This is probs why then

I don't think that is a valid comparison but o well

that's actually insane bruh

right

ohhhhh

i agree

ohhhh

I completely agree

facts

That is interesting because one of think that when you are once in the police department that they would teach some of the things you need to know yet they stull lack this ability

sad to see

interesting

That is actually crazy insane

interesting

I agree with this statement

How are they going to make police more efficient in controlling crime when they aren't receiving the sufficient funds to be fully trained and this does not go hand in hand with unintended harm or unnecessary imposition on the freedoms of individual citizens.

burden

means argument

Maybe the overall issue is the lack of community funding that they are receiving because then they feel the need to do these crimes in the first place.

A person's standing in society is often determined by his or her occupation, income or education level, or family background.

I wonder why? - For me it is because I come from a hispanic household and within my own small neighborhood we all have this fear that we are going ot get deported

As a caseworker for IDHS, I inform individuals receiving SNAP they are required to comply with Work Provisions and/or Work Requirements to continue to receive benefits. Individuals between 16 and 60 years old are subject to Work Provisions, meaning they must be registered to work, not voluntarily quit or reduce work hours to less than 30 hours a week. The Work Requirement applies to abled-bodied adults without dependents (ABAWD) between 18 and 52 years old, and they must work an average of 20 hours per week or 80 hours per month. Certain exemptions can be applied, and individuals can comply with alternative activities.

The takeaway from this section implies that the able-bodied need to demonstrate they are worthy of receiving aid. I strongly disagree that the able-bodied don’t deserve assistance if they are not working or not working hard enough. This concept doesn’t factor in that although able-bodied individuals physically appear capable of working, they could be dealing with emotional, mental, or psychological issues like low self-esteem, anxiety, depression, or any other mental disorder that limits them from working or completing daily activities.   

Find page number for this reference.

This is how she expresss the love between someone of course their Is drama there has to be , if it wasn't for this song I dont think I could tell what true love is.

tells me how Taylor has many hearts of the guy fan club too .

I could tel you this I how I felt when I saw Edward cullen movie for the first time this is fandom.

Tells us so much about how the feel of the concert was , such a great way to express .

nice tool

Hell yes

https://www.theguardian.com/commentisfree/article/2024/jul/03/caroline-lucas-green-party-standing-down-mp-climate-reasons-hopeful

https://www.theguardian.com/environment/article/2024/jul/04/far-right-using-climate-crisis-as-bogeyman-to-frighten-voters-and-build-higher-walls

https://www.liberation.fr/checknews/les-le-pen-lenvironnement-et-lenergie-vingt-ans-de-programmes-entre-continuite-et-reniements-20240704_T4ABBQVDFZGDTF3EKTLCWURHMM/

https://www.liberation.fr/checknews/les-le-pen-lenvironnement-et-lenergie-vingt-ans-de-programmes-entre-continuite-et-reniements-20240704_T4ABBQVDFZGDTF3EKTLCWURHMM/

https://www.liberation.fr/international/europe/la-serbie-sur-le-point-de-relancer-son-projet-controverse-de-mine-de-lithium-20240703_FM75CJYGB5DXJKUUZOVFQFITXU/

https://www.liberation.fr/environnement/climat/geo-ingenierie-solaire-une-fuite-en-avant-technosolutionniste-20240704_654IARTI75CKRIJDHNGLSOI3ZI/

No matter if you bind a ClusterRole, if bound locally only affect the user for a single project

Diesen Satz würde ich kürzen.

Hier würde ich noch diesen Text referenzieren: Andrew Piper: Can We Be Wrong? The Problem of Textual Evidence in a Time of Data. 2020.

Wie verhalten sich diese Perspektiven zu den folgenden Abschnitten 2,3 und 4? Hier entsteht beim Lesen der Eindruck, dass sich diese Perspektiven auch in einzelne Abschnitte manifestieren.

In Absatz 6 ist nicht von Perspektiven die Rede. An einer Stelle wird der Begriff "Dimension" verwendet. Ich würde vielleicht oben auch von Perspektiven sprechen.

Ich würde diesen Satz kürzen.

https://taz.de/Vorwuerfe-gegen-deutsche-Oelkonzerne/!6021613/

https://taz.de/Mehr-russisches-Erdgas-fuer-China/!6021655/

https://bari.repubblica.it/cronaca/2024/07/04/news/ilva_taranto_reati_vecchia_gestione-423357766/

https://bari.repubblica.it/cronaca/2024/07/04/news/ilva_taranto_reati_vecchia_gestione-423357766/

https://www.nytimes.com/2024/07/03/climate/gm-epa-pollution-settlement.html

note

eLife assessment

In this important study, the authors provide evidence that Treacle, a disease-relevant intrinsically disordered protein, undergoes biomolecular condensation to support the structure and function of the fibrillar center of the nucleolus. The findings, arising from complementary approaches, provide solid evidence for the role of Treacle condensation in supporting rDNA transcription, rRNA processing, and genome integrity. These findings may be of interest to the communities studying biomolecular condensates, nucleolar organization, and ribosome biogenesis.

Incredible!

[表記揺れ?] 全体的にこのPlotlyのチートシートでは名詞の plots は「グラフ」と訳しているようです （Matplotlibみたいに同じplotsでも実は別のオブジェクトを指しているなどだと用語を使い分けた方が良いかもしれませんが、Plotlyの構造詳しくないのでちょっとよくわかりません...

立方数のための新しいtraceを追加する

とかでしょうか

最高気温のための新しいtraceを追加する

とかでしょうか

方

方

好みかもしれませんが、ここは「集まり」などの方が自然かなと思いました。

[日本語] (1) 「ここには...ページです」だと主語（？）と動詞が対応していない感じがするので、「これは...ページです」の方が自然だと思います。

(2) 「示すリファレンスページ」より「確認できるリファレンスページ」などの方が日本語としては自然かなと思います。

代案:

これは、Plotlyで作成できる全ての種類のグラフを確認できるリファレンスページです。

原文:

The labels dictionary lets you specify which aspects of the plot will have custom labels.

原文だと labels がコード表記なので、引数または変数のlabelsを指していそうです。

代案:

辞書labelsを使うと、グラフのどの要素にカスタムラベルを設定するか指定できます。

などでしょうか。

の が連続してちょっと読みにくいので

その他可視化のためのフォーマットを指定する引数を受け取ることもできます。

などはどうでしょうか

(1) <br /> 「プロットが表示されたブラウザの」で区切って呼んじゃってちょっとわかりにくかったです。

(2) <br /> plots がひとつ前の文では「グラフ」、この文では「プロット」と書かれて別のものを指してるのか、表記揺れなのか悩みました（原文はどちらもplots）。意図的に使いわけているのでなければ統一した方が良いと思います。

代案:

コードに興味があれば、プロットが表示されたときにブラウザのインスペクターツールを開いてください。

[表記揺れ] 17章では 対話的 と書いていたような

Summary of Electric Closure Presentation

Overview and Purpose

• The presentation discusses the motivation, applications, and technical workings of Electric.
• Electric is designed for enterprise apps, specifically single-page applications with complex business rules and validations.
• It aims to solve issues encountered in CRUD apps by simplifying state management, network interaction, and rendering.

Application Examples

• Partner apps for internal operations, such as support apps for startups, demonstrate Electric's capabilities.
• Electric enables the creation of apps that look and function like spreadsheets, emphasizing strong user experience over styling.

Technical Approach

• Electric uses a DSL (domain-specific language) to encode operational CRUD apps.
• It allows the creation of live CRUD apps through a DSL that generates full-stack applications in real-time.
• The key innovation is the ability to compose functions directly with the DOM, simplifying the data flow and reducing the need for heavy frameworks.

Challenges and Solutions

• Traditional approaches to managing CRUD apps involve complex state and network interactions.
• Electric proposes a direct composition model, eliminating the need for frameworks like ORMs and simplifying the data flow.
• The system uses reactive programming to manage interleaved client-server data flows efficiently.

Architecture and Implementation

• Electric compiles programs into a directed acyclic graph (DAG), managing data flow between client and server.
• The DAG enables efficient rendering and state management, minimizing unnecessary computations and optimizing performance.
• Missionary, the functional effect system used by Electric, supports glitch-free rendering, cancellation, resource lifecycle management, and composability.

Practical Examples and Demos

• The chat app demo shows multiplayer capabilities by streaming state changes across clients.
• The 2D MVC demo illustrates full-stack applications created with Electric, highlighting its ability to handle real-time data entry and server-client synchronization.

Future Directions and Goals

• Electric aims to support more advanced use cases, such as offline-first applications and more complex data interactions.
• The project is committed to improving build times, optimizing network traffic, and refining the development experience.
• The team envisions Electric as a foundational technology for richer, more dynamic web applications, emphasizing strong composition and functional effects.

Key Takeaways

• Electric simplifies the development of enterprise applications by using a functional effect system to manage state and data flow.
• It provides a declarative approach to building CRUD apps, reducing accidental complexity and enhancing code readability and maintainability.
• The system's architecture ensures efficient real-time data synchronization and rendering, making it suitable for high-performance applications.

Relevant Quotes

• "We're going to talk about why we built this and what is for."
• "Electric is designed for enterprise apps, single-page applications."
• "The system uses reactive programming to manage interleaved client-server data flows efficiently."
• "Electric compiles programs into a directed acyclic graph (DAG), managing data flow between client and server."
• "Missionary, the functional effect system used by Electric, supports glitch-free rendering, cancellation, resource lifecycle management, and composability."

16章から

Reviewer #1 (Public Review):

Summary:

In this study, the authors developed an organoid system that contains smooth muscle cells (SMCs) and interstitial cells of Cajal (ICCs; pacemaker) but few enteric neurons, and generates rhythmic contractions as seen in the developing gut. The stereotypical arrangements of SMCs and ICCs in the organoid allowed the authors to identify these cell types in the organoid without antibody staining. The authors took advantage of this and used calcium imaging and pharmacology to study how calcium transients develop in this system through the interaction between the two types of cells. The authors first show that calcium transients are synchronized between ICC-ICC, SMC-SMC, and SMC-ICC. They then used gap junction inhibitors to suggest that gap junctions are specifically involved in ICC-to-SMC signaling. Finally, the authors used an inhibitor of myosin II to suggest that feedback from SMC contraction is crucial for the generation of rhythmic activities in ICCs. The authors also show that two organoids become synchronized as they fuse and SMCs mediate this synchronization.

Strengths:

The organoid system offers a useful model in which one can study the specific roles of SMCs and ICCs in live samples.

Weaknesses:

Since only one blocker each for gap junction and myosin II was used, the specificities of the effects were unclear.

eLife assessment

This valuable study reports the development of a novel organoid system for studying the emergence of autorhythmic gut peristaltic contractions through the interaction between interstitial cells of Cajal and smooth muscle cells. While the utility of the organoids for studying hindgut development is well illustrated by showing, for example, a previously unappreciated potential role for smooth muscle cells in regulating the firing rate of interstitial cells of Cajal, some of the functional analyses are incomplete. There are some concerns about the specificity and penetrance of perturbations and the reproducibility of the phenotypes. With these concerns properly addressed, this paper will be of interest to those studying the development and physiology of the gut.

