validity of our conclusions. With this in mind, we respectfully disagree with the assessment that our experiments and investigation are flawed. Please allow us to address these concerns and offer additional context to support the validity of our study.

Weaknesses:

The two main messages of the manuscript indicated in the title are not supported by the data. The title gives two messages that relate to CA1 pyramidal neurons in behaving head-fixed mice: (1) synchronous ensembles are associated with theta (2) synchronous ensembles are not associated with ripples.

There are two main methodological problems with the work:

(1) Experimentally, the theta and ripple signals were recorded using electrophysiology from the opposite hemisphere to the one in which the spiking was monitored. However, both signals exhibit profound differences as a function of location: theta phase changes with the precise location along the proximo-distal and dorso-ventral axes, and importantly, even reverses with depth. And ripples are often a local phenomenon - independent ripples occur within a fraction of a millimeter within the same hemisphere, let alone different hemispheres. Ripples are very sensitive to the precise depth - 100 micrometers up or down, and only a positive deflection/sharp wave is evident.

We appreciate the reviewer’s consideration regarding the collection of LFP from the contralateral hemisphere. While we acknowledge the limitation of this design, we believe that our findings still offer valuable insights into the dynamics of synchronous ensembles. Despite potential variations in theta phases with recording locations and depth, we find that the occurrence and amplitudes of theta oscillations are generally coordinated across hemispheres (Buzsaki et al., Neurosci., 2003). Therefore, the presence of prominent contralateral LFP theta around the times of synchronous ensembles in our study (see Figure 4A of the manuscript) strongly supports our conclusion regarding their association with theta oscillations, despite the collection of LFP from the opposite hemisphere.

In addition, in our manuscript, we specifically mentioned that the “preferred phases” varied from session to session, likely due to the variability of recording locations (see Line 254-256). Therefore, we think that the reviewer’s concern regarding theta phase variability has already been addressed in the present manuscript.

Regarding ripple oscillations, while we recognize that they can sometimes occur locally, the majority of ripples occur synchronously in both hemispheres (up to 70%, see Szabo et al., Neuron, 2022; Buzsaki et al., Neurosci., 2003). Therefore, using contralateral LFP to infer ripple occurrence on the ipsilateral side has been a common practice in the field, employed by many studies published in respectable journals (Szabo et al., Neuron, 2022; Terada et al., Nature, 2021; Dudok et al., Neuron, 2021; Geiller et al., Neuron, 2020). Furthermore, our observation that 446 synchronous ensembles during immobility do not co-occur with contralateral ripples, and the remaining 313 ensembles during locomotion are not associated with ripples, as ripples rarely occur during locomotion. Therefore, our conclusion that synchronous ensembles are not associated with ripple oscillations is supported by data.

(2) The analysis of the point process data (spike trains) is entirely flawed. There are many technical issues: complex spikes ("bursts") are not accounted for; differences in spike counts between the various conditions ("locomotion" and "immobility") are not accounted for; the pooling of multiple CCGs assumes independence, whereas even conditional independence cannot be assumed; etc.

We acknowledge the reviewer’s concern regarding spike train analysis. Indeed, complex bursts or different behavioral conditions can lead to differences in spike counts that could potentially affect the detection of synchronous ensembles. However, our jittering procedure (see Line 121-132) is designed to control for the variation of spike counts. Importantly, while the jittered spike trains also contain the same spike count variations, we found 7.8-fold more synchronous events in our data compared to jitter controls (see Figure 1G of the manuscript), indicating that these factors cannot account for the observed synchrony.

To explicitly demonstrate that complex bursts cannot account for the observed synchrony, we have performed additional analysis to remove all latter spikes in bursts and only count the single and the first spikes of bursts. Importantly, we found that this procedure did not change the rate and size of synchronous ensembles, nor did it significantly alter the grand-average CCG (see Author response image 3). The results of this analysis explicitly rule out a significant effect of complex spikes on the analysis of synchronous ensembles.

Author response image 3.

Population synchrony remains after the removal of spikes in bursts. (A) The grand-average cross correlogram (CCG) was calculated using spike trains without latter spikes in bursts. The gray line represents the mean grand average CCG between reference cells and randomly selected cells from different sessions. (B) Pairwise comparison of the event rates of population synchrony between spike trains containing all spikes and spike trains without latter spikes in bursts. Bar heights indicate group means (n=10 segments, p=0.036, Wilcoxon signed-rank test). (C) Histogram of the ensemble sizes as percentages of cells participating in the synchronous ensembles.

Beyond those methodological issues, there are two main interpretational problems: (1) the "synchronous ensembles" may be completely consistent with phase locking to the intracellular theta (as even shown by the authors themselves in some of the supplementary figures).

We agree with the reviewer that the synchronous ensembles are indeed consistent with theta phase locking. However, it is important to note that theta phase locking alone does not necessarily imply population synchrony. In fact, theta phase locking has been shown to “reduce” population synchrony in a previous study (Mizuseki et al., 2014, Phil. Trans. R. Soc. B.). Thus, the presence of theta phase locking cannot be taken as a simple alternative explanation of the synchronous ensembles.

To directly assess the contribution of theta phase locking to synchronous ensembles, we have performed a new analysis to randomize the specific theta cycles in which neurons spike, while keeping the spike phases constant. This manipulation disrupts spike co-occurrence while preserving theta phase locking, allowing us to test whether theta phase locking alone can explain the population synchrony, or whether spike co-occurrence in specific cycles is required. The grand-average CCG shows a much smaller peak compared to the original peak (Author response image 4A). Moreover, synchronous event rates show a 4.5-fold decrease in the randomized data compared to the original event rates (Author response image 4B). Thus, the new analysis reveals theta phase locking alone cannot account for the population synchrony.

Author response image 4.

Drastic reduction of population synchrony by randomizing spikes to other theta cycles while preserving the phases. (A) The grand-average cross correlogram (CCG) was calculated using original spike trains (black) and randomized spike trains where theta phases of the spikes are kept the same but spike timings were randomly moved to other theta cycles (red). (B) Pairwise comparison of the event rates of population synchrony between the original spike trains and randomized spike trains (n=10 segments, p=0.002, Wilcoxon signed-rank test). Bar heights indicate group means. ** p<0.01

(2) The definition of "synchrony" in the present work is very loose and refers to timescales of 20-30 ms. In previous literature that relates to synchrony of point processes, the timescales discussed are 1-2 ms, and longer timescales are referred to as the "baseline" which is actually removed (using smoothing, jittering, etc.).

Regarding the timescale of synchronous ensembles, we acknowledge that it varies considerably across studies and cell types. However, it is important to note that a timescale of dozens, or even hundreds of milliseconds is common for synchrony terminology in CA1 pyramidal neurons (see Csicsvari et al., Neuron, 2000; Harris et al., Science, 2003; Malvache et al., Science, 2016; Yagi et al., Cell Reports, 2023). In fact, a timescale of 20-30 ms is considered particularly important for information transmission and storage in CA1, as it matches the membrane time constant of pyramidal neurons, the period of hippocampal gamma oscillations, and the time window for synaptic plasticity. Therefore, we believe that this timescale is relevant and in line with established practices in the field.

eLife assessment

The authors perform voltage imaging of CA1 pyramidal cells in head-fixed mice running on a track while local field potentials (LFPs) are recorded. They suggest that synchronous ensembles of neurons are differentially associated with different types of LFP patterns, namely theta and ripples. However, evidence for the potentially useful findings is currently incomplete due to major weaknesses in the experimental and analytical approach.

Reviewer #1 (Public Review):

Summary:

For many years, there has been extensive electrophysiological research investigating the relationship between local field potential patterns and individual cell spike patterns in the hippocampus. In this study, using state-of-the-art imaging techniques, they examined spike synchrony of hippocampal cells during locomotion and immobility states. In contrast to conventional understanding of the hippocampus, the authors demonstrated that hippocampal place cells exhibit prominent synchronous spikes locked to theta oscillations.

Strengths:

The voltage imaging used in this study is a highly novel method that allows recording not only suprathreshold-level spikes but also subthreshold-level activity. With its high frame rate, it offers time resolution comparable to electrophysiological recordings. Moreover, it enables the visualization of actual cell locations, allowing for the examination of spatial properties (e.g., Figure 4G).

Weaknesses:

There is a notable deviation from several observations obtained through conventional electrophysiological recordings. Particularly, as mentioned below in detail, the considerable differences in baseline firing rates and no observations of ripple-triggered firing patterns raise some concerns about potential artifacts from imaging and analsyis, such as cell toxicity, abnormal excitability, and false detection of spikes. While these findings are intriguing if the validity of these methods is properly proven, accepting the current results as new insights is challenging.

Reviewer #2 (Public Review):

Summary:

This study employed voltage imaging in the CA1 region of the mouse hippocampus during the exploration of a novel environment. The authors report synchronous activity, involving almost half of the imaged neurons, occurred during periods of immobility. These events did not correlate with SWRs, but instead, occurred during theta oscillations and were phased-locked to the trough of theta. Moreover, pairs of neurons with high synchronization tended to display non-overlapping place fields, leading the authors to suggest these events may play a role in binding a distributed representation of the context.

Strengths:

Technically this is an impressive study, using an emerging approach that allows single-cell resolution voltage imaging in animals, that while head-fixed, can move through a real environment. The paper is written clearly and suggests novel observations about population-level activity in CA1.

Weaknesses:

The evidence provided is weak, with the authors making surprising population-level claims based on a very sparse data set (5 data sets, each with less than 20 neurons simultaneously recorded) acquired with exciting, but less tested technology. Further, while the authors link these observations to the novelty of the context, both in the title and text, they do not include data from subsequent visits to support this. Detailed comments are below:

(1) My first question for the authors, which is not addressed in the discussion, is why these events have not been observed in the countless extracellular recording experiments conducted in rodent CA1 during the exploration of novel environments. Those data sets often have 10x the neurons simultaneously recording compared to these present data, thus the highly synchronous firing should be very hard to miss. Ideally, the authors could confirm their claims via the analysis of publicly available electrophysiology data sets. Further, the claim of high extra-SWR synchrony is complicated by the observation that their recorded neurons fail to spike during the limited number of SWRs recorded during behavior- again, not agreeing with much of the previous electrophysiological recordings.

(2) The authors posit that these events are linked to the novelty of the context, both in the text, as well as in the title and abstract. However, they do not include any imaging data from subsequent days to demonstrate the failure to see this synchrony in a familiar environment. If these data are available it would strengthen the proposed link to novelty if they were included.

(3) In the discussion the authors begin by speculating the theta present during these synchronous events may be slower type II or attentional theta. This can be supported by demonstrating a frequency shift in the theta recording during these events/immobility versus the theta recording during movement.

(4) The authors mention in the discussion that they image deep-layer PCs in CA1, however, this is not mentioned in the text or methods. They should include data, such as imaging of a slice of a brain post-recording with immunohistochemistry for a layer-specific gene to support this.

Reviewer #3 (Public Review):

Summary:

In the present manuscript, the authors use a few minutes of voltage imaging of CA1 pyramidal cells in head-fixed mice running on a track while local field potentials (LFPs) are recorded. The authors suggest that synchronous ensembles of neurons are differentially associated with different types of LFP patterns, theta and ripples. The experiments are flawed in that the LFP is not "local" but rather collected in the other side of the brain, and the investigation is flawed due to multiple problems with the point process analyses. The synchrony terminology refers to dozens of milliseconds as opposed to the millisecond timescale referred to in prior work, and the interpretations do not take into account theta phase locking as a simple alternative explanation.

Weaknesses:

The two main messages of the manuscript indicated in the title are not supported by the data. The title gives two messages that relate to CA1 pyramidal neurons in behaving head-fixed mice: (1) synchronous ensembles are associated with theta (2) synchronous ensembles are not associated with ripples.

There are two main methodological problems with the work: (1) experimentally, the theta and ripple signals were recorded using electrophysiology from the opposite hemisphere to the one in which the spiking was monitored. However, both signals exhibit profound differences as a function of location: theta phase changes with the precise location along the proximo-distal and dorso-ventral axes, and importantly, even reverses with depth. And ripples are often a local phenomenon - independent ripples occur within a fraction of a millimeter within the same hemisphere, let alone different hemispheres. Ripples are very sensitive to the precise depth - 100 micrometers up or down, and only a positive deflection/sharp wave is evident. (2) The analysis of the point process data (spike trains) is entirely flawed. There are many technical issues: complex spikes ("bursts") are not accounted for; differences in spike counts between the various conditions ("locomotion" and "immobility") are not accounted for; the pooling of multiple CCGs assumes independence, whereas even conditional independence cannot be assumed; etc.

Beyond those methodological issues, there are two main interpretational problems: (1) the "synchronous ensembles" may be completely consistent with phase locking to the intracellular theta (as even shown by the authors themselves in some of the supplementary figures). (2) The definition of "synchrony" in the present work is very loose and refers to timescales of 20-30 ms. In previous literature that relates to synchrony of point processes, the timescales discussed are 1-2 ms, and longer timescales are referred to as the "baseline" which is actually removed (using smoothing, jittering, etc.).