Reviewer #2 (Public Review):

Summary:

In this study, Yagasaki et al. describe an organoid system to study the interactions between smooth muscle cells (SMCs) and interstitial cells of Cajal (ICCs). While these interactions are essential for the control of rhythmic intestinal contractility (i.e., peristalsis), they are poorly understood, largely due to the complexity of and access to the in vivo environment and the inability to co-culture these cell types in vitro for long term under physiological conditions. The "gut contractile organoids" organoids described herein are reconstituted from stromal cells of the fetal chicken hindgut that rapidly reorganize into multilayered spheroids containing an outer layer of smooth muscle cells and an inner core of interstitial cells. The authors demonstrate that they contract cyclically and additionally use calcium imagining to show that these contractions occur concomitantly with calcium transients that initiate in the interstitial cell core and are synchronized within the organoid and between ICCs and SMCs. Furthermore, they use several pharmacological inhibitors to show that these contractions are dependent upon non-muscle myosin activity and, surprisingly, independent of gap junction activity. Finally, they develop a 3D hydrogel for the culturing of multiple organoids and found that they synchronize their contractile activities through interconnecting smooth muscle cells, suggesting that this model can be used to study the emergence of pacemaking activities. Overall, this study provides a relatively easy-to-establish organoid system that will be of use in studies examining the emergence of rhythmic peristaltic smooth muscle contractions and how these are regulated by interstitial cell interactions. However, further validation and quantification will be necessary to conclusively determine show the cellular composition of the organoids and how reproducible their behaviors are.

Strengths:

This work establishes a new self-organizing organoid system that can easily be generated from the muscle layers of the chick fetal hindgut to study the emergence of spontaneous smooth muscle cell contractility. A key strength of this approach is that the organoids seem to contain few cell types (though more validation is needed), namely smooth muscle cells (SMCs) and interstitial cells of Cajal (ICCs). These organoids are amenable to live imaging of calcium dynamics as well as pharmacological perturbations for functional assays, and since they are derived from developing tissues, the emergence of the interactions between cell types can be functionally studied. Thus, the gut contractile organoids represent a reductionist system to study the interactions between SMCs and ICCs in comparison to the more complex in vivo environment, which has made studying these interactions challenging.

Weaknesses:

The study falls short in the sense that it does not provide a rigorous amount of evidence to validate that the gut organoids are made of bona fide smooth muscle cells and ICCs. For example, only two "marker" proteins are used to support the claims of cell identity of SMCs and ICCs. At the same time, certain aspects of the data are not quantified sufficiently to appreciate the variance of organoid rhythmic contractility. For example, most contractility plots show the trace for a single organoid. This leads to a concern for how reproducible certain aspects of the organoid system (e.g. wavelength between contractions/rhythm) might be, or how these evolve uniquely over time in culture. Furthermore, while this study might be able to capture the emergence of ICC-SMC interactions as they related to muscle contraction and pacemaking, it is unclear how these interactions relate to adult gastrointestinal physiology given that the organoids are derived from fetal cells that might not be fully differentiated or might have distinct functions from the adult. Finally, despite the strength of this system, discoveries made in it will need to be validated in vivo.

Reviewer #3 (Public Review):

Summary:

The paper presents a novel contractile gut organoid system that allows for in vitro studying of rudimentary peristaltic motions in embryonic tissues by facilitating GCaMP-live imaging of Ca2+<br /> dynamics, while highlighting the importance and sufficiency of ICC and SMC interactions in generating consistent contractions reminiscent of peristalsis. It also argues that ENS at later embryonic stages might not be necessary for coordination of peristalsis.

Strengths:

The manuscript by Yagasaki, Takahashi, and colleagues represents an exciting new addition to the toolkit available for studying fundamental questions in the development and physiology of the hindgut. The authors carefully lay out the protocol for generating contractile gut organoids from chick embryonic hindgut, and perform a series of experiments that illustrate the broader utility of these organoids for studying the gut. This reviewer is highly supportive of the manuscript, with only minor requests to improve confidence in the findings and broader impact of the work. These are detailed below.

Weaknesses:

(1) Given that the literature is conflicting on the role GAP junctions in potentiating communication between intestinal cells of Cajal (ICCs) and smooth muscle cells (SMCs), the experiments involving CBX and 18Beta-GA are well-justified. However, because neither treatment altered contractile frequency or synchronization of Ca++ transients, it would be important to demonstrate that the treatments did indeed inhibit GAP junction function as administered. This would strengthen the conclusion that GAP junctions are not required, and eliminate the alternative explanation that the treatments themselves failed to block GAP junction activity.

(2) Given that 5uM blebbistatin increases the frequency of contractions but 10uM completely abolishes contractions, confirming that cell viability is not compromised at the higher concentration would build confidence that the phenotype results from inhibition of myosin activity. One could either assay for cell death, or perform washout experiments to test for recovery of cyclic contractions upon removal of blebbistatin. The latter may provide access to other interesting questions as well. For example, do organoids retain memory of their prior setpoint or arrive at a new firing frequency after washout?

(3) Regulation of contractile activity was attributed to ICCs, with authors reasoning that Tuj1+ enteric neurons were only present in organoids in very small numbers (~1%). However, neuronal function is not strictly dependent on abundance, and some experimental support for the relative importance of ICCs over Tuj1+ cells would strengthen a central assumption of the work that ICCs the predominant cell type regulating organoid contraction. For example, one could envision forming organoids from embryos in which neural crest cells have been ablated via microdissection or targeted electroporation. Another approach would be ablation of Tuj1+ cells from the formed organoids via tetrodotoxin treatment. The ability of organoids to maintain rhythmic contractile activity in the total absence of Tuj1+ cells would add confidence that the ICCs are indeed the driver of contractility in these organoids.

(4) Given the implications of a time lag between Ca++ peaks in ICCs and SMCs, it would be important to quantify this, including standard deviations, rather than showing representative plots from a single sample.

(5) To validate the organoid as a faithful recreation of in vivo conditions, it would be helpful for authors to test some of the more exciting findings on explanted hindgut tissue. One could explant hindguts and test whether blebbistatin treatment silences peristaltic contractions as it does in organoids, or following RCAS-GCAMP infection at earlier stages, one could test the effects of GAP junction inhibitors on Ca++ transients in explanted hindguts. These would potentially serve as useful validation for the gut contractile organoid, and further emphasize the utility of studying these simplified systems for understanding more complex phenomena in vivo.

(6) Organoid fusion experiments are very interesting. It appears that immediately after fusion, the contraction frequency is markedly reduced. Authors should comment on this, and how it changes over time following fusion. Further, is there a relationship between aggregate size and contractile frequency? There are many interesting points that could be discussed here, even if experimental investigation of these points is left to future work.

(7) Minor: As seen in Movie 6 and Figure 6A, 5uM blebbistatin causes a remarkable increase in the frequency of contractions. Given the regular periodicity of these contractions, it is a surprising and potentially interesting finding, but authors do not comment on it. It would be helpful to note this disparity between 5 and 10 uM treatments, if not to speculate on what it means, even if it is beyond the scope of the present study to understand this further.

(8) Minor: While ENS cells are limited in the organoid, it would be helpful to quantify the number of SMCs for comparison in Supplemental Figure S2. In several images, the number of SMCs appears quite limited as well, and the comparison would lend context and a point of reference for the data presented in Figure S2B.

(9) Minor: additional details in the Figure 8 legend would improve interpretation of these results. For example, what is indicated in orange signal present in panels C, G and H? Is this GCAMP?

Author response:

Generals:

We deeply appreciate the efforts by the Senior and Reviewing Editors, and also thank the three reviewers for their careful reading of the MS and their constructive comments, which are very helpful to improve our MS. We agree that we extend our efforts to elaborate the pharmacological analyses including clarification of the penetrance of GAP junction inhibitor(s), and effectiveness and specificity of the drugs. We plan to test at least L-type calcium channel blocker nifedipine. Concerning the reproducibility of the phenotypes, we indeed repeated experiments at multiple times for each of the analyses. While we demonstrated in the current version a series of representative data for simplicity along with explanation in the text that we conducted multiple times of experiments,  in a revised version we will improve the demonstration so that readers/reviewers can be convinced with the reproducibility of the data. We will also try to test other markers to look into cell types constituting the gut contractile organoid

Specifics:

Our provisional responses to “The weakness” raised by the reviewers are as follows:

Reviewer #1:

Please see the responses shown above (“Generals”).

Reviewer #2:

In addition to the responses in “Generals”, our response also includes the followings: We will look into wavelength between contractions/rhythm of the orgnaoid. We agree that our organoids derived from embryonic hind gut (E15) might not necessarily recapitulate the cell function in adult. However, it has well been accepted in the field of developmental biology that studies with embryonic tissue/cells make a huge contribution to unveil how complicated physiological cell function is underpinned. Nevertheless, we will carefully consider in the revised version so that the MS would not send misleading messages. Recent advances have also shown that 3D organoids can somehow “replace/substitute for” a complicated in vivo specimen when a particular cellular function is a focus of study.

Reviewer #3:

We appreciate a strong support of our findings.

(1) We plan to perform positive control experiments, for example, to test if the drugs we use would interfere cardiac muscle functions.

(2) We plan to do wach-out experiment to  confirm 10uM blebbistatin does not kill the cells. Thank you for this suggestion.

(3) We plan to conduct tetrodotoxin treatment. Since experiments with such toxic reagents are not enouraged by our institute, we will perform experiments with a necessary-minimum amount.

(4) We plant to address this point properly

5) It is well predictable that blebbistatin would stop the gut movement in an explanted hindgut, and it is also well established that gut contractions (movements) are concomitant with Ca2+ transients. It would indeed be interesting to see how GJ inhibitors affect such in vivo gut movement. However, since all the reviewers and the Reviewing Editor pointed out, sensitivity (concentration) and penetrance of the drug is an important point of concern, we think that the in vivo analyses will be a next step to go in near future.

(6) We have indeed noticed that contraction frequency is reduced after organoidal fusion. It seems as if cells communicate with each other to decide which rhythm they need to be adjusted to. Furthermore, contraction frequency tends to be slow down when the organoid becomes larger in size. It might be attributed to a delay in conductance between cells over growing distance. We plan to either quantify these potentially interesting phenomena or make a concise speculation in the revised version.

(7)-(10) Thank you for these comments. We will fix them.

Points forts de la vidéo "La fabrique de la connaissance territoriale en région" avec timestamps:

00:00 - 05:00 Introduction et présentation des intervenants * Alix Roche, vice-président de l'Unadel, présente le contexte et les objectifs de la rencontre. * Jean-Baptiste Chabert, directeur de la connaissance de la région Sud Provence-Alpes-Côte d'Azur, présente son service et son approche de la connaissance territoriale. * Joseph Compera, chef du service prospective en région Bourgogne-Franche-Comté, présente son service et son approche de la prospective territoriale. * Stéphane Ambert, chef du service prospective en région Hauts-de-France, présente son service et son approche de la prospective territoriale.

05:00 - 25:00 Le rôle de la connaissance territoriale dans les politiques publiques * Les intervenants discutent de l'importance de la connaissance territoriale pour éclairer les politiques publiques locales et régionales. * Ils soulignent que la connaissance territoriale doit être accessible et partagée par tous les acteurs du territoire. * Ils évoquent les défis de la production et de la diffusion de la connaissance territoriale.

25:00 - 40:00 Les outils et méthodes pour la fabrique de la connaissance territoriale * Les intervenants présentent les différents outils et méthodes utilisés pour produire de la connaissance territoriale. * Ils évoquent notamment les enquêtes, les études documentaires, la modélisation et la simulation. * Ils soulignent l'importance de la collaboration entre les chercheurs et les acteurs du territoire.

40:00 - 50:00 Les enjeux de la prospective territoriale * Les intervenants discutent des enjeux de la prospective territoriale, notamment face aux transitions en cours (climatique, numérique, etc.). * Ils soulignent l'importance de la prospective pour anticiper les changements et préparer l'avenir des territoires. * Ils évoquent les défis de la construction de scénarios prospectifs.

50:00 - 53:00 Conclusion et perspectives * Les intervenants appellent à un renforcement de la coopération entre les acteurs de la connaissance territoriale. * Ils soulignent le rôle essentiel de la connaissance territoriale pour construire des politiques publiques durables et justes.

Remarques

• La vidéo est riche en informations et en exemples concrets.
• Les intervenants sont des experts reconnus dans le domaine de la connaissance territoriale.
• La vidéo est bien structurée et facile à suivre.

J'espère que ces points forts vous seront utiles. N'hésitez pas à me contacter si vous avez d'autres questions.

Notez que

• Les timestamps sont approximatifs et peuvent varier légèrement en fonction de la façon dont vous regardez la vidéo.
• Je n'ai pu inclure que les points forts les plus importants dans ce résumé. Si vous souhaitez des informations plus détaillées sur un sujet particulier, n'hésitez pas à me le faire savoir.

Souhaitez-vous que je vous fournisse un résumé d'une autre vidéo ? http://googleusercontent.com/youtube_content/1

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public Review):

The goal of Knudsen-Palmer et al. was to define a biological set of rules that dictate the differential RNAi-mediated silencing of distinct target genes, motivated by facilitating the long-term development of effective RNAi-based drugs/therapeutics. To achieve this, the authors use a combination of computational modeling and RNAi function assays to reveal several criteria for effective RNAi-mediated silencing. This work provides insights into how (1) cis-regulatory elements influence the RNAi-mediated regulation of genes; (2) it is determined that genes can "recover" from RNAi-silencing signals in an animal; and 3) pUGylation occurs exclusively downstream of the dsRNA trigger sequence, suggesting 3º siRNAs are not produced. In addition, the authors show that the speed at which RNAi-silencing is triggered does not correlate with the longevity of the silencing. These insights are significant because they suggest that if we understand the rules by which RNAi pathways effectively silence genes with different transcription/processing levels then we can design more effective synthetic RNAi-based

therapeutics targeting endogenous genes. The conclusions of this study are mostly supported by the data, but there are some aspects that need to be clarified.

We thank the reviewer for their kind words and for appreciating the practical utility of our approach and discoveries. 

(1) The methods do not describe the "aged RNAi plates feeding assay" in Figure 2E. The figure legend states that "aged RNAi plates" were used to trigger weaker RNAi, but the detail explaining the experiment is insufficient. How aged is aged? If the goal was to effectively reduce the dsRNA load available to the animals, why not quantitatively titrate the dsRNA provided? Were worms previously fed on the plates, or was simply a lawn of bacteria grown until presumably the IPTG on the plate was exhausted?

We have elaborated our methods section to describe that the plates were left at 4ºC for about 4 months before adding bacteria and performing the assay, with one possible reason for the weaker knockdown being that perhaps the IPTG in the RNAi plates is less effective. However, it is worth noting that the robustness of a feeding RNAi assay can vary from culture to culture and/or batch of plates. We therefore always perform RNAi assays with wild-type animals alongside test strains to gauge the strength of the RNAi assay for a given culture and batch of plates. We called the data in Figure 2E “weak” because of the response of wild-type animals was weak as evidenced by weak twitching in levamisole. Despite this reduced effect, we observed 100% penetrance in wild-type animals, enabling us to sensitively detect the reduced responses of the mutants. 

(2) Is the data presented in Figure 2F completed using the "aged RNAi plates" to achieve the partial silencing of dpy-7 observed? Clarification of this point would be helpful.

No. The only occasion when plates were older was as in response to comment 1 above.

(3) Throughout the manuscript the authors refer to "non-dividing cells" when discussing animals' ability to recover from RNA silencing. It is not clear what the authors specifically mean with the phrase "non-dividing cells", but as this is referred to in one of their major findings, it should be clarified. Do they mean the cells are somatic cells in aged animals, thus if they are "non-dividing" the siRNA pools within the cells cannot be diluted by cell division? Based on the methods, the animals of RNAi assays were L4/Young adults that were scored over 8 days after the initial pulse of dsRNA feeding. If this is the case, wouldn't these animals be growing into gravid adults after the feeding, and thus have dividing cells as they grew?

We thank the reviewer for highlighting the need to explain this point further. Our experiment test the silencing of the unc-22 gene, which is expressed and functions in body-wall muscle cells. Most of the body wall muscles in C. elegans are developed by the L1 stage (reviewed in Krause and Liu, 2012), and they do not divide between the L4 and adult stages. Therefore, during the duration of the experiment where we delivered a pulse of dsRNA and examined responses over days, none of these cells divide. We have added a statement in the main text to explicitly say that the recovery from silencing by dsRNA that we observed cannot be explained by dilution during cell divisions.

(4) What are the typical expression levels/turnover of unc-22 and bli-1? Based on the results from the altered cis-regulatory regions of bli-1 and unc-22 in Figure 5, it seems like the transcription/turnover rates of each of these genes could also be used as a proof of principle for testing the model proposed in Figure 4. The strength of the model would be further increased if the RNAi sensitivity of unc-22 reflects differences in its transcription/turnover rates compared to bli-1.

We can get a sense of the relative abundances of unc-22 and bli-1 across development from the RNA-seq experiments that have been performed by others in the field (see below). However, these data cannot be used to infer either the production or the turnover rates. Future experiments that measure production (the combined rate of transcriptional run-on, splicing, export from the nucleus, etc.) will be required to define the production rates. Similarly, assays that detect the rate of degradation of transcripts without confounding presence from continued production will be needed to establish turnover rates. Future efforts to obtain values for these in vivo rates for multiple genes will help further test the model.

Author response image 1.

Expression data for unc-22:

Author response image 2.

Expression data for bli-1:

Reviewer #2 (Public Review):

Summary:

This manuscript by Knudsen-Palmer et al. describes and models the contribution of MUT-16 and RDE-10 in the silencing through RNAi by the Argonaute protein NRDE-3 or others. The authors show that MUT-16 and RDE-10 constitute an intersecting network that can be redundant or not depending on the gene being targeted by RNAi. In addition, the authors provide evidence that increasing dsRNA processing can compensate for NRDE-3 mutants. Overall, the authors provide convincing evidence to understand the factors involved in RNAi in C. elegans by using a genetic approach.

Major Strengths:

The author's work presents a compelling case for understanding the intricacies of RNA interference (RNAi) within the model organism Caenorhabditis elegans through a meticulous genetic approach. By harnessing genetic manipulation, they delve into the role of MUT-16 and RDE-10 in RNAi, offering a nuanced understanding of the molecular mechanisms at play in two independent case study targets (unc-22 and bli-1).

We thank the reviewer for their kind words and for appreciating our genetic analysis.

Major Weaknesses:

(1) It is unclear how the molecular mechanisms of amplification are different under the MUT-16 and RDE-10 branches of the regulatory pathway, since they are clearly distinct proteins structurally. It would be interesting to do some small-RNA-seq of products generated from unc-22 and bli-1, on wild-type conditions and some of the mutants studied (eg. mut-16, rde-10 and mut16 + rde-10). That would provide some insights into whether the products of the 2 amplifications are the same in all conditions, just changing in abundance, or whether they are distinct in sequence patterns.

As we highlight in the paper, MUT-16 and RDE-10 are indeed very different proteins. One possible hypothesis suggested by this difference is that different kinds of small RNAs are made when the underlying mechanism relies on MUT-16 versus on RDE-10. However, postulating such a difference is not necessary for explaining the data. Furthermore, since the amounts of 2º siRNAs do not have to be correlated with the strength of silencing (Figure 4E), this work raises caution against the over-reliance on small RNA sequencing for inferring gene silencing. Nevertheless, it is indeed an attractive possibility that the amounts of small RNA, their distributions along mRNA sequence, and/or the sequence biases of the accumulating small RNAs could be different when relying on MUT-16- or RDE-10-dependent mechanisms. Future work that directly examine the small RNAs that accumulate in different mutant strains after initiating RNAi can shed light on these possibilities.

(2) In the same line, Figure 5 aims to provide insights into the sequence determinants that influence the RNAi of bli-1. It is unclear whether the changes in transcript stability dictated by the 3'UTR are the sole factor governing the preference for the MUT-16 and RDE-10 branches of the regulatory pathway. In line with the mutant jam297, it might be interesting to test whether factors like codon optimality, splicing, ... of the ORF region upstream from bli-1-dsRNA can affect its sensitivity to the MUT-16 and RDE-10 branches of the regulatory pathway.

In Figure 5, we eliminated the possibility that any gene that is transcribed using the bli-1 promoter would require NRDE-3, and showed using jam297 that modifications to the 3’ cis regulatory regions of a target can alter the dependence on NRDE-3 for knockdown. We agree that future experiments that control individual aspects of bli-1, potentially one feature at a time, can reveal the separate contributions of each characteristic of the gene to the observed dependence on NRDE-3 of the wild-type bli-1 gene. However, given the many ways that the same level of transcript knockdown can be achieved in our modeling (Figure 4 and its supplemental figures) we expect that multiple characteristics could contribute to NRDE-3 dependence. 

Recommendations For The Authors:

Reviewer #1 (Recommendations For The Authors):

(1) On page 5, the authors state that "MUT-16 and RDE-10 are redundantly or additively required for silencing unc-22"; however, based on their data in Figure 1D, it seems nearly 100% silencing of unc-22 is achieved in single mut-16 or rde-10 mutants. If this is the case, wouldn't it suggest that redundancy of MUT-16 and RDE-10, and not an "additive effect" of MUT-16 and RDE-10 function? Although, as the mutator complex nucleates around MUT-16, the data in Figure 1D suggests it is possible that the presence of MUT-16 or RDE-10 is sufficient for the recruitment of one or more factors that triggers the silencing of unc-22, and thus only one of these factors is necessary.