Author response:

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

eLife assessment

Connelly and colleagues provide convincing genetic evidence that importation from mainland Tanzania is a major source of Plasmodium falciparum lineages currently circulating in Zanzibar. This study also reveals ongoing local malaria transmission and occasional near-clonal outbreaks in Zanzibar. Overall, this research highlights the role of human movements in maintaining residual malaria transmission in an area targeted for intensive control interventions over the past decades and provides valuable information for epidemiologists and public health professionals.

Reviewer #1 (Public Review):

Zanzibar archipelago is close to achieving malaria elimination, but despite the implementation of effective control measures, there is still a low-level seasonal malaria transmission. This could be due to the frequent importation of malaria from mainland Tanzania and Kenya, reservoirs of asymptomatic infections, and competent vectors. To investigate population structure and gene flow of P. falciparum in Zanzibar and mainland Tanzania, they used 178 samples from mainland Tanzania and 213 from Zanzibar that were previously sequenced using molecular inversion probes (MIPs) panels targeting single nucleotide polymorphisms (SNPs). They performed Principal Component Analysis (PCA) and identity by descent (IBD) analysis to assess genetic relatedness between isolates. Parasites from coastal mainland Tanzania contribute to the genetic diversity in the parasite population in Zanzibar. Despite this, there is a pattern of isolation by distance and microstructure within the archipelago, and evidence of local sharing of highly related strains sustaining malaria transmission in Zanzibar that are important targets for interventions such as mass drug administration and vector control, in addition to measures against imported malaria.

Strengths:

This study presents important samples to understand population structure and gene flow between mainland Tanzania and Zanzibar, especially from the rural Bagamoyo District, where malaria transmission persists and there is a major port of entry to Zanzibar. In addition, this study includes a larger set of SNPs, providing more robustness for analyses such as PCA and IBD. Therefore, the conclusions of this paper are well supported by data.

Weaknesses:

Some points need to be clarified:

(1) SNPs in linkage disequilibrium (LD) can introduce bias in PCA and IBD analysis. Were SNPs in LD filtered out prior to these analyses?

Thank you for this point. We did not filter SNPs in LD prior to this analysis. In the PCA analysis in Figure 1, we did restrict to a single isolate among those that were clonal (high IBD values) to prevent bias in the PCA. In general, disequilibrium is minimal only over small distances <5-10kb without selective forces at play. This is much less than the average spacing of the markers in the panel. If there is minimal LD, the conclusions drawn on relative levels and connections at high IBD are unlikely to be confounded by any effects of disequilibrium.

( 2) Many IBD algorithms do not handle polyclonal infections well, despite an increasing number of algorithms that are able to handle polyclonal infections and multiallelic SNPs. How polyclonal samples were handled for IBD analysis?

Thank you for this point. We added lines 157-161 to clarify. This section now reads:

“To investigate genetic relatedness of parasites across regions, identity by descent (IBD) estimates were assessed using the within sample major alleles (coercing samples to monoclonal by calling the dominant allele at each locus) and estimated utilizing a maximum likelihood approach using the inbreeding_mle function from the MIPanalyzer package (Verity et al., 2020). This approach has previously been validated as a conservative estimate of IBD (Verity et al., 2020).”

Please see the supplement in (Verity et al., 2020) for an extensive simulation study that validates this approach.

Reviewer #1 (Recommendations For The Authors):

(3) I think Supplementary Figures 8 and 9 are more visually informative than Figure 2.

Thank you for your response. We performed the analysis in Figure 2 to show how IBD varies between different regions and is higher within a region than between.

Reviewer #2 (Public Review):

This manuscript describes P. falciparum population structure in Zanzibar and mainland Tanzania. 282 samples were typed using molecular inversion probes. The manuscript is overall well-written and shows a clear population structure. It follows a similar manuscript published earlier this year, which typed a similar number of samples collected mostly in the same sites around the same time. The current manuscript extends this work by including a large number of samples from coastal Tanzania, and by including clinical samples, allowing for a comparison with asymptomatic samples.

The two studies made overall very similar findings, including strong small-scale population structure, related infections on Zanzibar and the mainland, near-clonal expansion on Pemba, and frequency of markers of drug resistance. Despite these similarities, the previous study is mentioned a single time in the discussion (in contrast, the previous research from the authors of the current study is more thoroughly discussed). The authors missed an opportunity here to highlight the similar findings of the two studies.

Thank you for your insights. We appreciated the level of detail of your review and it strengthened our work. We have input additional sentences on lines 292-295, which now reads:

“A recent study investigating population structure in Zanzibar also found local population microstructure in Pemba (Holzschuh et al., 2023). Further, both studies found near-clonal parasites within the same district, Micheweni, and found population microstructure over Zanzibar.”

Strengths:

The overall results show a clear pattern of population structure. The finding of highly related infections detected in close proximity shows local transmission and can possibly be leveraged for targeted control.

Weaknesses:

A number of points need clarification:

(1) It is overall quite challenging to keep track of the number of samples analyzed. I believe the number of samples used to study population structure was 282 (line 141), thus this number should be included in the abstract rather than 391. It is unclear where the number 232 on line 205 comes from, I failed to deduct this number from supplementary table 1.

Thank you for this point. We have included 282 instead of 391 in the abstract. We added a statement in the results at lines 203-205 to clarify this point, which now reads:

“PCA analysis of 232 coastal Tanzanian and Zanzibari isolates, after pruning 51 samples with an IBD of greater than 0.9 to one representative sample, demonstrates little population differentiation (Figure 1A).”

(2) Also, Table 1 and Supplementary Table 1 should be swapped. It is more important for the reader to know the number of samples included in the analysis (as given in Supplementary Table 1) than the number collected. Possibly, the two tables could be combined in a clever way.

Thank you for this advice. Rather than switch to another table altogether, we appended two columns to the original table to better portray the information (see Table 1).

Methods

(3) The authors took the somewhat unusual decision to apply K-means clustering to GPS coordinates to determine how to combine their data into a cluster. There is an obvious cluster on Pemba islands and three clusters on Unguja. Based on the map, I assume that one of these three clusters is mostly urban, while the other two are more rural. It would be helpful to have a bit more information about that in the methods. See also comments on maps in Figures 1 and 2 below.

Cluster 3 is a mix of rural/urban while the clusters 2, 4 and 5 are mostly rural. This analysis was performed to see how IBD changes in relation to local context within different regions in Zanzibar, showing that there is higher IBD within locale than between locale.

(4) Following this point, in Supplemental Figure 5 I fail to see an inflection point at K=4. If there is one, it will be so weak that it is hardly informative. I think selecting 4 clusters in Zanzibar is fine, but the justification based on this figure is unclear.

The K-means clustering experiment was used to cluster a continuous space of geographic coordinates in order to compare genetic relatedness in different regions. We selected this inflection point based on the elbow plot and based the number to obtain sufficient subsections of Zanzibar to compare genetic relatedness. This point is added to the methods at lines 174-178, which now reads:

“The K-means clustering experiment was used to cluster a continuous space of geographic coordinates in order to compare genetic relatedness in different regions. We selected K = 4 as the inflection point based on the elbow plot (Supplemental Figure 5) and based the number to obtain sufficient subsections of Zanzibar to compare genetic relatedness.”

(5) For the drug resistance loci, it is stated that "we further removed SNPs with less than 0.005 population frequency." Was the denominator for this analysis the entire population, or were Zanzibar and mainland samples assessed separately? If the latter, as for all markers <200 samples were typed per site, there could not be a meaningful way of applying this threshold. Given data were available for 200-300 samples for each marker, does this simply mean that each SNP needed to be present twice?

Population frequency is calculated based on the average within sample allele frequency of each individual in the population, which is an unbiased estimator. Within sample allele frequency can range from 0 to 1. Thus, if only one sample has an allele and it is at 0.1 within sample frequency, the population allele frequency would be 0.1/100 = 0.001. This allele is removed even though this would have resulted in a prevalence of 0.01. This filtering is prior to any final summary frequency or prevalence calculations (see MIP variant Calling and Filtering section in the methods). This protects against errors occurring only at low frequency.

Discussion:

(6) I was a bit surprised to read the following statement, given Zanzibar is one of the few places that has an effective reactive case detection program in place: "Thus, directly targeting local malaria transmission, including the asymptomatic reservoir which contributes to sustained transmission (Barry et al., 2021; Sumner et al., 2021), may be an important focus for ultimately achieving malaria control in the archipelago (Björkman & Morris, 2020)." I think the current RACD program should be mentioned and referenced. A number of studies have investigated this program.

Thank you for this point. We have added additional context and clarification on lines 275-280, which now reads:

“Thus, directly targeting local malaria transmission, including the asymptomatic reservoir which contributes to sustained transmission (Barry et al., 2021; Sumner et al., 2021), may be an important focus for ultimately achieving malaria control in the archipelago (Björkman & Morris, 2020). Currently, a reactive case detection program within index case households is being implemented, but local transmission continues and further investigation into how best to control this is warranted (Mkali et al. 2023).”

(7) The discussion states that "In Zanzibar, we see this both within and between shehias, suggesting that parasite gene flow occurs over both short and long distances." I think the term 'long distances' should be better defined. Figure 4 shows that highly related infections rarely span beyond 20-30 km. In many epidemiological studies, this would still be considered short distances.

Thank you for this point. We have edited the text at lines 287-288 to indicate that highly related parasites mainly occur at the range of 20-30km, which now reads:

“In Zanzibar, highly related parasites mainly occur at the range of 20-30km.”

(8) Lines 330-331: "Polymorphisms associated with artemisinin resistance did not appear in this population." Do you refer to background mutations here? Otherwise, the sentence seems to repeat lines 324. Please clarify.

We are referring to the list of Pfk13 polymorphisms stated in the Methods from lines 146-148. We added clarifying text on lines 326-329:

“Although polymorphisms associated with artemisinin resistance did not appear in this population, continued surveillance is warranted given emergence of these mutations in East Africa and reports of rare resistance mutations on the coast consistent with spread of emerging Pfk13 mutations (Moser et al., 2021). “

(9) Line 344: The opinion paper by Bousema et al. in 2012 was followed by a field trial in Kenya (Bousema et al, 2016) that found that targeting hotspots did NOT have an impact beyond the actual hotspot. This (and other) more recent finding needs to be considered when arguing for hotspot-targeted interventions in Zanzibar.

We added a clarification on this point on lines 335-345, which now reads:

“A recent study identified “hotspot” shehias, defined as areas with comparatively higher malaria transmission than other shehias, near the port of Zanzibar town and in northern Pemba (Bisanzio et al., 2023). These regions overlapped with shehias in this study with high levels of IBD, especially in northern Pemba (Figure 4). These areas of substructure represent parasites that differentiated in relative isolation and are thus important locales to target intervention to interrupt local transmission (Bousema et al., 2012). While a field cluster-randomized control trial in Kenya targeting these hotspots did not confer much reduction of malaria outside of the hotspot (Bousema et al. 2016), if areas are isolated pockets, which genetic differentiation can help determine, targeted interventions in these areas are likely needed, potentially through both mass drug administration and vector control (Morris et al., 2018; Okell et al., 2011). Such strategies and measures preventing imported malaria could accelerate progress towards zero malaria in Zanzibar.”

Figures and Tables:

(10) Table 2: Why not enter '0' if a mutation was not detected? 'ND' is somewhat confusing, as the prevalence is indeed 0%.

Thank you for this point. We have put zero and also given CI to provide better detail.

(11) Figure 1: Panel A is very hard to read. I don't think there is a meaningful way to display a 3D-panel in 2D. Two panels showing PC1 vs. PC2 and PC1 vs. PC3 would be better. I also believe the legend 'PC2' is placed in the wrong position (along the Y-axis of panel 2).

Supplementary Figure 2B suffers from the same issue.

Thank you for your comment. A revised Figure 1 and Supplemental Figure 2 are included, where there are separate plots for PC1 vs. PC2 and PC1 vs. PC3.

(12) The maps for Figures 1 and 2 don't correspond. Assuming Kati represents cluster 4 in Figure 2, the name is put in the wrong position. If the grouping of shehias is different between the Figures, please add an explanation of why this is.

Thank you for this point. The districts with at least 5 samples present are plotted in the map in Figure 1B. In Figure 2, a totally separate analysis was performed, where all shehias were clustered into separate groups with k-means and the IBD values were compared between these clusters. These maps are not supposed to match, as they are separate analyses. Figure 1B is at the district level and Figure 2 is clustering shehias throughout Zanzibar.

The figure legend of Figure 1B on lines 410-414 now reads:

“B) A Discriminant Analysis of Principal Components (DAPC) was performed utilizing isolates with unique pseudohaplotypes, pruning highly related isolates to a single representative infection. Districts were included with at least 5 isolates remaining to have sufficient samples for the DAPC. For plotting the inset map, the district coordinates (e.g. Mainland, Kati, etc.) are calculated from the averages of the shehia centroids within each district.”

The figure legend of Figure 2 on lines 417-425 now reads:

“Figure 2. Coastal Tanzania and Zanzibari parasites have more highly related pairs within their given region than between regions. K-means clustering of shehia coordinates was performed using geographic coordinates all shehias present from the sample population to generate 5 clusters (colored boxes). All shehias were included to assay pairwise IBD between differences throughout Zanzibar. Pairwise comparisons of within cluster IBD (column 1 of IBD distribution plots) and between cluster IBD (column 2-5 of IBD distribution plots) was done for all clusters. In general, within cluster IBD had more pairwise comparisons containing high IBD identity.”