Because we are seeing 100% silencing in wild-type, mut-16(-), or rde-10(-) animals in Figure 1D, this assay (where the silencing response is strong) does not allow us to discriminate between differing levels of silencing. The “weak” RNAi assay in Figure 2E provides the opportunity to observe differences in the contributions made by MUT-16 or RDE-10, supporting the idea that the 2º siRNAs and relative contributions to silencing can indeed be additive, explaining the complete loss of silencing only in the double mutant. While MUT-16 has been shown to be required for the recruitment of other Mutators in the germline, Mutator foci are not detectable in the soma. Given that unc-22 and bli-1 are somatic targets, we are hesitant to assume a mechanism for the production of small RNAs that requires a similar MUT-16-dependent nucleation in somatic cells. MUT-16 is clearly required for full silencing. But, if it functions similarly in the soma and the germline remains an open question. Indeed the mechanism(s) for producing small RNAs in somatic cells could be different from that used for production of small RNAs in the germline because of known differences in the use of RNA-dependent RNA polymerases (e.g. Ravikumar et al., Nucleic Acids Res. 2019). Future studies that determine the subcellular localization(s) and potential biochemical function(s) of RDE-10 and MUT-16 in somatic cells are needed to further delineate mechanisms.

(2) On page 10, "rather than one that looks a frequency" - the "a" should be "at".

We thank the reviewer and have fixed this typo. 

(3) Figure 4 is very crowded, further dividing 4A (right) and 4B into subpanels would help the readability of the figure.

We thank the reviewer for identifying these figures as being particularly crowded. These panels are presented as single units because the left and right portions of each panel are intimately connected. In Fig. 4A, the outline of mechanism deduced on the left is based on experiments at various scales shown on the right. We have now clarified this in the figure legend. In Fig. 4B, the equations on the right define and use the constants depicted on the left and the definitions below apply to both parts. We have now adjusted both figure parts to make these connections clearer. 

(4) References to the subpanels of Figure 4 in the text on page 12 are off from the figure and figure legend.

For example:

"Overall, τkd and tkd were uncorrelated..." refers to 4C when it should refer to 4D. "However, the maximal amount of 2ºsiRNAs..." refers to 4D when it should refer to 4E. "Additionally, an increase in transcription..." refers to 4E when it should refer to 4F.

"When a fixed amount of dsRNA was exposed..." refers to 4F when it should refer to 4G.

We thank the reviewer for catching these errors and we have corrected these figure references.

Reviewer #2 (Recommendations For The Authors):

I would encourage the authors to follow up on some of the more mechanistic comments made above, that would strengthen and complement the genetic part of the work presented.

We agree that additional work is needed to elucidate differences in molecular mechanisms for amplifying small RNAs in an MUT-16-dependent vs. RDE-10-dependent manner. We hope to address these extensions of our work in future manuscripts that focus on the biochemistry of these proteins and the populations of small RNAs generated using them.

I appreciate the efforts to computationally model the dynamics of the system, but I am not sure that it helps that the mathematical modelling treats both branches of the pathway as functionally equals, since they could have some mechanistic specialisation that is not yet elucidated by the current work.

Our assumption that both branches are equivalent is the most parsimonious. If we allowed for differences, even more values for the parameters of the model will agree with experimental data. The strength of the model is that despite such conservative assumptions, it agrees with experimental data. Biochemical elaborations that make the MUT-16 and RDE-10 branches qualitatively different could exist in vivo as suggested by the reviewer. Even with such qualitative differences in detail, the overall impact on gene silencing is a quantitative and additive one as demonstrated by our experiments. Future experimental work focused on biochemistry could elucidate how a Maelstrom domain-containing protein (RDE-10) and an intrinsically disordered protein (MUT-16) act differently to ultimately promote small RNA production.

eLife assessment

This valuable study shows how an intersecting network of regulators acting on genes with differences in their RNA metabolism explains why the loss of some regulators of RNAi in C. elegans can selectively impair the silencing of some target genes. The evidence presented is convincing, as the authors use a combination of computational modeling and RNAi assays to support their conclusions.

Reviewer #1 (Public Review):

SUMMARY:

The goal of Knudsen-Palmer et al. was to define a biological set of rules that dictate the differential RNAi-mediated silencing of distinct target genes, motivated by facilitating the long-term development of effective RNAi-based drugs/therapeutics. This work provides insights into how 1) cis-regulatory elements influence the RNAi-mediated regulation of genes; 2) determines that genes can "recover" from RNAi-silencing signals in an animal; and 3) pUGylation occurs exclusively downstream of the dsRNA trigger sequence, suggesting 3º siRNAs are not produced. In addition, the authors show that the speed at which RNAi-silencing is triggered does not correlate with the longevity of the silencing. Overall, the work presented supports the conclusions of the authors. The insights are significant because they suggest that if we understand the rules by which RNAi pathways effectively silence genes with different transcription/processing levels then we can design more effective synthetic RNAi-based therapeutics targeting endogenous genes.

MAJOR STRENGTH:

The authors use a combination of computational modeling, genetics, and RNAi function assays to reveal several criteria for effective RNAi-mediated silencing of two distinct targets.

WEAKNESS:

It may be beyond the scope of this study, but it would be interesting to know the typical expression levels and turnover rates of unc-22 and bli-1. Based on the results from the altered cis-regulatory regions of bli-1 and unc-22 in Fig 5, it seems like the transcription/turnover rates of each of these genes could also be used as a proof of principle for testing the model proposed in Figure 4. The strength of the model would be further increased if the RNAi sensitivity of unc-22 reflects differences in its transcription/turnover rates compared to bli-1.

Reviewer #2 (Public Review):

SUMMARY

This manuscript by Knudsen-Palmer et al. describes and models the contribution of MUT-16 and RDE-10 in the silencing through RNAi by the Argonaute protein NRDE-3 or others. The authors show that MUT-16 and RDE-10 constitute an intersecting network that can be redundant or not depending on the gene being targeted by RNAi. In addition, the authors provide evidence that increasing dsRNA processing can compensate for NRDE-3 mutants. Overall, the authors provide convincing evidence to understand the factors involved in RNAi in C. elegans by using a genetic approach.

MAJOR STRENGTHS

The author's work presents a compelling case for understanding the intricacies of RNA interference (RNAi) within the model organism Caenorhabditis elegans through a meticulous genetic approach. By harnessing genetic manipulation, they delve into the role of MUT-16 and RDE-10 in RNAi, offering a nuanced understanding of the molecular mechanisms at play in two independent case study targets (unc-22 and bli-1).

MAJOR WEAKNESSES

(1) It is unclear how the molecular mechanisms of amplification are different under the MUT-16 and RDE-10 branches of the regulatory pathway, since they are clearly distinct proteins structurally. It would be interesting to do some small-RNA-seq of products generated from unc-22 and bli-1, on wild-type conditions and some of the mutants studied (eg. mut-16, rde-10 and mut-16 + rde-10). That would provide some insights on whether the products of the 2 amplifications are the same in all conditions, just changing in abundance, or whether they are distinct in sequence patterns.

(2) In the same line, Figure 5 aims to provide insights to the sequence determinants that influence on the RNAi of bli-1. It is unclear whether the changes in transcript stability dictated by the 3'UTR are the sole factor governing the preference for the MUT-16 and RDE-10 branches of the regulatory pathway. In line with the mutant jam297, it might be interesting to test whether factors like codon optimality, splicing, ... of the ORF region upstream from bli-1-dsRNA can affect its sensitivity to the MUT-16 and RDE-10 branches of the regulatory pathway.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

TMC7 knockout mice were generated by the authors and the phenotype was analyzed. They found that Tmc7 is localized to Golgi and is needed for acrosome biogenesis.

Strengths:

The phenotype of infertility is clear, and the results of TMC7 localization and the failed acrosome formation are highly reliable. In this respect, they made a significant discovery regarding spermatogenesis.

In the original version, I pointed out the gap between their pH/calcium imaging data and the hypothesis of ion channel function of TMC7 in the Golgi. Now the author agrees and has changed the description to be reasonable. Additional experiments were also performed, and I can say that they have answered my concern adequately.

I would say it is good to add any presumed mechanism for the observed changes in pH and calcium concentration in the cytoplasm this time.

We appreciate your positive comments on our revised manuscript.

Reviewer #2 (Public Review):

Summary:

This study presents a significant finding that enhances our understanding of spermatogenesis. TMC7 belongs to a family of transmembrane channel-like proteins (TMC1-8), primarily known for their role in the ear. Mutations to TMC1/2 are linked to deafness in humans and mice and were originally characterized as auditory mechanosensitive ion channels. However, the function of the other TMC family members remains poorly characterized. In this study, the authors begin to elucidate the function of TMC7 in acrosome biogenesis during spermatogenesis. Through analysis of transcriptomics datasets, they identify TMC7 as a transmembrane channel-like protein with elevated transcript levels in round spermatids in both mouse and human testis. They then generate Tmc7-/- mice and find that male mice exhibit smaller testes and complete infertility. Examination of different developmental stages reveals spermatogenesis defects, including reduced sperm count, elongated spermatids, and large vacuoles. Additionally, abnormal acrosome morphology is observed beginning at the early-stage Golgi phase, indicating TMC7's involvement in proacrosomal vesicle trafficking and fusion. They observed localization of TMC7 in the cis-Golgi and suggest that its presence is required for maintaining Golgi integrity, with Tmc7-/- leading to reduced intracellular Ca2+, elevated pH, and increased ROS levels, likely resulting in spermatid apoptosis. Overall, the work delineates a new function of TMC7 in spermatogenesis and the authors suggest that its ion channel activity is likely important for Golgi homeostasis. This work is of significant interest to the community and is of high quality.

Strengths:

The biggest strength of the paper is the phenotypic characterization of the TMC7-/- mouse model, which has clear acrosome biogenesis/spermatogenesis defects. This is the main claim of the paper and it is supported by the data that are presented.

Weaknesses:

The claim is that TMC7 functions as an ion channel. It is reasonable to assume this given what has been previously published on the more well-characterized TMCs (TMC1/2), but the data supporting this is preliminary here, and more needs to be done to solidify this hypothesis. The authors are careful in their interpretation and present this merely as a hypothesis supporting this idea.

We appreciate this constructive suggestion.

Reviewer #3 (Public Review):

Summary:

In this study, Wang et al. have demonstrated that TMC7, a testis-enriched multipass transmembrane protein, is essential for male reproduction in mice. Tmc7 KO male mice are sterile due to reduced sperm count and abnormal sperm morphology. TMC7 co-localizes with GM130, a cis-Golgi marker, in round spermatids. The absence of TMC7 results in reduced levels of Golgi proteins, elevated abundance of ER stress markers, as well as changes of Ca2+ and pH levels in the KO testis. However, further confirmation is required because the analyses were performed with whole testis samples in spite of the differences in the germ cell composition in WT and KO testis. In addition, the causal relationships between the reported anomalies await thorough interrogation

Strengths:

By using PD21 testes, the revised assays have consolidated that depletion of TMC7 leads to a reduced level of Ca2+ and an elevated level of ROS in the male germ cells. The immunohistochemistry analyses have clearly indicated the reduced abundance of GM130, P115, and GRASP65 in the knockout testis.

Weaknesses:

The Discussion section contains sentences reiterating the Introduction and Results of this manuscript (e.g., Lines 79-85 and 231-236; Lines 175-179 and 259-263). Those read repetitive and can be removed.

We thank the reviewer for this import comment. We have modified the text according to your suggestion.

Future studies are required to decipher how TMC7 stabilizes Golgi structure, coordinates vesicle transport, and maintains the germ cell homeostasis.

Thanks. We appreciate this constructive suggestion. We totally agree the reviewer that future studies are required to decipher how TMC7 stabilizes Golgi structure, coordinates vesicle transport, and maintains the germ cell homeostasis.

Recommendations for the authors

Reviewer #1 (Recommendations For The Authors):

1. In Fig S6d, the bar of Tmc7-/- is broken in the middle for P-EIF2.

Thanks. We have remade Fig S6d according to your suggestion in the revised manuscript.

Reviewer #2 (Recommendations For The Authors):

None. The reviewers have adequately answered my points. Many thanks!

We thank the reviewer for accepting our revisions as sufficient.

Reviewer #3 (Recommendations For The Authors):

In the revised manuscript, the authors have addressed most of my concerns.

We are pleased that we were able to adequately address the reviewer’s concerns. We appreciate your suggestions to further improve our study.

eLife assessment

This study reports an important discovery highlighting the essential role of the putative ion channel, TMC7, in acrosome formation during sperm development and thus male fertility. The evidence for the requirement of TMC7 in acrosome biogenesis and sperm function is convincing, although its function as an ion channel remains to be further determined. Overall, this work will be of great interest to developmental biologists and ion channel physiologists alike.

Reviewer #1 (Public Review):

Summary:

TMC7 knockout mice were generated by the authors and the phenotype was analyzed. They found that Tmc7 is localized to Golgi and is needed for acrosome biogenesis.

Strengths:

The phenotype of infertility is clear, and the results of TMC7 localization and the failed acrosome formation are highly reliable. In this respect, they made a significant discovery regarding spermatogenesis.

In the original version, I pointed out the gap between their pH/calcium imaging data and the hypothesis of ion channel function of TMC7 in the Golgi. Now the author agrees and has changed the description to be reasonable. Additional experiments were also performed, and I can say that they have answered my concern adequately.

I would say it is good to add any presumed mechanism for the observed changes in pH and calcium concentration in the cytoplasm this time.

Reviewer #2 (Public Review):

Summary:

This study presents a significant finding that enhances our understanding of spermatogenesis. TMC7 belongs to a family of transmembrane channel-like proteins (TMC1-8), primarily known for their role in the ear. Mutations to TMC1/2 are linked to deafness in humans and mice and were originally characterized as auditory mechanosensitive ion channels. However, the function of the other TMC family members remains poorly characterized. In this study, the authors begin to elucidate the function of TMC7 in acrosome biogenesis during spermatogenesis. Through analysis of transcriptomics datasets, they elevated levels of TMC7 in round spermatids in both mouse and human testis. They then generate Tmc7-/- mice and find that male mice exhibit smaller testes and complete infertility. Examination of different developmental stages reveals spermatogenesis defects, including with reduced sperm count, elongated spermatids and large vacuoles. Additionally, abnormal acrosome morphology are observed beginning at the early-stage Golgi phase, indicating TMC7's involvement in proacrosomal vesicle trafficking and fusion. They observed localization of TMC7 in the cis-Golgi and suggest that its presence is required for maintaining Golgi integrity, with Tmc7-/- leading to reduced intracellular Ca2+, elevated pH and increased ROS levels, likely resulting in spermatid apoptosis. Overall, the work delineates a new function of TMC7 in spermatogenesis and the authors propose that its ion channel and/or scramblase activity is likely important for Golgi homeostasis. This work is of significant interest to the community and is of high quality.

Strengths:

The biggest strength of the paper is the phenotypic characterization of the TMC7-/- mouse model, which has clear acrosome biogenesis/spermatogenesis defects. This is the main claim of the paper and it is supported with the data that are presented.

Weaknesses:

It isn't clear whether TMC7 functions as an ion channel from the current data presented in this paper, but the authors are careful in their interpretation and present this merely as a hypothesis supporting this idea.

Reviewer #3 (Public Review):

Summary:

In this study, Wang et al. have demonstrated that TMC7, a testis-enriched multipass transmembrane protein, is essential for male reproduction in mice. Tmc7 KO male mice are sterile due to reduced sperm count and abnormal sperm morphology. TMC7 co-localizes with GM130, a cis-Golgi marker, in round spermatids. The absence of TMC7 results in reduced levels of Golgi proteins, elevated abundance of ER stress markers, as well as changes of Ca2+ and pH levels in the KO testis. However, further confirmation is required because the analyses were performed with whole testis samples in spite of the differences in the germ cell composition in WT and KO testis. In addition, the causal relationships between the reported anomalies await thorough interrogation

Strengths:

By using PD21 testes, the revised assays have consolidated that depletion of TMC7 leads to a reduced level of Ca2+ and an elevated level of ROS in the male germ cells. The immunohistochemistry analyses have clearly indicated the reduced abundance of GM130, P115, and GRASP65 in the knockout testis.

Weaknesses:

Future studies are required to decipher how TMC7 stabilizes Golgi structure, coordinates vesicle transport, and maintains the germ cell homeostasis.

Author response:

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

Reviewer #1

Summary:

In this paper, the authors performed molecular dynamics (MD) simulations to investigate the molecular basis of the association of alpha-synuclein chains under molecular crowding and salt conditions. Aggregation of alpha-synuclein is linked to the pathogenesis of Parkinson's disease, and the liquid-liquid phase separation (LLPS) is considered to play an important role in the nucleation step of the alpha-synuclein aggregation. This paper re-tuned the Martini3 coarse-grained force field parameters, which allows long-timescale MD simulations of intrinsically disordered proteins with explicit solvent under diverse environmental perturbation. Their MD simulations showed that alpha-synuclein does not have a high LLPS-forming propensity, but the molecular crowding and salt addition tend to enhance the tendency of droplet formation and therefore modulate the alpha-synuclein aggregation. The MD simulation results also revealed important intra- and inter-molecule conformational features of the alpha-synuclein chains in the formed droplets and the key interactions responsible for the stability of the droplets. These MD simulation data add biophysical insights into the molecular mechanism underlying the association of alpha-synuclein chains, which is important for understanding the pathogenesis of Parkinson's disease.

Strengths:

(1) The re-parameterized Martini 3 coarse-grained force field enables the large-scale MD simulations of the intrinsically disordered proteins with explicit solvent, which will be useful for a more realistic description of the molecular basis of LLPS.

(2) This paper showed that molecular crowding and salt contribute to the modulation of the LLPS through different means. The molecular crowding minimally affects surface tension, but adding salt increases surface tension. It is also interesting to show that the aggregation pathway involves the disruption of the intra-chain interactions arising from C-terminal regions, which potentially facilitates the formation of inter-chain interactions.

We thank the reviewer for pointing out the strengths of our study.

Weaknesses:

(1) Although the authors emphasized the advantage of the Martini3 force field for its explicit description of solvent, the whole paper did not discuss the water's role in the aggregation and LLPS.

We thank the reviewer for pointing this out. We agree that we have not explored or discussed the role of water in aS aggregation or LLPS. We would like to convey that we would like to explore that in detail in a separate study altogether. However we have updated the “Discussion” section with the following lines to convey to the readers the importance water plays in aggregation and LLPS of aS.

Page 24: “The significance of the solvent in alpha-synuclein (αS) aggregation remains underexplored. Recent studies [26, 55] underscore the pivotal role of water as a solvent in LLPS. It suggests that comprehending the solvent’s role, particularly water, is essential for attaining a deeper grasp of the thermodynamic and physical aspects of αS LLPS and aggregation. By delving into the solvent’s contribution, researchers can uncover additional factors influencing αS aggregation. Such insights hold the potential to advance our comprehension of protein aggregation phenomena, crucial for devising strategies to address diseases linked to protein misfolding and aggregation, notably Parkinson’s disease. Future investigations focusing on elucidating the interplay between αS, solvent (especially water), and other environmental elements could yield valuable insights into the mechanisms underlying LLPS and aggregation. Ultimately, this could aid in the development of therapeutic interventions or preventive measures for Parkinson’s and related diseases.”

(2) This paper discussed the effects of crowders and salt on the surface tension of the droplets.

The calculation of the surface tension relies on the droplet shape. However, for the formed clusters in the MD simulations, the typical size is <10, which may be too small to rigorously define the droplet shape. As shown in previous work cited by this paper [Benayad et al., J. Chem. Theory Comput. 2021, 17, 525−537], the calculated surface tension becomes stable when the chain number is larger than 100.

We appreciate the insightful feedback from the reviewer. However, we would like to emphasize that the αS droplets exhibit a highly liquid-like behavior, characterized by frequent exchanges of chains between the dense and dilute phases, alongside a slow aggregation process. In the study by Benayad et al. (2020, JCTC) [ref. 30], FUS-LCD was the protein of choice at concentrations in the (mM) range. FUS-LCD is known to undergo very rapid LLPS at concentrations lower than 100 (μM) where for αS the critical concentration for LLPS is 500 (μM) and undergoes slower aggregation than FUS. Moreover, the diffusion constant of αS inside newly formed droplets (no liquid to solid phase transition has occurred) has been estimated to be 0.23-0.58 μm2/s (Ray et al, 2020, Nat. Comm.). The value of diffusion constant for FUS-LCD inside LLPS droplets has been estimated to be 0.17 μm2/s (Murthy et al. 2023, Nat. Struct. and Mol. Biol.). These prove that αS forms droplets that are less viscous than that formed by FUS-LCD. This dynamic nature impedes the formation of large droplets in the simulations, making it challenging to rigorously calculate surface tension from interfacial width, which, in turn, necessitates the computation of g(r) between water and the droplet.

Furthermore, it's essential to note that our primary aim in calculating surface tension was not to determine its absolute value. Rather, we aimed to compare surface tensions obtained for the three distinct environments explored in this study. Hence, our primary objective is to compare the distributions of surface tensions rather than focusing solely on the mean values obtained. The distributions shown in Figure 4a clearly show a trend which we have stated in the article.