(13) Figure 2: In the main panel, please clarify what the lines indicate (median and quartiles?). It is very difficult to see anything except the outliers. I wonder whether another way of displaying these data would be clearer. Maybe a table with medians and confidence intervals would be better (or that data could be added to the plots). The current plots might be misleading as they are dominated by outliers.

Thank you for this point and it greatly improved this figure. We changed the plotting mechanisms through using a beeswarm plot, which plots all pairwise IBD values within each comparison group.

(14) In the insert, the cluster number should not only be given as a color code but also added to the map. The current version will be impossible to read for people with color vision impairment, and it is confusing for any reader as the numbers don't appear to follow any logic (e.g. north to south).

Thank you very much for these considerations. We changed the color coding to a color blind friendly palette and renamed the clusters to more informative names; Pemba, Unguja North (Unguja_N), Unguja Central (Unguja_C), Unguja South (Unguja_S) and mainland Tanzania (Mainland).

(15) The legend for Figure 3 is difficult to follow. I do not understand what the difference in binning was in panels A and B compared to C.

Thank you for this point. We have edited the legend to reflect these changes. The legend for Figure 3 on lines 427-433 now reads:

“Figure 3. Isolation by distance is shown between all Zanzibari parasites (A), only Unguja parasites (B) and only Pemba parasites (C). Samples were analyzed based on geographic location, Zanzibar (N=136) (A), Unguja (N=105) (B) or Pemba (N=31) (C) and greater circle (GC) distances between pairs of parasite isolates were calculated based on shehia centroid coordinates. These distances were binned at 4km increments out to 12 km. IBD beyond 12km is shown in Supplemental Figure 8. The maximum GC distance for all of Zanzibar was 135km, 58km on Unguja and 12km on Pemba. The mean IBD and 95% CI is plotted for each bin.”

(16) Font sizes for panel C differ, and it is not aligned with the other panels.

Thank you for pointing this out. Figure 3 and Supplemental Figure 10 are adjusted with matching formatting for each plot.

(17) Why is Kusini included in Supplemental Figure 4, but not in Figure 1?

In Supplemental Figure 4, all isolates were used in this analysis and isolates with unique pseudohaplotypes were not pruned to a single representative infection. That is why there are additional isolates in Kusini. The legend for Supplemental Figure 4 now reads:

“Supplemental Figure 4. PCA with highly related samples shows population stratification radiating from coastal Mainland to Zanzibar. PCA of 282 total samples was performed using whole sample allele frequency (A) and DAPC was performed after retaining samples with unique pseudohaplotypes in districts that had 5 or more samples present (B). As opposed to Figure 1, all isolates were used in this analysis and isolates with unique pseudohaplotypes were not pruned to a single representative infection.”

(18) Supplemental Figures 6 and 7: What does the width of the line indicate?

The sentence below was added to the figure legends of Supplemental Figures 6 and 7 and the legends of each network plot were increased in size:

“The width of each line represents higher magnitudes of IBD between pairs.”

(19) What was the motivation not to put these lines on the map, as in Figure 4A? This might make it easier to interpret the data.

Thank you for this comment. For Supplemental Figure 8 and 9, we did not put these lines that represent lower pairwise IBD to draw the reader's attention to the highly related pairs between and within shehias.

Reviewer #2 (Recommendations For The Authors):

(1) There is a rather long paragraph (lines 300-323) on COI of asymptomatic infections and their genetic structure. Given that the current study did not investigate most of the hypotheses raised there (e.g. immunity, expression of variant genes), and the overall limited number of asymptomatic samples typed, this part of the discussion feels long and often speculative.

Thank you for your perspective. The key sections highlighted in this comment, regarding immunity and expression of variant genes, were shortened. This section on lines 300-303 now reads:

“Asymptomatic parasitemia has been shown to be common in falciparum malaria around the globe and has been shown to have increasing importance in Zanzibar (Lindblade et al., 2013; Morris et al., 2015). What underlies the biology and prevalence of asymptomatic parasitemia in very low transmission settings where anti-parasite immunity is not expected to be prevalent remains unclear (Björkman & Morris, 2020).”

(2) As a detail, line 304 mentions "few previous studies" but only one is cited. Are there studies that investigated this and found opposite results?

Thank you for this comment. We added additional studies that did not find an association between clinical disease and COI. These changes are on lines 303-308, which now reads:

“Similar to a few previous studies, we found that asymptomatic infections had a higher COI than symptomatic infections across both the coastal mainland and Zanzibar parasite populations (Collins et al., 2022; Kimenyi et al., 2022; Sarah-Matio et al., 2022). Other studies have found lower COI in severe vs. mild malaria cases (Robert et al., 1996) or no significant difference between COI based on clinical status (Earland et al. 2019; Lagnika et al. 2022; Conway et al. 1991; Kun et al. 1998; Tanabe et al. 2015)”

(3) Table 2: Percentages need to be checked. To take one of several examples, for Pfk13-K189N a frequency of 0.019 for the mutant allele is given among 137 samples. 2/137 equals to 0.015, and 3/137 to 0.022. 0.019 cannot be achieved. The same is true for several other markers. Possibly, it can be explained by the presence of polyclonal infections. If so, it should be clarified what the total of clones sequenced was, and whether the prevalence is calculated with the number of samples or number of clones as the denominator.

Thank you for this point. We mistakenly reported allele frequency instead of prevalence. An updated Table 2 is now in the manuscript. The method for calculating the prevalence is now at lines 148-151:

“Prevalence was calculated separately in Zanzibar or mainland Tanzania for each polymorphism by the number of samples with alternative genotype calls for this polymorphism over the total number of samples genotyped and an exact 95% confidence interval was calculated using the Pearson-Klopper method for each prevalence.”

eLife assessment

Connelly and colleagues provide convincing genetic evidence that importation from mainland Tanzania is a major source of Plasmodium falciparum lineages currently circulating in Zanzibar. This study also reveals ongoing local malaria transmission and occasional near-clonal outbreaks in Zanzibar. Overall, the manuscript effectively highlights the role of human movements in maintaining residual malaria transmission in an area targeted for intensive control interventions over the past decades and provides clear and valuable information for epidemiologists and public health professionals.

Reviewer #2 (Public Review):

Summary:

This manuscript describes P. falciparum population structure in Zanzibar and mainland Tanzania. 282 samples were typed using molecular inversion probes. The manuscript is overall well written and shows clear population structure. It follows a similar manuscript published earlier this year, which typed a similar number of samples collected mostly in the same sites around the same time. The current manuscript extends this work by including a large number of samples from coastal Tanzania, and by including clinical samples, allowing for a comparison with asymptomatic samples.

The two studies made overall very similar findings, including strong small-scale population structure, related infections on Zanzibar and the mainland, near-clonal expansion on Pemba, and frequency of markers of drug resistance.

Strengths:

The overall results show a clear pattern of population structure. The finding of highly related infections detected in close proximity shows local transmission and can possibly be leveraged for targeted control.

Comments on revised version:

The authors have addressed my comments.

add a new ref

Author response:

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

Reviewer #1 (Public Review):

Granados-Aparici et al., investigate somatic-germline interactions in female mice. Mammalian oocytes are nurtured in multi-cellular ovarian follicles and communication with surrounding somatic cells is critical for oocyte development. This study focused on transzonal projections (TZP) extending from granulosa cells to the surface of oocytes and documented the importance of SMAD4, a TGF- β mediator, in regulating the TZPs. They propose a model in which individual TZPs contact the surface of the oocyte and stably attach if there is sufficient N-cadherin. In SMAD4-depleted cells, there is insufficient N-cadherin to stabilize the attachment. The TZP continues to elongate but eventually retracts. Their model is well supported by their experimental evidence and the manuscript is both well-formulated and written.

Reviewer #2 (Public Review):

Summary:

This study proposed a new mechanism by which the TGF-beta signaling pathway promotes contacts between oocytes and the surrounding somatic cells in mice, by regulating the numbers of transzonal projections (TZPs).

Strengths:

The conditional Smad4 knockout and three-dimensional observation of transzonal projections are solid and sufficiently support the major conclusions.

Weaknesses:

The physiological significance of SMAD4-dependent formation of transzonal projection networks is not assessed in this study.

Previous studies have shown that physical contact and gap junctional communication with the granulosa cells is essential for normal oocyte development. A recent study has also shown that depleting Myo10 in granulosa cells reduces the number of TZPs and leads to abnormalities in oocyte and embryo development. Thus, the importance of TZPs is well-established. These findings, which were insufficiently brought out in the Introduction of the original manuscript, have now been made more clearly (Introduction, 2nd paragraph). We recognize that these reports do not directly test a role for SMAD4-dependent TZPs. Unfortunately, it is beyond our technical capacity to obtain embryos following meiotic maturation and fertilization of oocytes that have grown in vitro, which wold be necessary for us to fully test the physiological role of SMAD4-dependent TZPs.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) The authors switch from Amhr2-cre to ER-cre to increase the number of GFP-positive granulosa cells in 12 d/o ovaries. To avoid disruption of FSH secretion by SMAD4, they use an in vitro model that requires 6 days in GEO culture (1 d tamoxifen + 5 d). Could it be that Amhr2-cre didn't work because most follicles would not have reached the atretic preantral stage in 12 d/o ovaries? Did the authors consider 6 days in vitro GEO culture to determine if Amhr2-cre would be efficient and avoid exposure to tamoxifen?

Please see below.

When is Amhr2 expressed?

Previous studies (Jorgez et al, 2004; Pangas et al, 2006) report that Amhr2 is expressed in growing follicles that have progressed beyond a single layer of granulosa cells (often defined as secondary and primary follicles, respectively). As shown in Fig. 1C, we did not observe evidence of widespread Cre activity in multilayer follicles. At least two factors may contribute why we observed relatively weak Cre activity. One possibility is that, on the genetic background our mice, Amhr2 is expressed relatively late during follicular growth. Thus, we might have observed more GFP-positive granulosa cells in antral or pre-ovulatory follicles. Because the granulosa cells of these late-stage follicles would already have produced many TZPs, the number of new TZPs generated in wild-type but not SMAD4-depleted cells after Amhr2 activation would be a relatively small proportion of the total population. This would make it more difficult to detect a reduction in TZP number in the absence of SMAD4.

A second point is that we used pre-puberal mice whereas Jorgez et al examined Amhr2 expression in ovaries of adult mice. Pangas et al evaluated both prepuberal and adult females. It may be that Amhr2 is expressed earlier or more strongly in granulosa cells of adult mice. Regarding the suggestion to culture complexes obtained from mice on the Amhr2-Cre background, as this might allow widespread expression of Cre without the need for tamoxifen, this is an excellent idea. If there is considerable heterogeneity among cells in the timing of Amhr2-Cre activity, though, this may further cloud efforts to uncover the role of SMAD4 in the production or stability of TZPs, as noted above.

(2) Did most of the GEO cultured in vitro reach the antral follicle stage after 6 days?

Since GOCs were treated with collagenase, the thecal layer was removed. Therefore, development of an antrum does not occur. We observed that, in some cases, the oocyte was extruded from the granulosa cell mass. These abnormal complexes were discarded.

(3). Was the development/diameter of the oocyte in the GEO comparable to the oocyte growing in vivo?

We did not compare the diameter of the oocytes grown in vitro to those grown in vivo. Thus, we cannot say whether the oocytes grown in vitro reached the same size as those grown in vivo. We did, however, compare the diameter of the oocytes in the wt and ko groups and observed no difference (Figure 2). This indicates that depletion of SMAD4 in the granulosa cells does not impair oocyte growth. Importantly for our studies, it excludes the possibility that the reduction in TZP-number is simply due to a smaller surface area of the oocyte.

(4) SMAD4 depletion in granulosa cells disrupts steroidogenesis leading to increased progesterone levels and precocious luteinization of granulosa cells (Pangas et al., 2006). Did the authors determine the expression level of luteal markers of granulosa cells in the in vitro GEO culture Smad4 knockout model? Are their observations direct effects of the absence of SMAD4?

This is an excellent point. We checked our previously performed RNA-seq analysis of the wild-type and knockout granulosa cells, but found no difference in the quantities of Cyp11a1, Sfrp4, Star or Ptgfr. This is now described in the Discussion (4th paragraph). One potentially important difference between our study and that of Pangas et al (2006) is that they observed premature luteinization when prepuberal (3-week old) mice were injected with the FSH analogue, equine serum gonadotropin, whereas we studied granulosa-oocyte complexes cultured in vitro. This could underlie the apparent differences with respect to luteinization.

(5) Could the reduced number of TZPs in ER-cre+; Smad4fl/fl GOCs be explained by luteinization?

This interesting and logical possibility is related to the previous point. In other words, luteinization could be considered as a default pathway of differentiation that is suppressed by SMAD signaling. It is possible that luteinized cells are unable to generate or maintain TZPs. This model offers a potential mechanistic basis for our observation, and we now raise it in the Discussion (3rd paragraph).