(3) In this work, the Martini 3 force field was modified by rescaling the LJ parameters \epsilon and \sigma with a common factor \lambda. It has not been very clearly described in the manuscript why these two different parameters can be rescaled by a common factor and why it is necessary to separately tune these two parameters, instead of just tuning the coefficient \epsilon as did in a previous work [Larsen et al., PLoS Comput Biol 16: e1007870].

We thank the reviewer for the comment. We think that the distance of the first hydration layer also should have an impact on aggregation/LLPS. Here we are scaling both the epsilon and sigma. A higher epsilon of water-protein interactions mean higher the energy required for removal of water molecules (dehydration) when a chain goes from the dilute to the dense phase. A higher sigma on the other hand means that the hydration shell will also be at a larger distance making dehydration easier. Moreover, tuning both (either by same or different parameter) required a change of the overall protein-water interaction by only 1%, thereby requiring only considerably minimal change in forcefield parameters (compared to the case where only epsilon is being tuned which required 6-10% change in epsilon from its original values.) . Thus we think one of the ways of tuning water-protein interactions which requires minimal retuning of Martini 3 is by optimizing both epsilon and sigma. However whether a single scaling parameter is good enough requires further exploration and is outside the scope of the current study. More importantly it would introduce another free parameter into the system and the lesser the number of free parameters, the better. For this study, a single parameter sufficed as depicted in Figure 9. To inform the readers of why we chose to scale both sigma and epsilon, we have added the following in the main text:

Page 25-26: “Increasing the ϵ value of water-protein interactions results in a higher energy demand for removing water molecules (dehydration) as a chain transitions from the dilute to the dense phase. Conversely, a higher σ value implies that the hydration shell will be at a greater distance, facilitating dehydration if a chain moves into the dilute phase. Therefore, adjusting water-protein interactions based on the protein’s single-chain behavior may not significantly influence the protein’s phase behavior. Furthermore, fine-tuning both ϵ and σ parameters only requires a minimal change in the overall protein-water interaction (1%). As a result, this adjustment minimally alters the force field parameters.”

(4) Both the sizes and volume fractions of the crowders can affect the protein association. It will be interesting to perform MD simulations by adding crowders with various sizes and volume fractions. In addition, in this work, the crowders were modelled by fullerenes, which contribute to protein aggregation mainly by entropic means as discussed in the manuscript. It is not very clear how the crowder effect is sensitive to the chemical nature of the crowders (e.g., inert crowders with excluded volume effect or crowders with non-specific attractive interactions with proteins, etc) and therefore the force field parameters.

We thank the reviewer for a potential future direction. In this investigation our main focus was to simulate the inertness features of crowders only, to ensure that only entropic effect of the crowders are explored. Although this study focuses on the factors that enable aS to form an aggregates/LLPS under different environmental conditions, it would be interesting to explore in a systematic way the mechanism of action of crowders of varying shapes, sizes and interactions. Therefore we added the following lines in the “Discussion” section to let the readers know that this is also a future prospect of investigation.

Page 22: “Under physiological conditions, crowding effects emerge prominently. While crowders are commonly perceived to be inert, as has been considered in this investigation, the morphology, dimensions, and chemical interactions of crowding agents with αS in both dilute and dense phases may potentially exert considerable influence on its LLPS. Hence, a comprehensive understanding through systematic exploration is another avenue that warrants extensive investigation.”

Reviewer #1 (Recommendations For The Authors):

(1) Figure S1. The title of the figure and the description in the figure caption are inconsistent?

We thank the reviewer for the comment and we have updated the article with the correct caption.

(2) Page 14, line 3, the authors may want to provide more descriptions of the "ms1", "ms2", and "ms3" for better understanding.

We are grateful to the reviewer for pointing this out. We have added a line describing in brief what “ms1”, “ms2” and “ms3” represent. It reads “Subsequent to the investigation, we utilize three representative conformations, each corresponding to one of the macrostates. We designate these macrostates as 1 (ms1), 2 (ms2), and 3 (ms3) (Figure S7)” (Page 28)

(3) Page 20, the authors may want to briefly explain how the normalized Shannon entropy was calculated.

We thank the reviewer for pointing this out. This is plain Shannon Entropy and the word “normalized” should not have been there. To avoid confusion we have provided the equation we have used to calculate the Shannon entropy (Eq 8) (Page 21).

Reviewer #2 (Public Review):

In the manuscript "Modulation of α-Synuclein Aggregation Amid Diverse Environmental Perturbation", Wasim et al describe coarse-grained molecular dynamics (cgMD) simulations of α-Synuclein (αS) at several concentrations and in the presence of molecular crowding agents or high salt. They begin by bench-marking their cgMD against all-atom simulations by Shaw. They then carry 2.4-4.3 µs cgMD simulations under the above-noted conditions and analyze the data in terms of protein structure, interaction network analysis, and extrapolated fluid mechanics properties. This is an interesting study because a molecular scale understanding of protein droplets is currently lacking, but I have a number of concerns about how it is currently executed and presented.

We thank the reviewer for finding our study interesting.

(1) It is not clear whether the simulations have reached a steady state. If they have not, it invalidates many of their analysis methods and conclusions.

We have used the last 1 μs (1.5-2.5 1 μs) from each simulation for further analysis in this study. To understand whether the simulations have reached steady state or not, we plot the time profile of the concentration of the protein in the dilute phase for all three cases.

Author response image 1.

Except for the scenario of only αS (Figures a and b), the rest show very steady concentrations across various sections of the trajectory (Figures c-f). The larger sudden fluctuations observed inFigures a and b are due to the fact that only αS undergo very slow spontaneous aggregation and owing to the fact that the dense phase itself is very fluxional, addition/removal of a few chains to/from the dense to dilute phase register themselves as large fluctuations in the protein concentration in the dilute phase. For the other two scenarios (Figures c-f) aggregation has been accelerated due to the presence of crowders/salt. This causes larger aggregates to be formed. Therefore addition/removal of one or two chains does not significantly affect the concentration and we do not see such sudden large jumps. In summary, the large jumps seen in Figures a and b are due to slow, fluxional aggregation of pure αS and finite size effects. However as these still are only fluctuations, we posit that the systems have reached steady states. This claim is further supported by the following figure where the time profile of a few useful system wide macroscopic properties show no change between 1.5-2.5 µs.

We also have added a brief discussion in the Methods section (Page 29-30) with these figures in the Supplementary Information.

Author response image 2.

“In this study, we utilized the final 1 µs from each simulation for further analysis. To ascertain whether the simulations have achieved a steady state, we plotted the time profile of protein concentration in the dilute phase for all three cases. Except for minor intermittent fluctuation involving only αS in neat water (Figures S8a and S8b), the remaining cases exhibit notably stable concentrations throughout various segments of the trajectory (Figures S8 c-f). The relatively higher fluctuations observed in Figures S8a and b stem from the slow, spontaneous aggregation of αS alone, compounded by the inherently ambiguous nature of the dense phase.

Consequently, the addition or removal of a few chains from the dense to the dilute phase results in significant fluctuations in protein concentration within the dilute phase. Conversely, in the other two scenarios (Figures S8c-f), aggregation is expedited by the presence of crowders/salt, leading to the formation of larger aggregates. Consequently, the addition or removal of one or two chains has negligible impact on concentration, thereby mitigating sudden large jumps. In summary, the conspicuous jumps depicted in Figures S8a and b arise from the gradual, fluctuating aggregation of pure αS and finite size effects. However, since these remain within the realm of fluctuations, we assert that the systems have indeed reached steady states. This assertion is bolstered by the subsequent figure, where the time profile of several pertinent system-wide macroscopic properties reveals no discernible change between 1.5-2.5 µs (Figures S9).”

(2) The benchmarking used to validate their cgMD methods is very minimal and fails to utilize a large amount of available all-atom simulation and experimental data.

We disagree with the reviewer on this point. We have cited multiple previous studies [26, 27] that have chosen Rg as a metric of choice for benchmarking coarse-grained model and have used a reference (experimental or otherwise) to tune Martini force fields. Majority of the notable literature where Rg was used as a benchmark during generation of new coarse-grained force fields are works by Dignon et al. (PLoS Comp. Biol.) [ref. 25], Regy et al (Protein Science. 2021) [ref. 26], Joseph et al.(Nature Computational Science. 2021) [ref. 27] and Tesei et al (Open Research Europe, 2022) [ref. 28]. From a polymer physics perspective, tuning water-protein interactions is simply changing the solvent characteristics for the biopolymer and Rg has been generally considered a suitable metric in the case of coarse-grained model. Moreover we try to match the distribution of the Rg rather than only the mean value. This suggests that at a single molecule level, the cgMD simulations at the optimum water of water-protein interactions would allow the protein to sample the conformations present in the reference ensemble. We use the extensively sampled 70 μs all-atom data from DE Shaw Research to obtain the reference Rg distribution. Also we perform a cross validation by comparing the fraction of bound states in all-atom and cgMD dimer simulations which also seem to corroborate well with each other at optimum water-protein interactions. To let the readers understand the rationale behind choosing Rg we have added a section in the Methods section (Page 25) that explains why Rg is plausibly a good metric for tuning water-protein interactions in Martini 3, at least when dealing with IDPs.

Our optimized model is further supported by the FRET experiments by Ray et al. [6]. They found that interchain NAC-NAC interactions drive LLPS. Residue level contact maps obtained from our simulations also show decreased intrachain NAC-NAC interactions with an increased interchain NAC-NAC interactions inside the droplet. This corroborates well with the experimental observations and furthermore validates the metrics we have used for optimization of the water-protein interactions. However the comparison with the FRET data by Ray et al. was not present earlier and we have added the following lines in the updated draft.

Page17: “Thus we observed that increased inter-chain NAC-NAC regions facilitate the formation of αS droplets which also have previously been seen from FRET experiments on αS LLPS

droplets[6].”

(3) They also miss opportunities to compare their simulations to experimental data on aSyn protein droplets.

We thank the reviewer for pointing this out. We have tried to compare the results from our simulations to existing experimental FRET data on αS. Please see the previous response where we have described our comparison with FRET observations.

(4) Aspects such as network analysis are not contextualized by comparison to other protein condensed phases.

For a proper comparison between other protein condensed phases, we would require the position phase space of such condensates which is not readily available. Therefore we tried to explain it in a simpler manner to paint a picture of how αS forms an interconnecting network inside the droplet phase.

(5) Data are not made available, which is an emerging standard in the field.

We thank the reviewer for mentioning this. We have provided the trajectories between 1.5-2.5 μs, which we used for the analysis presented in the article, via a zenodo repository along with other relevant files related to the simulations (https://zenodo.org/records/10926368).

Firstly, it is not clear that these systems are equilibrated or at a steady state (since protein droplets are not really equilibrium systems). The authors do not present any data showing time courses that indicate the system to be reaching a steady state. This is problematic for several of their data analysis procedures, but particularly in determining free energy of transfer between the condensed and dilute phases based on partitioning.

We have addressed this concern as stated previously in the response. We have updated the article accordingly.

Secondly, the benchmarking that they perform against the 73 µs all-atom simulation of aSyn monomer by Shaw and coworkers provides only very crude validation of their cgMD models based on reproducing Rg for the monomer. The authors should make more extensive comparisons to the specific conformations observed in the DE Shaw work. Shaw makes the entire trajectory publicly available. There are also a wealth of experimental data that could be used for validation with more molecular detail. See for example, NMR and FRET data used to benchmark Monte Carlo simulations of aSyn monomer (as well as extensive comparisons to the Shaw MD trajectory) in Ferrie at al: A Unified De Novo Approach for Predicting the Structures of Ordered and Disordered Proteins, J. Phys. Chem. B 124 5538-5548 (2020)

DOI:10.1021/acs.jpcb.0c02924

I note that NMR measurements of aSyn in liquid droplets are available from Vendruscolo: Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies, Journal of Molecular Cell Biology, Volume 13, Issue 4, April 2021, Pages 282-294, https://doi.org/10.1093/jmcb/mjaa075.

In addition, there are FRET studies by Maji: Spectrally Resolved FRET Microscopy of α-Synuclein Phase-Separated Liquid Droplets, Methods Mol Biol 2023:2551:425-447. doi: 10.1007/978-1-0716-2597-2_27.

So the authors are missing opportunities to better validate the simulations and place their structural understanding in greater context. This is just based on my own quick search, so I am sure that additional and possibly better experimental comparisons can be found.

We have performed a comparison with existing FRET measurements by Ray et al. (2020) as discussed in a previous response and also updated the same in the article. The doi (10.1007/978-1-0716-2597-2_27) provided by the reviewer is however for a book on Methods to characterize protein aggregates and does not contain any information regarding the observations from FRET experiments. The other doi (https://doi.org/10.1093/jmcb/mjaa075) for the article from Vendrusculo group does not contain information directly relevant to this study. Moreover NMR measurements cannot be predicted from cgMD since full atomic resolution is lost upon coarse-graining of the protein . A past literature survey by the authors found very little scientific literature on molecular level characterization of αS LLPS droplets.

Thirdly, the small word network analysis is interesting, but hard to contextualize. For instance, the 8 Å cutoff used seems arbitrary. How does changing the cutoff affect the value of S determined? Also, how does the value of S compare to other condensed phases like crystal packing or amyloid forms of aSyn?

The 8 Å cutoff is actually arbitrary since a distance based clustering always requires a cutoff which is empirically decided. However 8 Å is quite large compared to other cutoffs used for distance based clustering. For example in ref 26, 5 Å was used as a cutoff for calculation of protein clusters. Larger cutoffs will lead to sparser network structures. However we used the same cutoff for all distance based clustering which makes the networks obtained comparable. We wanted to perform a comparison among the networks formed by αS under different environmental conditions.

Fourthly, I see no statement on data availability. The emerging standard in the computational field is to make all data publicly available through Github or some similar mechanism.

We thank the reviewer for pointing this out and we have provided the raw data between 1.5-2.5 μs for each scenario along with other relevant files via a zenodo repository (https://zenodo.org/records/10926368).

Finally, on page 16, they discuss the interactions of aSyn(95-110), but the sequence that they give is too long (seeming to contain repeated characters, but also not accurate). aSyn(95-110) = VKKDQLGKNEEGAPQE. Presumably this is just a typo, but potentially raises concerns about the simulations (since without available data, one cannot check that the sequence is accurate) and data analysis elsewhere.

This indeed is a typographical error. We have updated the article with the correct sequence. The validity of the simulations can be verified from the data we have shared via the zenodo repository (https://zenodo.org/records/10926368).

eLife assessment

This study provides important biophysical insights into the molecular mechanism underlying the association of alpha-synuclein chains, which is essential for understanding the pathogenesis of Parkinson's disease. The data analysis is solid, and the methodology can help investigate other molecular processes involving intrinsically disordered proteins.

Reviewer #1 (Public Review):

Summary:

In this paper, the authors performed molecular dynamics (MD) simulations to investigate the molecular basis of association of alpha-synuclein chains under molecular crowding and salt conditions. Aggregation of alpha-synuclein is linked to the pathogenesis of Parkinson's disease, and the liquid-liquid phase separation (LLPS) is considered to play an important role in the nucleation step of the alpha-synuclein aggregation. This paper re-tuned the Martini3 coarse-grained force field parameters, which allows long-timescale MD simulations of intrinsically disordered proteins with explicit solvent under diverse environmental perturbation. Their MD simulations showed that alpha-synuclein does not have a high LLPS-forming propensity, but the molecular crowding and salt addition tend to enhance the tendency of droplet formation and therefore modulate the alpha-synuclein aggregation. The MD simulation results also revealed important intra and inter-molecule conformational features of the alpha-synuclein chains in the formed droplets and the key interactions responsible for the stability of the droplets. These MD simulation data add biophysical insights into the molecular mechanism underlying the association of alpha-synuclein chains, which may be useful for understanding the pathogenesis of Parkinson's disease.

Strengths:

(1) The re-parameterized Martini 3 coarse-grained force field enables the large-scale MD simulations of the intrinsically disordered proteins with explicit solvent, which will be useful for a more realistic description of the molecular basis of LLPS.

(2) This paper showed that the molecular crowding and salt contribute to the modulation of the LLPS through different means. The molecular crowding minimally affects surface tension, but adding salt increases surface tension. It is also interesting to show that the aggregation pathway involves the disruption of the intra-chain interactions arising from C-terminal regions, which potentially facilitates the formation of inter-chain interactions.

Weaknesses:

(1) Although the authors emphasized the advantage of the Martini3 force field for its explicit description of solvent, this paper did not analyze the water behavior contained in the simulation trajectories and discuss the water's role in the aggregation and LLPS.

(2) This paper discussed the effects of crowders and salt on the surface tension of the droplets. The calculation of the surface tension relies on the droplet shape. However, for the formed clusters in the MD simulations, the typical size is <10, which may be too small to rigorously define the droplet shape. As shown in previous work cited by this paper [Benayad et al., J. Chem. Theory Comput. 2021, 17, 525−537], the calculated surface tension becomes stable when the chain number is larger than 100.

(3) Both the sizes and volume fractions of the crowders can affect the protein association. It will be interesting to perform MD simulations by adding crowders with various sizes and volume fractions. In addition, in this work the crowders were modelled by fullerenes, which contribute to protein aggregation mainly by entropic means as discussed in the manuscript. It is not very clear how the crowder effect is sensitive to the chemical nature of the crowders (e.g., inert crowers with excluded volume effect or crowders with non-specific attractive interactions with proteins, etc).

Reviewer #2 (Public Review):

In the manuscript "Modulation of α-Synuclein Aggregation Amid Diverse Environmental Perturbation", Wasim et al describe coarse-grained molecular dynamics (cgMD) simulations of α-Synuclein (aSyn) at several concentrations and in the presence of molecular crowding agents or high salt. They begin by bench-marking their cgMD against all-atom simulations by Shaw. They then carry 2.4-4.3 µs cgMD simulations under the above-noted conditions and analyze the data in terms of protein structure, interaction network analysis, and extrapolated fluid mechanics properties. This is an interesting study because a molecular scale understanding of protein droplets is currently lacking.

個人的にはここに読点入れたほうが読みやすいかなと思いました。<br /> Ex) 画面上に得点をきれいに表示するために、文字の色を

Author response:

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

Reviewer #1 (Public Review): 

Summary:

In this manuscript, Fister et. al. investigate how amputational and burn wounds affect sensory axonal damage and regeneration in a zebrafish model system. The authors discovered that burn injury results in increased peripheral axon damage and impaired regeneration. Convincing experiments show altered axonal morphology and increased Ca2+ fluxes as a result of burn damage. Further experimental proof supports that early removal of the burnt tissue by amputation rescues axonal damage. Burn damage was also shown to markedly increase keratinocyte migration and increase localized ROS production as measured by the dye Pfbsf. These responses could be inhibited by Arp 2/3 inhibition and isotonic treatment. 

Strengths: 

The authors use state-of-the-art methods to study and compare transection and burn-induced tissue damage. Multiple experimental approaches (morphology, Ca2+ fluxing, cell membrane labeling) confirm axonal damage and impaired regeneration time. Furthermore, the results are also accompanied by functional response tests of touch sensitivity. This is the first study to extend the role of tissue-damage-related osmotic exposure beyond wound closure and leukocyte migration to a novel layer of pathology: axonal damage and regeneration. 

Weaknesses: 

The conclusions of the paper claiming a link between burn-induced epithelial cell migration, spatial redox signaling, and sensory axon regeneration are mainly based on correlative observations. Arp 2/3 inhibition impairs cell migration but has no significant effect on axon regeneration and restoration of touch sensitivity. 

We agree with the reviewer. We have tried many experiments to address this question. The data show that Arp 2/3 inhibition with CK666 is an effective way to inhibit initial keratinocyte migration. However, later migration still proceeds. What is interesting is that just inhibition of the early migration is sufficient to restore localized ROS production in the wound area in the first  hour post-burn, even if this is not sufficient to prevent ROS accumulation over time. There is also a trend toward improved sensory neuron function late after this early treatment. However, this is not statistically significant. We think it is likely that both migration and tissue scale ROS influence the regeneration defect of sensory neurons after burn. The data using isotonic solution supports this conclusion. We have tried many other ways to limit keratinocyte migration including depletion of talin and expression of a dominant negative Rac in basal epithelial cells, but these treatments were not compatible with survival of the fish after burn.

Pharmacological or genetic approaches should be used to prove the role of ROS production by directly targeting the known H2O2 source in the system: DUOX. 

We agree that pharmacologic or genetic approaches to directly manipulate ROS production would provide substantial support to the hypothesis that ROS, along with keratinocyte migration, is a main factor contributing to poor burn outcomes. To address this, we first tried using a morpholino to deplete DUOX. However, the combination of DUOX morpholino and burn injury was lethal to larvae. We also used pharmacologic inhibition of ROS production using DPI (Diphenyleneiodonium). With this treatment, ROS is inhibited for only the first hour post-burn as treatment is lethal for longer periods of time. Burned larvae have marginally improved axon density and touch sensitivity, suggesting the importance of ROS in burn outcomes, however it was not statistically significant. It is likely that an increased effect would be observed with longer treatment, but treatment for more than 1 hour was toxic. We have added a supplemental figure with this new DPI data.

While the authors provide clear and compelling proof that osmotic responses lie at the heart of the burn-induced axonal damage responses, they did not consider the option of further exploring any biology related to osmotic cell swelling. Could osmotic ATP release maybe play a role through excitotoxicity? Could cPLA2 activation-dependent eicosanoid production relate to the process? Pharmacological tests using purinergic receptor inhibition or blockage of eicosanoid production could answer these questions. 