Reviewer #3 (Recommendations For The Authors):

The expression and localization of N-cadherin should be observed in Smad4 and control granulosa cell-oocyte complexes.

We agree that this would be an excellent approach to confirm the decreased expression of N-cadherin in the granulosa cells that was observed by immunoblotting. We were confronted by two challenges, however. First, we were unable to consistently obtain strong staining of granulosa cell membranes in the inner layers of multilayer granulosa-oocyte complexes. Other antibodies are able to stain structures at the oocyte surface, indicating that antibodies are not physically blocked from penetrating the complex. More likely, the anti-N-cadherin does not bind its target strongly enough to generate a robust signal that can be detected through multiple overlying layers of cells. Second, whereas for immunoblotting we collect all granulosa cells from culture complexes, for immunofluorescence we are only able to examine those that remain in the complex. This means that, for immunofluorescence, we essentially but unavoidably select against cells that are only loosely attached – as would be expected for N-cadherin-deficient cells – to their neighbours. Given these challenges, we believe that the immunoblotting approach, which produced highly reproducible results over six biological replicates (Fig. 6), is the most reliable.

Reviewer #1 (Public Review):

Granados-Aparici et al., investigate somatic-germline interactions in female mice. Mammalian oocytes are nurtured in multi-cellular ovarian follicles and communication with surrounding somatic cells is critical for oocyte development. This study focused on transzonal projections (TZP) extending from granulosa cells to the surface of oocytes and document the importance of SMAD4, a TGF- β mediator, in regulating the TZPs. They propose a model in which individual TZPs contact the surface of the oocyte and stably attaches if there is sufficient N-cadherin. In SMAD4-depleted cells, there is insufficient N-cadherin to stabilize the attachment. The TZP continues to elongate but eventually retracts. Their model is well supported by their experimental evidence and the manuscript is both well-formulated and written.

Comments on revised version:

The authors have addressed the issues raised in the original review.

Reviewer #2 (Public Review):

Summary:

This study proposed a new mechanism by which TGF-beta signaling pathway promotes contacts between oocyte and the surrounding somatic cells in mouse, by regulating the numbers of transzonal projections (TZPs).

Strengths:

The conditional Smad4 knockout and three-dimensional observation of transzonal projections are solid and sufficiently support the major conclusions.

Comments on revised version:

The authors have adequately addressed the reviewers' questions and comments.

Author response:

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

eLife assessment

This study presents useful findings regarding the role of formin-like 2 in mouse oocyte meiosis. The submitted data are supported by incomplete analyses, and in some cases, the conclusions are overstated. If these concerns are addressed, this paper would be of interest to reproductive biologists.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The presented study focuses on the role of formin-like 2 (FMNL2) in oocyte meiosis. The authors assessed FMNL2 expression and localization in different meiotic stages and subsequently, by using siRNA, investigated the role of FMNL2 in spindle migration, polar body extrusion, and distribution of mitochondria and endoplasmic reticulum (ER) in mouse oocytes.

Strengths:

Novelty in assessing the role of formin-like 2 in oocyte meiosis.

Weaknesses:

Methods are not properly described.

Overstating presented data.

It is not clear what statistical tests were used.

My main concern is that there are missing important details of how particular experiments and analyses were done. The material and methods section are not written in the way that presented experiments could be repeated - it is missing basic information (e.g., used mouse strain, timepoints of oocytes harvest for particular experiments, used culture media, image acquisition parameters, etc.). Some of the presented data are overstated and incorrectly interpreted. It is not clear to me how the analysis of ER and mitochondria distribution was done, which is an important part of the presented data interpretation. I'm also missing important information about the timing of particular stages of assessed oocytes because the localization of both ER and mitochondria differs at different stages of oocyte meiosis. The data interpretation needs to be justified by proper analysis based on valid parameters, as there is considerable variability in the ER and mitochondria structure and localization across oocytes based on their overall quality and stage.

Thank you for your comment. We regret the oversight of omitting critical information in the manuscript. In the revised manuscript, we have included essential details such as mouse strains, culture media, stages of oocyte and statistical methods in the materials and methods section. Please find our details responses in the “Recommendations for the authors” part.

Reviewer #2 (Public Review):

Summary:

This research involves conducting experiments to determine the role of Fmnl2 during oocyte meiosis I.

Strengths:

Identifying the role of Fmnl2 during oocyte meiosis I is significant.

Weaknesses:

The quantitative analysis and the used approach to perturb FMNL2 function are currently incomplete and would benefit from more confirmatory approaches and rigorous analysis.

(1) Most of the results are expected. The new finding here is that FMNL2 regulates cytoplasmic F-actin in mouse oocytes, which is also expected given the role of FMNL2 in other cell types. Given that FMNL2 regulates cytoplasmic F-actin, it is very expected to see all the observed phenotypes. It is already established that F-actin is required for spindle migration to the oocyte cortex, extruding a small polar body and normal organelle distribution and functions.

Thank you for your comment. In the recent decade, Arp2/3 complex (Nat Cell Biol 2011), Formin2 (Nat Cell Biol 2002, Nat Commun 2020), and Spire (Curr Biol 2011) were reported to be 3 key factors to involve into this process. These factors regulate actin filaments in different ways. However, how they cross with each other for the subcellular events were still fully clear. Our current study identified that FMNL2 played a critical role in coordinating these molecules for actin assembly in oocytes. Our findings demonstrate that FMNL2 interacts with both the Arp2/3 complex and Formin2 to facilitate actin-based meiotic spindle migration. Additionally, we discovered a novel role for FMNL2 in determining the distribution and function of the endoplasmic reticulum and mitochondria, which may in turn influence meiotic spindle migration in oocytes. Our results not only uncover the novel functions of FMNL2-mediated actin for organelle distribution, but also extend our understanding of the molecular basis for the unique meiotic spindle migration in oocyte meiosis.

(2) The authors used Fmnl2 cRNA to rescue the effect of siRNA-mediated knockdown of Fmnl2. It is not clear how this works. It is expected that the siRNA will also target the exogenous cRNA construct (which should have the same sequence as endogenous Fmnl2) especially when both of them were injected at the same time. Is this construct mutated to be resistant to the siRNA?

Thank you for your question. We regret any misunderstanding that may have been caused by the inappropriate description in our manuscript. In the rescue experiments, we initially injected FMNL2 siRNA into oocytes, followed by the microinjection of FMNL2 mRNA 18-20 hours later. After conducting our previous experiments, we have verified through Western blotting that endogenous FMNL2 is effectively suppressed 18-20 hours following the microinjection of FMNL2 siRNA. Additionally, we observed a significant increase in exogenous FMNL2 protein expression 2 hours after the injection of FMNL2 mRNA. We believe that the exogenous FMNL2 could compensate the decrease by FMNL2 knockdown, and this approach was adopted in many oocyte studies.

(3) The authors used only one approach to knockdown FMNL2 which is by siRNA. Using an additional approach to inhibit FMNL2 would be beneficial to confirm that the effect of siRNA-mediated knockdown of FMNL2 is specific.

Thank you for your question. Yes, the specificity is always the concern for siRNA or morpholino microinjection due to the off-target issue. Due to the limitation we could not generate the knock out model, and there are no known inhibitors with specific targeting capabilities for FMNL2. To solve this, we performed the rescue study with exogenous mRNA to confirm the effective knock down of FMNL2. These measures provide reassurance regarding the credibility of the experimental outcomes, and this is also the general way to avoid the off-target of siRNA or morpholino.

Reviewer #3 (Public Review):

Summary:

The authors focus on the role of formin-like protein 2 in the mouse oocyte, which could play an important role in actin filament dynamics. The cytoskeleton is known to influence a number of cellular processes from transcription to cytokinesis. The results show that downregulation of FMNL2 affects spindle migration with resulting abnormalities in cytokinesis in oocyte meiosis I.

Weaknesses:

The overall description of methods and figures is overall dismissively poor. The description of the sample types and number of replicate experiments is impossible to interpret throughout, and the quantitative analysis methods are not adequately described. The number of data points presented is unconvincing and unlikely to support the conclusions. On the basis of the data presented, the conclusions appear to be preliminary, overstated, and therefore unconvincing.

Thank you for your comment. We regret the oversight of omitting critical information in the manuscript. In the revised manuscript, we have incorporated your suggestions for modification, particularly regarding the Materials and Methods section. Please see the detailed revision and responses in the “Recommendations for the authors” part.

Recommendations for the authors:

Reviewer #1 (Recommendations for The Authors):

My main concern is that there are missing important details of how particular experiments and analyses were done. The material and methods section is not written in the way that presented experiments could be repeated - it is missing basic information (e.g., used mouse strain, timepoints of oocytes harvest for particular experiments, used culture media, image acquisition parameters, etc.). Some of the presented data are overstated and incorrectly interpreted. It is not clear to me how the analysis of ER and mitochondria distribution was done, which is an important part of the presented data interpretation. I'm also missing important information about the timing of particular stages of assessed oocytes because the localization of both ER and mitochondria differs at different stages of oocyte meiosis. The data interpretation needs to be justified by proper analysis based on valid parameters, as there is considerable variability in the ER and mitochondria structure and localization across oocytes based on their overall quality and stage. My specific comments are listed below.

(1) Information about statistical tests that were used needs to be provided for all quantification experiments.

Thank you for your suggestion. Based on your suggestions, we revised the statistical analysis description in the Materials and Methods section. Additionally, we also included a description of the statistical methods in the legends of the relevant result figures.

(2) I recommend replacing the plunger plots, used in most quantification data, with alternatives allowing evaluation of the distribution of the data (dot plots, box plots, whisker plots).

Thank you for your suggestion. Following your suggestion, we replaced the plunger plots in Fig 2C, D, H, I and Fig3 B, C with dot plots.

(3) Can the authors provide information about particular time points when were individual oocyte stages (GVBD, meiosis I, and meiosis II) harvested/used for immunofluorescence protein detection, western blotting, microinjection, and ER and mitochondria staining? Were the time points always the same in all presented experiments and experimental vs control group? If not, this needs to be clarified.

Thank you for your suggestion. We used oocytes in the metaphase I (MI) stage for the statistical analysis of spindle migration, actin filament aggregation, endoplasmic reticulum localization, and mitochondrial localization. In the Western blot analysis, GV stage oocytes were utilized to evaluate the efficiency of knockdown and rescue experiments. The protein expression levels of Arp2, Formin2, INF2, Cofilin, Grp78, and Chop in different treatment groups were detected using MI-stage oocytes. In the revised version, we provided all the detailed information about the stages.

(4) Figure 1B: Can the authors comment on why there is a missing representative image of MII oocyte FMBL2-Ab? I recommend including this in the figure to have a complete view of comparing overexpressed and endogenous FMNL2 localization in oocyte meiosis.

Thank you for your suggestion. In the revised manuscript, we added immunostaining images of FMNL2 antibody in MII stage oocytes.

(5) Figure 1C: The figure legend says, "FMNL2 and actin overlapped in cortex and spindle surrounding". In MI oocytes, there is usually no accumulated actin signal around the spindle, which is also true in the presented images, so there cannot be overlapping with the FMNL2 signal. The interpretation should be changed.

We apologize for this inappropriate description that was used, and we deleted this sentence.

(6) Figure 2B: What were the parameters of the "large" and "normal" polar bodies for performing the analysis?

Thank you for your question. In order to assess the size of the polar body, we conducted a comparison between the diameter of the polar body and that of the oocyte. If the diameter of the polar body was found to be less than 1/3 of the oocyte's diameter, we categorized it as normal-sized polar body. Conversely, if the polar body's diameter exceeded 1/3 of the oocyte's diameter, we categorized it as a large polar body. We have included these details in the Results section of the manuscript.

(7) Figure 2F: Can the authors comment on what can be the second band in the rescue group?

Thank you for your question. In the rescue experiment, we microinjected exogenous FMNL2-EGFP mRNA into the oocytes. As a result, compared to endogenous FMNL2, the protein size increased due to the addition of the EGFP tag, approximately 27 kDa. Hence, in the Western blot bands of the rescue group, the upper band represents the expression of exogenous FMNL2-EGFP, while the lower band corresponds to the expression of endogenous FMNL2. We have provided annotations in the revised Figure 2F to clarify this.

(8) Can the authors comment on the variability of PBE between 2C and 2H in the FMNL2-KD groups? In panel C, the PBE in the KD group was 59.5 {plus minus} 2.82%; in panel H, the PBE in the KD group was 48.34 {plus minus} 4.2%, and in the rescue group, the PBE was 62.62 {plus minus} 3.6%. The rescue group has a similar PBE rate as the KD group in panel C. How consistent was the FMNL2 knockdown across individual replicates? Can the authors provide more details on how the rescue experiment was performed?

Thank you for your question. We believe that the difference in PBE observed in Figure 2C and 2H of the FMNL2-KD group was due to the microinjection times and the duration of in vitro arrest. The results shown in Figure 2C depict the outcome of a single injection of FMNL2 siRNA into GV stage oocytes, followed by 18 hours of in vitro arrest; the results shown in Figure 2H contain a subsequent additional injection of FMNL2-EGFP mRNA with another 2 hours of arrest. The two rounds of microinjection and the extended period of in vitro arrest both affect oocyte maturation rates.

(9). Figure 2J and K: What groups were compared together? The used statistic needs to be properly described.

Thank you for your question. The FMNL2-KD, FMNL3-KD, and FMNL2+3-KD groups were all compared to the Control group, therefore, t-test was used for analysis. We have provided explanations in the revised manuscript.

(10) Figure 4B and C: Can the authors provide representative images without oversaturated actine signal?

Thank you for your question. For the analysis of oocyte F-actin, the F-actin are divided into cortex actin and cytoplasmic actin. Due to the contrast during imaging, the strong cortex actin signals affected the detection of cytoplasmic actin, therefore, it is necessary to increase the scanning index, which will cause the overexpose the cortex actin signal. This is for the better observation of the cytoplasmic signals.

(11) Figure 4G + 5H: Can the authors comment on why they used as a housekeeping gene actin instead of tubulin, which was used in the rest of the WB experiments?

Thank you for your question. In most of the western blot experiments conducted in this study, we used tubulin as a housekeeping gene. However, due to the supply of antibodies by delivery period, we had GAPDH and actin as well for some experiments. These housekeeping genes were all valid for the study.

(12) Based on what parameters was ER considered normally or abnormally distributed, and what stages of oocytes were assessed?

Thank you for your question. In this study, we employed oocytes at the MI stage for the analysis of ER localization. In the MI stage, the ER localized around the spindle, which is regarded as the typical localization pattern. The ER displayed a dispersed distribution throughout the cytoplasm or clustered were categorized as aberrant positioning. We included relevant descriptions in the revised version of the manuscript.

(13) Figure 5H: As a housekeeping gene was used actin - the quantification is labeled as a Grp78 to tubulin ratio.

Thank you for pointing out the error. This is a label mistake and we corrected it.

(14) Information about how JC-1 staining was done needs to be provided.

Thank you for your carefully reading. We included a description of JC1 staining in the Materials and Methods section.

(15). Line 231-232: "As shown in Figure 4A" - the text doesn't correspond to the figure.

Thank you for pointing out the error. We revised this mistake in the revised manuscript by correcting "Fig3A" to "Fig4A."

(16) Line 265: there is probably a missing word "Formin2".

Thank you and we corrected the error and made the necessary changes in the revised manuscript.

Reviewer #2 (Recommendations for The Authors):

(1) Quantification and analysis:

• Fig. 3B: The rate of spindle migration should be quantified based on the distance from the spindle to the cortex. Also, the orientation of the spindle (Z-position) needs to be taken into consideration.

• Fig. 5C, D: It is unclear how the rate of ER distribution was calculated.

• Western blot: In many experiments (such as Fig. 5H), the bands are saturated which will prevent accurate intensity measurements and quantifications.

For spindle migration, we specifically focused on spindles exhibiting a distinctive spindle-like shape with clear bipolarity to eliminate any statistical discrepancies potentially caused by variations in Z-axis alignment. Our criterion for determining successful migration was based on the contact between the spindle pole and the cortical region of the oocyte. Therefore, we think that the rate is better to reflect the phenotype than the distance.

For the examination of ER localization, Reviewer 1 also raised this issue. We utilized oocytes at the MI stage in this study. The ER localized around the spindle in MI stage. The ER displayed a dispersed distribution throughout the cytoplasm or clustered were categorized as aberrant positioning. We included relevant descriptions in the revised version of the manuscript.

For the bands of the western blot results, during the experimental procedure we typically capture multiple images at different exposure levels (3-5 images). In the revised manuscript, we have replaced the inappropriate images with more suitable ones.

(2) Given that all Immunoprecipitation experiments in this manuscript were performed on the whole ovary which contains more somatic cells than oocytes, the results do not necessarily reflect meiotic oocytes. Please consider this possibility during the interpretation.

Thank you for your suggestion. Yes, we agree with you. In the revised manuscript, we made appropriate modifications to the relevant descriptions.

(3) 351-365: The conclusion that Arp2/3 compensates for the decreased formin 2 in FMNL2 knockdown oocytes is a bit unconvincing. 1- In mouse oocytes, it is already known that Arp2/3 and formin 2 regulate different pools of F-actin nucleation. 2- The authors found an increase in Arp2/3 in FMNL2 knockdown oocytes compared to control oocytes without any change in cortical F-actin. Given that Arp2/3 is primarily promoting cortical F-actin, it is expected to see an increase in cortical F-actin in FMNL2 knockdown oocytes, which was not the case.

Thank you for your question. Yes, previous studies showed that formin2 localizes to the cytoplasm of oocytes and accumulates around the spindle, which facilitate cytoplasmic actin assembly. While Arp2/3 is primarily responsible for actin assembly at the cortex region of oocytes. In invasive cells, FMNL2 is mainly localized in the leading edge of the cell, lamellipodia and filopodia tips, to improve cell migration ability by actin-based manner (Curr Biol 2012). We showed that FMNL2 localized both at spindle periphery and cortex, but depletion of FMNL2 did not affect cortex actin intensity. We think that FMNL2 and Arp2/3 both contribute to the cortex actin dynamics, when FMNL2 decreased, ARP2 increased to compensate for this, which maintained the cortex actin level. In the revised manuscript, we have made modifications to avoid excessive extrapolation from our results, ensuring that our conclusions are presented in a more objective manner.

(4) Lines 195-197: The spindle is initially formed soon after the GVBD, so there is no spindle during GVBD. Also, I can't see oocytes at anaphase I or telophase I in this figure. Please revise.

Thank you for your suggestion. We apologize for the inappropriate descriptions that were used. In the revised manuscript, we have made modifications to the respective descriptions in the Results part.

(5) Fig. 2E: It seems that the control oocyte is abnormal with mild cytokinesis defects. Please replace or delete it since this information is already included in Fig. 3A.

Thank you for your suggestion. Based on our observations, during the extrusion of the first polar body in oocytes, there is a temporary occurrence of cellular morphological fragmentation due to cortical reorganization (11h in control oocyte from Fig 2E). However, after the extrusion of the first polar body, the oocyte morphology returns to normal. Figure 2E illustrates the meiotic division process of oocytes, while Figure 3A primarily focuses on the process of oocyte spindle migration. We think that it is better to retain both to present our results.

Reviewer #3 (Recommendations for The Authors):

In the case of the observed phenotype, the stage of GV is important. The phenotypes presented also occur in meiotic or developmentally incompetent oocytes. In addition, the images of GV oocytes appear as NSN, which also show the KD phenotype in Figs. 2 and 3.

Thank you for your concern. As the oocyte grows, the proportion of SN-type oocytes gradually increases. When the oocyte diameter reaches 70-80 μm, the proportion of SN oocytes is approximately 52.7% (Mol Reprod Dev. 1995). In our study, both the control and knockdown groups collected oocytes with a diameter of around 80 μm, which is considered as fully-grown oocytes, predominantly in the SN phase. Since the collection period and size of the oocytes were consistent, we can sure that the observed differences between the control and knockdown groups in phenotype analysis could be solid and reliable.

MII is absent in Fig. 1B.

In the revised manuscript, we added immunostaining images of FMNL2 in MII stage oocytes.

The result of KD is not convincing. Also, discuss whether the heterozygous effect of Fmnl2 deletion affects reproductive fitness.

Thank you for your concern. In our investigation, limited to the setup of knock out model, we employed siRNA to knockdown FMNL2 expression, to avoid the risk of off-target, we performed rescue experiment with exogenous mRNA, which we believe that it could solve this issue. When designing siRNA sequences, we ensured their specificity for binding to FMNL2 mRNA only, and we assessed the levels of FMNL2 and FMNL3 mRNA in oocytes after injection of FMNL2 siRNA. The results showed that, compared to the control group, the expression of FMNL2 mRNA decreased by approximately 70% after 18 hours of FMNL2 siRNA injection, while the level of FMNL3 mRNA was not decreased.

Fig. 2F rescue experiment with double bands. What bands are seen here? Did the authors inject tagged or untagged FMNL2? Or does endogenous FMNL2 appear higher in the sample after KD?

Thank you for your question. In the rescue experiment, we microinjected exogenous FMNL2-EGFP mRNA into the oocytes. As a result, compared to endogenous FMNL2, the protein size increased due to the addition of the EGFP tag, approximately 27 kDa. Hence, in the Western blot bands of the rescue group, the upper band represents the expression of exogenous FMNL2-EGFP, while the lower band corresponds to the expression of endogenous FMNL2. We provided annotations in the revised Figure 2F to clarify this.

Variability in mitochondria and ER distribution patterns is also known in healthy and developing oocytes, although the authors described only a single phenotype.

Thank you for your concern. Yes, mitochondria and ER show dynamic localization in different stage of oocyte maturation. However, in this study we employed oocyte MI stage for the analysis of ER and mitochondria localization, and in MI stage, both the ER and mitochondria localize around the spindle. This pattern is considered as the normal localization. Several studies showed that dispersed or clustered localization contributed to maturation defects. We included relevant descriptions in the revised manuscript.

What exactly is meant by input in the IP experiments? Why is the target missing in the input sample?

Thank you for your question. We subjected the input samples to electrophoresis on a single channel, all the analyzed proteins demonstrated normal expression, thereby confirming the viability of the input sample. However, upon simultaneous exposure with the IP samples, we observed a lack of clear signal for certain proteins in the input group. This phenomenon is due to the excessive signal intensity resulting from protein enrichment in the IP group, which caused the low exposure of proteins in input group.

Explain the rationale for using, actin or tubulin as loading or normalization controls in the study focusing on the cytoskeleton.

Thank you for your question. Actin and tubulin are both widely used as the control due to their stable expression. For actin, there are α-actin and β-actin isoforms. Formins and Arp2/3 complex regulate the polymerization of α-actin and β-actin to form F-actin, not isoform expression. In our study F-actin (the functional type) was examined. While α-tubulin and β-tubulin are two subtypes of tubulin, and they interact with each other to form stable α/β-tubulin heterodimers. The changes of cytoskeleton dynamics could not change the expression of α/β-tubulin. Therefore, β-actin and α-tubulin could be used as normalization controls.

Fig. 6E shows only , but the legend says *.

Thank you for pointing out the error. We correct the mistake in the revised manuscript.

Spindle positioning appears to differ between control and KD. Does this affect the quantification of Fig. 6F? Adequate nomenclature should be used here.

Thank you for your question. Yes, spindle positioning was affected by FMNL2 depletion. However, central spindle or cortex spindle all belong to MI stage, and JC1 is not related with the stage difference. To avoid misunderstanding we replaced the representative images and corresponding description in Figure 6F.

The description of the methods and legends should be significantly improved.

Thank you for your suggestion. Reviewer 1 and 2 also raised the similar concern. We enriched the description of methods and legends in the revised manuscript.

https://www.liberation.fr/environnement/climat/en-2023-leurope-a-connu-un-nombre-record-de-jours-de-stress-thermique-extreme-prejudiciables-pour-la-sante-20240422_UOESSBADENAHTGZ6ORSZWQ36DU/

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Extreme Regenfälle sorgen seit Monaten in großen Teilen Frankreichs für Überflutungen. Sie folgen auf eine lange Trockenperiode. Der Wechsel von Trockenheit und starken Niederschlägen entspricht den Prognosen der Klimaforschung für das Wetter in Frankreich später in diesem Jahrhundert. Ob die Extremwetter-Ereignisse der letzten Monate eine direkte Folge der globalen Erhitzung sind, ist wissenschaftlich umstritten.

https://www.liberation.fr/environnement/climat/pluies-record-en-france-depuis-octobre-six-mois-dhumidite-hors-norme-20240422_2P66AIF2PFHPHGRHTZQUWMB2BM/

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This is something I was not aware of, but totally makes sense!! This article is great for students because of tidbits like this that are so interesting, but may also be super relevant to their science classes!

Using this text with my students, I would certainly stop at the end of every, or every other paragraph, and check for comprehension. We would likely discuss what new information they learned, and as mentioned above, could certainly use a KWL chart!

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Author response:

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

eLife assessment

This study presents valuable new insights into HIV-associated nephropathy (HIVAN) kidney phenotype in the Tg26 transgenic mouse model and delineates the kidney cell types that express HIV genes and are injured in these HIV-transgenic mice. A series of compelling experiments demonstrated that PKR inhibition can ameliorate HIVAN with reversal of mitochondrial dysfunction (mainly confined to endothelial cells), a prominent feature shared in other kidney diseases. Although there are concerns regarding the specificity of C16 to PKR inhibition, as well as with the in situ hybridization studies, the data suggests that inhibition of PKR and mitochondrial dysfunction has potential clinical significance for HIVAN.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

HIV-associated nephropathy (HIVAN) is a rapidly progressing form of kidney disease that manifests secondary to untreated HIV infection, and is predominantly seen in individuals of African descent. Tg26 mice carrying an HIV transgene lacking gag and pol exhibit high levels of albuminuria and rapid decline in renal function that recapitulates many features of HIVAN in humans. HIVAN is seen predominantly in individuals carrying two copies of missense variants in the APOL1 gene, and the authors have previously shown that APOL1 risk variant mRNA induces activity of the double-strand RNA sensor kinase PKR. Because of the tight association between the APOL1 risk genotype and HIVAN, the authors hypothesized that PKR activation may mediate renal injury in Tg26 mice and tested this hypothesis by treating mice with a commonly used PKR inhibitory compound called C16. Treatment with C16 substantially attenuated renal damage in the Tg26 model as measured by urinary albumin/creatinine ratio, urinary NGAL/creatinine ratio, and improvement in histology. The authors then performed bulk and single-nucleus RNAseq on kidneys from mice from different treatment groups to identify pathways and patterns of cell injury associated with HIV transgene expression as well as to determine the mechanistic basis for the effect of C16 treatment. They show that proximal tubule nuclei from Tg26 mice appear to have more mitochondrial transcripts which was reversed by C16 treatment and suggest that this may provide evidence of mitochondrial dysfunction in this model. They explore this hypothesis by showing there is a decrease in the expression of nuclear-encoded genes and proteins involved in oxidative phosphorylation as well as a decrease in respiratory capacity via functional assessment of respiration in tubule and glomerular preparations from these mouse kidneys. All of these changes were reversed by C16 treatment. The authors propose the existence of a novel injured proximal tubule cell-type characterized by the leak of mitochondrial transcripts into the nucleus (PT-Mito). Analysis of HIV transgene expression showed high level expression in podocytes, consistent with the pronounced albuminuria that characterizes this model and HIVAN, but transcripts were also detected in tubular and endothelial cells. Because of the absence of mitochondrial transcripts in the podocytes, the authors speculate that glomerular mitochondrial dysfunction in this model is driven by damage to glomerular endothelial cells.

Strengths:

The strengths of this study include the comprehensive transcriptional analysis of the Tg26 model, including an evaluation of HIV transgene expression, which has not been previously reported. This data highlights that HIV transcripts are expressed in a subset of podocytes, consistent with the highly proteinuric disease seen in mice and humans. However, transcripts were also seen in other tubular cells, notably intercalated cells, principal cells and injured proximal tubule cells. Though the podocyte expression makes sense, the relevance of the tubular expression to human disease is still an open question.

The data in support of mitochondrial dysfunction are also robust and rely on combined evidence from downregulation of transcripts involved in oxidative phosphorylation, decreases in complex I and II as determined by immunoblot, and assessments of respiratory capacity in tubular and glomerular preparations. These data are largely consistent with other preclinical renal injury models reported in the literature as well as previous, less thorough assessments in the Tg26 model.

Weaknesses:

The key weakness of the study lies in the use of a PKR inhibitor with questionable specificity. C16 has been reported to inhibit numerous other kinases including cyclin CDKs and GSK3α and -β, and this means that the conclusions of this study with respect to the role of PKR are highly questionable. The rationale for the dose used was not provided (and is lower than used in other publications with C16), and in the absence of drug exposure data and assessment of target engagement, it is difficult to ascertain whether substantial inhibition of PKR was achieved.

A second key weakness lies in the identification of the PT-Mito cell cluster. Though the authors provide some rationale for the identification of this specific cell type, it seems equally plausible the cells merely reflect a high background capture of mitochondria in a subset of droplets. The IHC analysis that was provided is not convincing enough to support the claim and more careful high resolution imaging and in situ hybridization (with appropriate quantitation) will be needed to provide substantive support for the presence of a proximal tubule cell type with mitochondrial transcript that are trafficked to the nucleus.

We appreciate the reviewer’s thoughtful summary.

With regard to non-specificity of C16, we added to the Discussion a description and references that describe non-specificity of C16. as suggested by the reviewer. Of note, the C16 doses that we used were also used previously (Okamoto, CommBiol, 2018). Importantly, newly-added immunofluorescence images using a phospho-PKR specific antibody showed PKR inhibition (Supplemental Figure 1).

Identification of the PT-Mito cluster in tissues was challenging, mainly due to the absence of existence of know marker genes for newly-identified cluster. Finally, We added in situ hybridization images, with a negative control probe, to show specificity of target probes.

Reviewer #2 (Public Review):

Summary:

Numerous studies by the authors and other groups have demonstrated an important role for HIV gene expression kidney cells in promoting progressive chronic kidney disease, especially HIV-associated nephropathy. The authors had previously demonstrated a role for protein kinase R (PKR) in a non-HIV transgenic model of kidney disease (Okamoto, Commun Bio, 2021). In this study, the authors used innovative techniques including bulk and single nuclear RNAseq to demonstrate that mice expressing a replication-incompetent HIV transgene have prominent dysregulation of mitochondrial gene expression and activation of PKR and that treatment of these mice with a small molecule PKR inhibitor ameliorated the kidney disease phenotype in HIV-transgenic mice. They also identified STAT3 as a key upstream regulator of kidney injury in this model, which is consistent with previously published studies. Other important advances include identifying the kidney cell types that express the HIV transgene and have dysregulation of cellular pathways.

Strengths:

Major strengths of the study include the use of a wide variety of state-of-the-art molecular techniques to generate important new data on the pathogenesis of kidney injury in this commonly used model of kidney disease and the identification of PKR as a potential druggable target for the treatment of HIV-induced kidney disease. The authors also identify a potential novel cell type within the kidney characterized by high expression of mitochondrial genes.

Weaknesses:

Though the HIV-transgenic model used in these studies results in a phenotype that is very similar to HIV-associated nephropathy in humans, the model has several limitations that may prevent direct translation to human disease, including the fact that mice lack several genetic factors that are important contributors to HIV and kidney pathogenesis in humans. Additional studies are therefore needed to confirm these findings in human kidney disease.

We appreciate the succinct summary of the present work. We agree that the findings from the HIV Tg26 mouse model warrant additional investigation in human kidney disease samples. Further studies will be needed to confirm whether the mechanisms presented here are operative in human HIVAN or other RNA virus-associated kidney diseases.

Reviewer #1 (Recommendations For The Authors)

The specificity of the C16 tool has been called into question in 3 publications - Chen et al, 2008, PMID: 19046382; Lopez-Grancha et al, 2021, PMID: 34531308; and Cusak et al, 2023, PMID: 36400288. Lopez-Grancha et al have reported a novel, more selective PKR inhibitor with good pharmacological properties that might enable a more robust test of the PKR hypothesis. Regardless, compound exposures and target engagement (i.e. by monitoring phosphorylation of PKR targets such eIF2α) should accompany these studies. Alternatively, it may be easier to probe the role of PKR in Tg26 pathogenicity by crossing the Tg26 line to a PKR knockout mouse.

In response, we have added a description and references about the the possibility of non-specificity of C16 in the Discussion as a limitation as suggested. (Page 21).

“Third, we acknowledge possibility of a non-specific effect of C16 as an inhibitor of PKR.66-68”

Further, we added immunohistochemistry images of pPKR on kidney tissue as shown in Supplemental Figure 1A-D. Images showed PKR activation in Tg26 tubular cells, which was inhibited by C16 treatment.

Author response image 1.

Immunofluorescent images showing pPKR. (A-D) Immunofluorescent images showed PKR activation by detecting pPKR in Tg26 mouse kidney. pPKR was inhibited by C16 treatments.

The suggested PKR knockout mice experiment is an excellent idea for future work but we believe Is outside the scope of the current manuscript.

To enhance the evidentiary base for the PT-Mito cell type, it would be interesting to know whether these cells can also be found in human datasets like KPMP, though this might require reprocessing the original snRNAseq data. Further in situ hybridization in both mouse and human samples using fluorescent rather than colorimetric approaches should yield a more compelling dataset to provide evidence for this cell type. These approaches would also allow for more precise quantification of the PT-Mito cells compared to the population of proximal tubule cells. Again, the default assumption here should be that the mitochondrial transcripts represent a contamination, and the purpose of these additional experiments is to definitively rule out that explanation.

Authors: First, as suggested, we carried out additional analyses. We examined a publiclyavailable human kidney snRNA-seq dataset (GSE131882) and found in it the same PT-Mito cluster as shown in Supplemental Figure 6. The PT-Mito cluster was located in close proximity to the PT cluster in a UMAP plot. We added this finding in the Results as follows (Page 12):

“We also confirmed the existence of similar PT-Mito cluster in published human kidney single-nuclear RNA-seq data47 by the re-analysis of the original data. (Supplemental Figure 6A-C).”

Author response image 2.

PT-Mito cluster detection of publicly available human kidney single-nuclear RNA-seq data (GSE131882) (A) UMAP plot of human kidney single-nuclear RNA-seq data shows 16 clusters. Cluster 1, 4 are proximal tubule (PT) clusters, and cluster 7 is PT-Mito cluster. (B) Dot plot shows expression of PT marker genes and PT-Mito marker genes obtained from current manuscript data. PTMito markers including MT-CO1 and MT-CO2 had high expression in cluster 7. (C) UMAP plot shows all six samples are contributing to all cell clusters.

Second, as suggested, we also included negative control data from in situ hybridization studies (Supplementary Figure 5A, 5B), which shows that the signals in Figure 4B, 4C are true signals.

Author response image 3.

Additional in situ hybridization images. (A) In situ hybridization images probing dapB (negative control probe) showed no signals. (B) In situ hybridization images probing Ppib (positive control probe) showed strong signals.

Reviewer #2 (Recommendations For The Authors)

(1) The supplementary data file seems to have been uploaded twice but the supplementary methods were not available which would have been helpful when assessing some methods such as using PodoCount to count podocytes.

We acknowledge that we inadvertently failed to upload the Supplementary Methods section-thank you for pointing this out. The supplementary methods are now provided in the revised submission, including detailed methods about PodoCount. Corresponding descriptions are as follows:

“Estimation of glomerular podocyte count

PodoCount5, a computational tool for whole slide podocyte estimation from digitized histologic sections, was used to detect, enumerate, and characterize podocyte nuclear profiles in the glomeruli of immunohistochemically labeled (IHC-labeled) murine kidney sections. Formalin-fixed, paraffin embedded tissues (2 µm thickness) were IHC-labeled for p57kip2, a marker of podocyte terminal differentiation (ab75974, Abcam, Cambridge, UK), and detected with horse radish peroxidase (RU-HRP1000, Diagnostic BioSystems, Pleasanton, CA) and diaminobenzidine chromogen substrate (BSB0018A, Bio SB, Santa Barbara, CA). A periodic acid-Schiff post-stain was applied without hematoxylin counterstain. The tool uses a combination of stain deconvolution, digital image processing, and feature engineering to compute histologic podometrics6 with correction for section thickness7. In this study, PodoCount was used to assess mean glomerular podocyte count per mouse.“

(2) In the abstract, the authors give the impression that they know definitively the sequence of HIV gene expression, cytoskeletal dysregulation, dedifferentiation, then loss from glomeruli. Since they could only examine cells that were present in glomeruli, they can't definitively say much about the cells that were lost from glomeruli.

As suggested, deleted the following text: “and were lost from glomeruli tuft”

(3) The authors state that 56,976 cells were used for snRNAseq studies. Was the number of cells similar for each of the 8 mice (from 4 different groups)?

In response, we have created a new table summarizing numbers of nuclei from each sample (i.e. each mouse) added to the Supplemental Figure 2D as follows:

Author response table 1.

Pre-processing of single-nuclear RNA-seq data, Breakdown of nuclei numbers from each sample showed comparable numbers of nuclei analyzed.

(4) Please provide information on the assay that was used to measure creatinine since some methods can be unreliable in mice

This is now provided in the revised submission, including creatinine measurement methods (LC-MS/MS) on page 3 of Supplementary Material:

“Mouse chemistry measurements

Plasma creatinine was measured by isotope dilution LC-MS/MS at The University of Alabama at Birmingham O’Brien Center Core C (Birmingham, AL).”

(5) The authors state that expression of PKR (Eif2ak2) was expressed in all nephron segments. However, it appears on visual inspection of the UMAP in Fig S2B that the percentage of cells expressing Eif2ak2 was low. What percent of cells expressed Eif2ak2 and if it was a low percentage, what is the authors hypothesis for how expression in a small percentage of cells led to the kidney phenotype?

Supplemental Figure 2B (now 3B) does show modest expression of Eif2ak2, approximately 10%. The technique may lack sensitivity to detect low gene expression and even low gene expression may be sufficient to cause phenotypic change.

(6a) In figure 4B and C, it is not clear what genotype/treatment group is shown.

The legend for figure 4B, 4C has been modified to state that the group was wildtype mice

(B, C) In situ hybridization of mt-Co1 and mt-Atp6 genes showed signals inside nuclei of WT mice

(6b) Also, if these ISH images are from Tg26 mice, it would be helpful to do ISH in mice with/without C16 treatment.

These images of ISH for these two genes are from wild-type mice, as now stated in the revised legend. Our purpose was to show that these mitochondrial-encoded gene transcripts (mt-Co1 and mt-Atp6) are transported to nuclei from the cytoplasm. We believe it is not necessary to do ISH in Tg26 mice because these genes are not disease-specific.

(6c) Also, only 3-6% of cells express these "PT-mito" markers by snRNAseq, but it appears that far more are expressed by ISH, raising concerns for nonspecific binding of the ISH probe.

(6d) Also, nonsense controls should be included to demonstrate the specificity of the ISH data.

First (comment 6c), the PT-mito cluster does not have specific markers, to our knowledge. Second (comment 6d) , to address the concern for non-specific binding of the ISH probes, we have now added additional ISH images, together with a negative control probe (C. elegans gene dapB) and a positive control probe (mouse Ppib), as shown in Supplementary Figure 5A and 5B, respectively.

Author response image 4.

Additional in situ hybridization images. (A) In situ hybridization images probing dapB (negative control probe) showed no signals. (B) In situ hybridization images probing Ppib (positive control probe) showed strong signals.

(7) The authors state that "mitochondrial dysfunction was most pronounced in the PT-Mito cluster" but in Figure 4D, the oxidative phosphorylation activation Z score was most down in the PT-inj (injured PT cells) and the PT-Mito cells were the 4-most downregulated cell type.

We appreciate the careful reading and agree with reviewer’s comment. In the revision, we have deleted “most” from this description.

(8) In Fig 4F, please state what "Cp expression" means.

We have spelled out ceruloplasmin (Cp).

(9) It is not clear in immunohistochemistry images in Fig 5F where the p-stat3 was detected due to the hematoxylin counterstain which may have obscured subtle nuclear staining. Also, some of the strongest staining appears to be in peritubular capillaries, instead of tubular and glomerular epithelial cells.

We have added arrows to help readers see where we show that p-Stat3 was detected as faintly-brown and distinct cytoplasmic granules in injured tubular cells in Tg26 mice (panel F), as opposed to diffuse in tubular cytoplasmic color in wild-type mice (panel E).

Author response image 5.

(10) For the studies of mitochondrial oxygen consumption (Fig 6), it would be helpful to also provide data on the effect of C16 in wild-type kidneys, in case C16 somehow causes a primary increase in mitochondrial oxygen consumption rather than preventing HIV-induced loss in kidney cells from HIV-transgenic mice.

We did not include Seahorse data regarding oxygen consumption from WT mice treated with C16, as C16 did not affect either renal function or transcriptomes in WT mice, in contrast to the Tg26 mice (Figure 1A-G).

(11) The authors emphasize that podocytes had the highest expression of HIV genes (Fig 7). However, it appears that <2% of podocytes expressed HIV genes. How do the authors explain the severe renal phenotype given the relatively small number of cells expressing the HIV transgene? Also, did the same cells express all/most of the HIV transcripts, or did some cells express some HIV transcripts? For instance, since the authors state that vpr and nef have the most important role in kidney injury, were the same cells that expressed nef also expressing Vpr?

We know that snRNA-seq cannot detect the whole transcriptome in each cell, due to the well-known drop-out effect characteristic of the method. Several factors may contribute to this drop-out effect, including stochastic patterns of gene expression, low RNA amounts and inefficient mRNA capture (Qiu, Nature Comm, 2020; Ran, Bioinformatics, 2020).

Our interpretation is that HIV gene expressing-podocytes had higher expression of HIV genes, but it does not mean that other kidney cells entirely lack HIV gene expression. With regard to co-expression of other HIV transcripts, nef and vpr were more often coexpressed as shown in Figure 7J. Vpr was expressed in nef-positive podocytes and not detected in nef-negative podocytes.

(12) In figure 8, the authors emphasize the dysregulation of genes involved in cell-cell interaction, particularly PDGF-D. They show some data for the effect of C16 in this system in Fig 8 but it would be helpful if they can state the effect in the text of the Results section.

We have added text in the Results describing activating interactions in Tg26 mice, that were reduced by C16 exposure, as follows: (page 18)

“For example, platelet derived growth factor D (PDGF-D) was upregulated in PT-Inj in Tg26 mice and was downregulated by C16 treatment (Figure 8D). Further, PDGF-D may interact with PDGFR-B in fibroblasts.”

cf dequeue

first comment

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4 - emoji : app

Author response:

We extend our sincere gratitude to the editor and three reviewers for their invaluable feedback, which not only included positive comments but also provided constructive suggestions for enhancing the quality of our manuscript.

Of potential interest to you is our forthcoming investigation into vaccine efficacy, where we will compare the effectiveness of our live-attenuated vaccine with an mRNA-based alternative.

Moreover, we acknowledge and fully endorse the recommendation to elucidate why immunization with our live-attenuated vaccine confers protection against viral challenge, even in the absence of sufficient neutralizing antibodies. As pointed out by the reviewers, this phenomenon may be attributed to mucosal immunity. Consequently, we have outlined plans to investigate whether the attenuated live vaccine elicits mucosal immunity as part of our ongoing research.

We are currently working to gather the necessary data to address these inquiries comprehensively, and are aiming to resubmit our manuscript at the earliest opportunity.

Reviewer #1: We sincerely appreciate the insightful comments provided by Reviewer #1. In response to this feedback, we will conduct a comparative analysis of efficacy between our live-attenuated vaccine and an mRNA-based alternative. Furthermore, we will thoroughly examine and delineate the advantages and limitations of this/our live-attenuated vaccine in our discussion.

Reviewer #2: We express our sincere appreciation to Reviewer #2 for invaluable suggestions. In light of the insightful observation concerning the weakness of our study, related to the poor assessment/evaluation of the induction of mucosal immunity by our vaccine candidate, we have resolved to undertake a comprehensive analysis in this regard.

Furthermore, we will take into account this reviewer's recommendation to compare BK2102 results with those of an mRNA vaccine. We are currently in the process of planning additional experiments to thoroughly address this aspect.

Reviewer #3: We are very grateful to Reviewer #3 for the positive feedback and invaluable suggestions. In order to further explore the immune mechanisms underlying the protection against the Omicron variant in the absence of detectable neutralizing antibodies, we are currently devising plans for experiments focused on evaluating mucosal immunity.

Moreover, in accordance with Reviewer #3's suggestion, we are considering the incorporation of an ELISPOT assay experiment. However, we acknowledge uncertainties regarding the feasibility of establishing an experimental system for this purpose.

eLife assessment

This is a valuable study on the efficacy of a live attenuated vaccine that was tested in different animal models. The evidence is convincing, but it could be further strengthened by comparing the efficacy of their platform with an mRNA vaccine and further investigating mucosal protection.

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4 - emoji - snarf : ©🗐 - emoji - web.snarf

Author response:

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The authors collected genomic information from public sources covering 423 eukaryote genomes and around 650 prokaryote genomes. Based on pre-computed CDS annotation, they estimated the frequency of alternative splicing (AS) as a single average measure for each genome and computed correlations with this measure and other genomic properties such as genome size, percentage of coding DNA, gene and intergenic span, etc. They conclude that AS frequency increases with genome complexity in a somewhat directional trend from "lower" organisms to "higher" organisms.

Strengths:

The study covers a wide range of taxonomic groups, both in prokaryotes and eukaryotes.

Weaknesses:

The study is weak both methodologically and conceptually. Current high throughput sequencing technologies, coupled with highly heterogeneous annotation methods, can observe cases of AS with great sensitivity, and one should be extremely cautious of the biases and rates of false positives associated with these methods. These issues are not addressed in the manuscript.

We are aware of the bias that may exist in annotation files. Since the source of noise can be highly variable, we have assumed that most of the data has a similar bias. However, we agree with the reviewer that we could perform some analysis to test for these biases and their association to different methodologies. Thus, we will measure the uncertainty present in the data. From one side, we will be more explicit about the data limitations and the biases it can generate in the results. On the other side, while analyzing the false positives in the data is out of our scope, we will perform a statistical test to detect possible biases regarding different methods of sequencing and annotation, and types of organisms (model or non-model organisms). If positive, we will proceed, as far as possible, to normalize the data or to estimate a confidence interval.

Here, AS measures seem to be derived directly from CDS annotations downloaded from public databases, and do not account for differing annotation methods or RNA sequencing depth and tissue sample diversity.

Beyond taking into account the differential bias that may exist in the data, we do not consider that our AS measure is problematic. The NCBI database is one of the most reliable databases that we have to date and is continuously updated from all scientific community. So, the use of this data and the corresponding procedures for deriving the AS measure are perfectly acceptable for a comparative analysis on such a huge global scale. Furthermore, the proposal of a new genome-level measure of AS that allows to compare species spanning the whole tree of life is part of the novelty of the study. We understand that small-scale studies require a high specificity about the molecular processes involved in the study. However, this is not the case, where we are dealing with a large-scale problem. On the other side, as we have previously mention, we agree with the reviewer to analyze the degree of uncertainty in the data to better interpret the results.

There is no mention of the possibility that AS could be largely caused by random splicing errors, a possibility that could very well fit with the manuscript's data. Instead, the authors adopt early on the view that AS is regulated and functional, generally citing outdated literature.

There is no question that some AS events are functional, as evidenced by strongly supported studies. However, whether all AS events are functional is questionable, and the relative fractions of functional and non-functional AS are unknown. With this in mind, the authors should be more cautious in interpreting their data.

Many studies suggest that most of the AS events observed are the result of splicing errors and are therefore neither functional nor conserved. However, we still have limited knowledge about the functionality of AS. Just because we don’t have a complete understanding of its functionality, doesn’t mean there isn’t a fundamental cause behind these events. AS is a highly dynamic process that can be associated with processes of a stochastic nature that are fundamental for phenotypic diversity and innovation. This is one of the reasons why we do not get into a discussion about the functionality of AS and consider it as a potential measure of biological innovation. Nevertheless, we agree with the reviewer’s comments, so we will add a discussion about this issue with updated literature and look at any possible misinterpretation of the results.

The "complexity" of organisms also correlates well (negatively) with effective population size. The power of selection to eliminate (slightly) deleterious mutations or errors decreases with effective population size. The correlation observed by the authors could thus easily be explained by a non-adaptive interpretation based on simple population genetics principles.

We appreciate the observation of the reviewer. We know well the M. Lynch’s theory on the role of the effective population size and its eventual correlation with genomic parameters, but we want to emphasize that our objective is not to find an adaptive or non-adaptive explanation of the evolution of AS, but rather to reveal it. Nevertheless, as the reviewer suggests, we will look at the correlation between the AS and the effective population size and discuss about a possible non-adaptive interpretation.

The manuscript contains evidence that the authors might benefit from adopting a more modern view of how evolution proceeds. Sentences such as "... suggests that only sophisticated organisms optimize alternative splicing by increasing..." (L113), or "especially in highly evolved groups such as mammals" (L130), or the repeated use of "higher" and "lower" organisms need revising.

As the reviewer suggests, we will proceed with the corresponding linguistic corrections.

Because of the lack of controls mentioned above, and because of the absence of discussion regarding an alternative non-adaptive interpretation, the analyses presented in the manuscript are of very limited use to other researchers in the field. In conclusion, the study does not present solid conclusions.

Reviewer #2 (Public Review):

Summary:

In this contribution, the authors investigate the degree of alternative splicing across the evolutionary tree and identify a trend of increasing alternative splicing as you move from the base of the tree (here, only prokaryotes are considered) towards the tips of the tree. In particular, the authors investigate how the degree of alternative splicing (roughly speaking, the number of different proteins made from a single ORF (open reading frame) via alternative splicing) relates to three genomic variables: the genome size, the gene content (meaning the fraction of the genome composed of ORFs), and finally, the coding percentage of ORFs, meaning the ratio between exons and total DNA in the ORF. When correlating the degree of alternative splicing with these three variables, they find that the different taxonomic groups have a different correlation coefficient, and identify a "progressive pattern" among metazoan groups, namely that the correlation coefficient mostly increases when moving from flowering plants to arthropods, fish, birds, and finally mammals. They conclude that therefore the amount of splicing that is performed by an organismal group could be used as a measure of its complexity.

Weaknesses:

While I find the analysis of alternative splicing interesting, I also find that it is a very imperfect measure of organismal complexity and that the manuscript as a whole is filled with unsupported statements. First, I think it is clear to anyone studying evolution over the tree of life that it is the complexity of gene regulation that is at the origin of much of organismal structural and behavioral complexity. Arguably, creating different isoforms out of a single ORF is just one example of complex gene regulation. However, the complexity of gene regulation is barely mentioned by the authors.

We disagree with the reviewer with that our measure of AS is imperfect. Just as we responded to the first reviewer, we will quantify the uncertainty in the data and correct for differential biases caused by annotation and sequencing methods. Thus, beyond correcting relevant biases in the data, we consider that our measure is adequate for a comparative analysis at a global scale. A novelty of our study is the proposal of a genome-level measure of AS that takes into account data from the entire scientific community.

We want also to emphasize that we assume from the beginning that AS may reflect some kind of biological complexity, it is not a conclusion from the results. An argument in favor of such an assumption is that AS is associated with stochastic processes that are fundamental for phenotypic diversity and innovation. Of course, we agree with the reviewer that it is not the only mechanism behind biological complexity, so we will emphasize it in the manuscript. On the other side, we will be more explicit about the assumptions and objectives, and will correct any unsupported statement.

Further, it is clear that none of their correlation coefficients actually show a simple trend (see Table 3). According to these coefficients, birds are more complex than mammals for 3 out of 4 measures.

An evolutionary trend is broadly defined as the gradual change in some characteristic of organisms as they evolve or adapt to a specific environment. Under our context, we define an evolutionary trend as the gradual change in genome composition and its association with AS across the main taxonomic groups. If we look at Figure 4 and Table 3 we can conclude that there is a progressive trend. We will be more precise about how we define an evolutionary trend and correct any possible misinterpretation of the results. On the other side, we do not assume that mammals should be more complex than birds. First, we will emphasize that our results show that birds have the highest values of such a trend. Second, after reading the reviewer’s comments, we have decided that we will perform an additional analysis to correct for differences in the taxonomic group sizes, which will allow us to have more confidence in the results.

It is also not clear why the correlation coefficient between alternative splicing ratio and genome length, gene content, and coding percentage should display such a trend, rather than the absolute value. There are only vague mechanistic arguments.

The study analyzes the relationship of AS with genomic composition for the large taxonomic groups. We assume that significant differences in these relationships are indicators of the presence of different mechanisms of genome evolution. However, we agree with the reviewer that a correlation does not imply a causal relation, so we will be more cautious when interpreting the results.

To quantify the relationships we use correlation coefficients, the slopes of such correlations, and the relation of variability. Although the absolute values of AS are also illustrated in Table 4, we consider that they are less informative than if we include how it relates to the genomic composition. For example, we observe that plants have a different genome composition and relation with AS if compared to animals, which suggest that they follow different mechanisms of genome evolution. On the other hand, we observe a trend in animals, where high values of AS are associated to a large percentage of introns and a percentage of intergenic DNA of about the 50% of genomes.

Much more troubling, however, is the statement that the data supports "lineage-specific trends" (lines 299-300). Either this is just an ambiguous formulation, or the authors claim that you can see trends within lineages.

We agree with the reviewer that this statement is not correct, so we will proceed to correct it.

The latter is clearly not the case. In fact, within each lineage, there is a tremendous amount of variation, to such an extent that many of the coefficients given in Table 3 are close to meaningless. Note that no error bars or p-values are presented for the values shown in Table 3. Figure 2 shows the actual correlation, and the coefficient for flowering plants there is given as 0.151, with a p-value of 0.193. Table 3 seems to quote r=0.174 instead. It should be clear that a correlation within a lineage or species is not a sign of a trend.

The reviewer is not understanding correctly the results in Table 3. It is precisely the variation of the genome variables what we are measuring. Given the standardization of these values by the mean values, we have proceeded to compare the variability between groups, which is the result shown in Table 3. In this case there are no error bars or p-values associated. On the other hand, we agree that a correlation is not a sign of a trend. But the relations of variability, together with the results obtained in Figure 3, are indicators of a trend. As we mentioned before, we will proceed to analyze whether the variation in the group sizes is causing a bias in the results.

There are several wrong or unsupported statements in the manuscript. Early on, the authors state that the alternative splicing ratio (a number greater or equal to one that can be roughly understood as the number of different isoforms per ORF) "quantifies the number of different isoforms that can be transcribed using the same amount of information" (lines 51-52). But in many cases, this is incorrect, because the same sequence can represent different amounts of information depending on the context. So, if a changed context gives rise to a different alternative splice, it is because the genetic sequence has a different meaning in the changed context: the information has changed.

We agree that there are not well supported statements, so we will proceed to revise them.

In line 149, the authors state that "the energetic cost of having large genomes is high". No citation is given, and while such a statement seems logical, it does not have very solid support.

We will also revise the bibliography and support our statements with updated references.

If there was indeed a strong selective force to reduce genome size, we would not see the stunning diversity of genome sizes even within lineages. This statement is repeated (without support) several times in the manuscript, apparently in support of the idea that mammals had "no choice" to increase complexity via alternative splicing because they can't increase it by having longer genomes. I don't think this reasoning can be supported.

We agree with the reviewer in this issue, so we will carefully revise the statements that indirectly (or directly) assume the action of selective forces on the genome composition.

Even more problematic is the statement that "the amount of protein-coding DNA seems to be limited to a size of about 10MB" (line 219). There is no evidence whatsoever for this statement.

In Figure 1A we observe a one-to-one relationship between the genome size and the amount of coding. However, in multicellular organisms, although the genome size increases we observe that the amount of coding does not increase by more than 10Mb, which suggest the presence of some genomic limitation. Of course, this is not an absolute or general statement, but rather a suggestion. We are only describing our results.

The reference that is cited (Choi et al 2020) suggests that there is a maximum of 150GB in total genome size due to physiological constraints. In lines 257-258, the authors write that "plants are less restricted in terms of storing DNA sequences compared to animals" (without providing evidence or a citation).

We will revise the bibliography and add updated references.

I believe this statement is made due to the observation that plants tend to have large intergenic regions. But without examining the functionality of these interagency regions (they might host long non-coding RNA stretches that are used to regulate the expression of other genes, for example) it is quite adventurous to use such a simple measure as being evidence that plants "are less restricted in terms of storing DNA sequences", whatever that even means. I do not think the authors mean that plants have better access to -80 freezers. The authors conclude that "plant's primary mechanism of genome evolution is by expanding their genome". This statement itself is empty: we know that plants are prone to whole genome duplication, but this duplication is not, as far as we understand, contributing to complexity. It is not a "primary mechanism of genome evolution".

We will revise these statements.

In lines 293-294, the authors claim that "alternative splicing is maximized in mammalian genomes". There is no evidence that this ratio cannot be increased. So, to conclude (on lines 302-303) that alternative splicing ratios are "a potential candidate to quantify organismal complexity" seems, based on this evidence, both far-fetched and weak at the same time.

Our results show the highest values of AS in mammals, but we understand that the results are limited to the availability and accuracy of data, which we will emphasize in the manuscript. As we previously mention, we will also proceed to analyze the uncertainty in data and carry out the appropriate corrections.

I am also not very comfortable with the data analysis. The authors, for example, say that they have eliminated from their analysis a number of "outlier species". They mention one: Emmer wheat because it has a genome size of 900 Mb (line 367). Since 900MB does not appear to be extreme, perhaps the authors meant to write 900 Gb. When I consulted the paper that sequenced Triticum dicoccoides, they noted that 14 chromosomes are about 10GB. Even a tetraploid species would then not be near 900Gb. But more importantly, such a study needs to state precisely which species were left out, and what the criteria are for leaving out data, lest they be accused of selecting data to fit their hypothesis.

The reviewer is right, we wanted to say 900Mb, which is approximately 7.2Gb. We had a mistake of nomenclature. This value is extreme compared to the typical values, so it generates large deviations when applying measures of central tendency and dispersion. We want to obtain mean values that are representative of the most species composing the taxonomic groups, so we find appropriate to exclude all outlier values in the study. Nevertheless, we will specify the criteria that we have used to select the data in a rigorous way.

I understand that Methods are often put at the end of a manuscript, but the measures discussed here are so fundamental to the analysis that a brief description of what the different measures are (in particular, the "alternative splicing ratio") should be in the main text, even when the mathematical definition can remain in the Methods.

We agree with the reviewer, so we will add a brief description of the genomic variables at the beginning of the Results section.

Finally, a few words on presentation. I understand that the following comments might read differently after the authors change their presentation. This manuscript was at the border of being comprehensible. In many cases, I could discern the meaning of words and sentences in contexts but sometimes even that failed (as an example above, about "species-specific trends", illustrates). The authors introduced jargon that does not have any meaning in the English language, and they do this over and over again.

Note that I completely agree with all the comments by the other reviewer, who alerted me to problems I did not catch, including the possible correlation with effective population size: a possible non-adaptive explanation for the results.

Broacdast Channel API works on the same origin.

CTRL + / marche très bien pour commenter la ligne

What examples of online profiles can you think of?

Sometimes I feel like visual disabilities are not willing to be always caring about. They would be more happy if people just treat them just as same as how they treat normal people so that they don't feel any discrimination. Thus sometimes too much caring may not be the best solution.

I remember in my learning of INFO360, our instructor mentioned the word, "empathy" in designs. One group of people that we should empathize is people with disability. I'm wondering the disability mentioned in this book would also count as a disability in design? Are they similar?

驚喜到吃手手

改天來如法炮製，看如何下prompt讓它儘量用全漢的台文輸出，減少羅馬拼音，除非基本字型出不來的字。這只是我的個人偏好。

太興奮了。我會希望AI用台文書寫，儘量減少tailo或POJ。以後來研究這個system prompt怎麼下。

AI

Taigi

Chhit Am-goat · 《 用台文直接共 AI 討台文內容 》 昨昏有朋友問講 AI 出無按家己向望彼號型。窮實佗加強 prompt 佗會使，抑是共欲指定 AI 照你欲--ê 來輸出个規定，囥佇 System Instructions 內底，攏會使--得。 下跤圖--ni̍h 是 ka 囥佇 Gemini-pro-1.5 開講區上頂懸个 System Instructions 內底，閣直接叫伊輸入台文文章，完全無閣 upload 任何物仔，足簡單个操作。 輸出愛檢查，家己愛改，毋過完成度是不止仔懸--矣，而且直接佗出台文內容，毋免閣過翻譯。 Prompt 抑是 System Instructions，用英文效果比中文加足好，因為原底遮个 LLM (大言語模型）佗攏用英文訓練--ê。 罔參考，會使按家己需要閣改，內底个例攏會換--得，例个長度小可長，效果較好。 若想欲輸出 全 POJ、全 TL、抑是全漢，會使改內底个規則佮例，試驗看覓。 《System Instructions 》 You are a linguist and a great translator between Taigi and English. Translate English text into Taigi I give you, and vise versa．Or chat with me in Taigi. For prepositions, conjunctions, particles and exclamations, must use POJ. For NERs such as country, place or human names keep the original name.English. Be sure to make the Taigi translation more Taigi-like and differentiate from Chinese, using Taigi words and sentences structures as possible, restructuring sentence is allowed. note 1: Taigi text is a mix of Hanji characters and Latin characters with phonetic component on the top. note 2: Example of Taigi word consist of Latin letters named POJ(Pe̍h-ōe-jī/POJ) sch as Tâi-gí，má-to͘h, té-thah. Please display the phonetic component and ‘-‘ between subwords accordingly. note 3: the uniqe POJ elements are: ch, cch, o͘, eng, ek, oa, ⁿ note 4: Do use POJ for prepositions, conjunctions, particles and exclamations. Output as format&example. <format&example>

• Leonardo da Vinci ê 無限好玄

Leonardo da Vinci 是文藝復興時期 ê 代表人物, 伊 ê 人生 kah 作品 lóng 浸透彼个時代 ê 精神。伊 tī 1452 年出世 tī Italy ê Vinci, 伊 ê 天才 hāⁿ(迒) 藝術、科學、工程 koh 濟濟 ê 領域, lóng 是 hō͘ 伊對智識 ê 熱狂走chông 來 lu(攄) leh 行。

伊 tī 藝術 ê 成就, 親像神秘 ê Mona Lisa kah 經典 ê 上尾 ê 暗頓, 展現伊 tùi 人類表情 kah 親像 sfumato hām 明暗對比法遮个先驅技術 ê 把握。 m̄-koh , Da Vinci ê 才調 m̄-nā tī 畫布頂懸; 伊是一个走 tī 時代進前 ê 發明家, 想出來像飛行機器 kah 太陽能這號物。

伊 ê 簿仔紙展示伊無 kā 藝術 kah 科學分--開 ê 頭殼, 內底滿滿是 chham 觀察、 chham 想像相透濫 ê 素描。Da Vinci ê 哲學是「研究藝術 ê 科學。研究科學 ê 藝術... 認捌 tio̍h 萬物 lóng 連連鬥陣」, 這反映伊相信所有學科 lóng 是 kap(合)做伙--ê。

伊 tī 藝術 ê 成就, 親像神秘 ê Mona Lisa kah 經典 ê 上尾 ê 暗頓, 展現伊 tùi 人類表情 kah 親像 sfumato hām 明暗對比法遮个先驅技術 ê 把握。 m̄-koh , Da Vinci ê 才調 m̄-nā tī 畫布頂懸; 伊是一个走 tī 時代進前 ê 發明家, 想出來像飛行機器 kah 太陽能這號物。

Gemini-1.5-pro: https://aistudio.google.com/

eLife assessment

This important study indicates a significant role for individual let-7 miRNA clusters in regulating generation of Tc17 CD8 cells and emphysema severity in a mouse model. The authors provide convincing evidence for let-7-mediated repression of the transcription factor RORgt and consequent modulation of IL-17-producing CD8 T cells, with correlated data from human emphysema material, though some of the effective let-7 clusters remain to be tested for the ability to modulate disease. The findings, which substantially advance the understanding of roles that let-7 miRNA clusters play in modulating both T cell responses and emphysematous lung disease, will be of interest to T cell and lung disease researchers.

eLife assessment

This important study reports on the genome evolution of a poorly studied fungal group. By combining long-read sequencing and different bioinformatic analyses, the authors show that the giant genome of Entomophthora muscae expanded due to extensive transposable element activity. The strength of evidence is compelling and the authors are to be commended for their multiple comparative analyses of gene content along with transparently written and visualized techniques, data curation, and methods. This paper will be of relevance to fungal biologists as well as to evolutionary biologists interested in the study of genome size dynamics.

from - search - single level memory https://hyp.is/1BeKTgIWEe-VNVeItxpMMg/www.google.com/search?q=%22single+level+memory%22

single-level memory/storage

single-level memory/storage

single level memory

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from

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