We agree that the role of osmotic cell swelling in the burn response is an interesting avenue for future study. However, we make use of isotonic treatment in this study specifically for its effect on keratinocyte migration and broad-scale wound healing. As a result, we feel that pursuing the biology of this swelling phenomenon is outside the scope of this paper.

The authors provide elegant experiments showing that early removal of the burnt tissue can rescue damage-induced axonal damage, which could also be interpreted in an osmotic manner: tail fin transections could close faster than burn wounds, allowing for lower hypotonic exposure time. Axonal damage and slow regeneration in tail fin burn wounds could be a direct consequence of extended exposure time to hypotonic water. 

We have done experiments using FM dye to test how long it takes burn and transection wounds to close (shown below). In these experiments, dye entry into wounded tissue is used as a readout of wound closure. Dye is only able to enter wounded tissue when the epithelial barrier is disrupted. Our data reveal that transections take approximately 10 minutes to fully close, while burns take approximately 20 minutes to close.

Author response image 1.

To test if this difference in wound closure time would have an effect on axon outcomes, we repeated, but slightly modified, the dual-wound experiment. We increased the amount of time the burn condition was exposed to hypotonic conditions by 10 additional minutes (by transecting burned tissue at 15 minutes post burn, shortly before closure) and compared axon outcomes to the 5 mpw control transection. These results show there was no difference in axon regeneration or function when secondary transection was performed at 5 or 15 minutes post burn, suggesting that increased exposure to hypotonic solution is not the reason for defects in axon outcomes after burn injury.

Author response image 2.

Reviewer #2 (Public Review): 

This is an interesting study in which the authors show that a thermal injury leads to extensive sensory axon damage and impaired regrowth compared to a mechanical transection injury. This correlates with increased keratinocyte migration. That migration is inhibited by CK666 drug treatment and isotonic medium. Both restrict ROS signalling to the wound edge. In addition, the isotonic medium also rescues the regrowth of sensory axons and recovery of sensory function. The findings may have implications for understanding non-optimal re-innervation of burn wounds in mammals. 

The interpretation of results is generally cautious and controls are robust. 

Here are some suggestions for additional discussion: 

The study compares burn injury which produces a diffuse injury to a mechanical cut injury which produces focal damage. It would help the reader to give a definition of wound edge in the burn situation. Is the thermally injured tissue completely dead and is resorbed or do axons have to grow into damaged tissue? The two-cut model suggests the latter. Also giving timescales would help, e.g. when do axons grow in relation to keratinocyte movement? An introductory cartoon might help. 

We thank the reviewer for these insightful comments and questions. The burn wound is defined as the area that is directly damaged as a result of increased heat (labeled by FM dye entry), and the burn wound edge as the first line of healthy cells adjacent to the burned cells. These definitions have been added to the text to clarify the areas referenced. Recent experiments lead us to believe the wound area is composed almost completely of dead cells, but we are currently working to discover the fate of these dead cells as well as the wound adjacent cells that migrate to the wound edge after burn. As a result, we do not know whether axons grow into damaged tissue or if the damaged tissue is extruded, but we do see growth cone formation within a few hours after wounding suggesting the axons are actively trying to regenerate after a burn.

Could treatment with CK666 or isotonic solution influence sensory axons directly, or through other non-keratinocyte cell types, such as immune cells? 

We have done experiments looking at the density of caudal fin innervation in CK666, isotonic, or DPI treated fins. The axon density is unchanged in all these treatments compared to control treated larvae, so we do not believe these treatments affect axon health homeostatically. These data have been added to supplemental figure 3. Additionally, one of the benefits of the larval zebrafish burn model is the simplicity of the system – the epidermis is primarily composed of sensory axons, mesenchymal cells and keratinocytes. The burn environment is proinflammatory so it does promote immune cell recruitment, but we do not believe the immune cells are interacting directly with sensory axons besides clearing axonal debris. Previous papers by our lab have shown that peak immune cell recruitment occurs at 6 hpw, but they localize to the damaged tissue in the burn area and not the wound edge.

Reviewer #3 (Public Review): 

Fister and colleagues use regeneration of the larval zebrafish caudal fin to compare the effects of two modes of tissue damage-transection and burn-on cutaneous sensory axon regeneration. The authors found that restoration of sensory axon density and function is delayed following burn injury compared to transection. 

The authors hypothesized that thermal injury triggers signals within the wound microenvironment that impair sensory neuron regeneration. The authors identify differences in the responses of epithelial keratinocytes to the two modes of injury: keratinocytes migrate in response to burn but not transection. Inhibiting keratinocyte migration with the small-molecule inhibitor of Arp2/3 (CK666) resulted in decreased production of reactive oxygen species (ROS) at early, but not late, time points. Preventing keratinocyte migration by wounding in isotonic media resulted in increased sensory function 24 hours after burn. 

Strengths of the study include the beautiful imaging and rigorous statistical approaches used by the authors. The ability to assess both axon density and axon function during regeneration is quite powerful. The touch assay adds a unique component to the paper and strengthens the argument that burns are more damaging to sensory structures and that different treatments help to ameliorate this. 

A weakness of the study is the lack of genetic and cell-autonomous manipulations. Additional comparisons between transection and burns, in particular with manipulations that specifically modulate ROS generation or cell migration without potentially confounding effects on other cell types or processes would help to strengthen the manuscript.

The use of genetic and cell-autonomous approaches would strengthen our study, however, we were unable to do this due to the lethality of these genetic approaches (or cell autonomous approaches). Basal epithelial migration is necessary for embryonic development. We attempted to circumvent this by generation of larvae transiently expressing a dominant-negative form of Rac, a protein crucial to the migratory process. The chimeric expression of the dominant negative Rac was either damaging to the larvae or the mosaicism was too low to observe any effects on migration phenotype.

We also attempted a genetic approach to manipulate ROS production, as discussed above. We found that the DUOX morpholino was lethal to burned larvae. Finally, we attempted pharmacological inhibition of ROS production using the inhibitor DPI (Diphenyleneiodonium). With this treatment, burned larvae have marginally improved axon density and touch sensitivity, suggesting that dampening ROS may improve outcome. The DPI data have been added to the manuscript.

In terms of framing their results, the authors refer to "sensory neurons" and "sensory axons" throughout the text - it should be made clear what type of neuron(s)/axon(s) are being visualized/assayed. Along these lines, a broader discussion of how burn injuries affect sensory function in other systems - and how the authors' results might inform our understanding of these injury responses - would be beneficial to the reader. 

In summary, the authors have established a tractable vertebrate system to investigate different sensory axon wound healing outcomes in vivo that may ultimately allow for the identification of improved treatment strategies for human burn patients. Although the study implicates differences in keratinocyte migration and associated ROS production in sensory axon wound healing outcomes, the links between these processes could be more rigorously established. 

The inconsistency between “neuron” and “axon” has been noted and the text has been corrected accordingly. “Neuron” is used when referring to the cell as a whole, while “axon” is used when referring to the sensory processes in the caudal fin. We added information about burn in the introduction as suggested: “While epithelial tissue is well adapted to repair from mechanical damage, burn wounds heal poorly. Thermal injury results in chronic pain and lack of sensation in the affected tissue, suggesting that an abnormal sensory neuron response contributes to burn wound pathophysiology.”

We thank the reviewer’s for their comments.

Recommendations For The Authors:

Reviewer #1 (Recommendations For The Authors): 

Suggested experiments: 

(1) ROS measurements with the dye Pfbsf should be validated with more established ROS probes such as HyPer. 

Pfbsf has been used previously as a readout of ROS production, and its use is documented in zebrafish (Maeda et al., Angew Chem Int Ed Engl, 2004, and Niethammer et al, Nature, 2009). These sources have been added as references when introducing Pfbsf to provide context for its use. The probe was validated and compared to HyPer in Niethammer’s 2009 paper. In our hands, we have used both probes and have similar results with tail transection.

(2) To better support claims on ROS and H2O2 playing a central role in mediating axonal damage, the authors should consider pharmacological approaches such as rescue experiments with H2O2 and experiments using inhibitors such as DPI ar apocynin. 

While the above reagents and drugs have limitations and non-specific side effects, more convincing proof could result from genetic approaches including experiments on DOUX knockdown or knockout lines. 

To further dissect the role of ROS in the burn response, we conducted experiments using DPI, a potent ROS inhibitor that is well-documented in the literature. We found that 20 uM treatment of DPI (1 hour pretreatment, 1 hour post-burn) marginally improved axon density when quantified 24 hpw. Any higher dose, when in combination with a burn, proved to be lethal. Longer treatment with DPI was also not tolerated.

In addition to experiments with DPI, we attempted to burn larvae that were injected with DUOX morpholino. The combined use of burn and DUOX MO was lethal. We have dampened the conclusions and include the new data with the DPI in the revised manuscript.

Minor corrections: 

(1)A phrase/expression in the abstract is confusing: isotonic treatment does not "induce osmotic regulation". Cells exposed to hypo- or hypertonicity will respond by regulatory volume decrease or increase, respectively. Isotonic treatment maintains homeostasis. 

We appreciate this point and agree with the distinction. Revisions have been made in the text accordingly.

(2) Figures 4E and 5E would be better to show as an average of multiple experiments with statistical significance. 

The purpose of figures 4E and 5E are to demonstrate changes in fluorescence intensity and localization of ROS using the representative time series shown in 4D and 5D. The figure legend has been updated accordingly.

Reviewer #2 (Recommendations For The Authors): 

Figure 3D How can one distinguish between the two cellular elements that randomly meet or that there is actual coordination? Can the interactions be quantified? It is also unclear what the authors mean by "sensory neuron movement". The authors show that the neuronal cell bodies stay in their position, so only the axons change position. Do they do this by growth, i.e. the neuronal growth cones follow the keratinocytes or do keratinocytes displace the axon shafts? 

We have included supplemental movies that address this question in the new uploaded document. Figure 3D is comprised of still images taken from supplemental movie 2, which is a timelapse of keratinocytes/axons moving together after a burn injury.  This movie clearly shows keratinocytes and their ensheathed axons moving simultaneously, so keratinocytes are mechanically pulling sensory axon shafts with them. We have revised the text to say axon movement, not sensory neuron movement.

Over the time course of axonal movement (1 hour post-burn), it is not possible that neuronal growth cones contribute to movement, as this is too slow – previous work by other labs has shown that it takes several hours for axons to fully regenerate into amputated tissue, with movement not even noticeable until about 3 hours post-wound (Rieger and Sagasti, PLOS Biology, 2011).

Regarding the second point, “neuron” vs. “axon” is an inconsistency in the text that has been corrected. “Neuron” is used when referring to the cell as a whole, “axon” is used when referring to the processes that innervate the caudal fin. The axons are physically pulled along with keratinocytes as they migrate after burn application. From our observations, growth cones appear closer to the wound site after the movement has stopped.

Figure 4G It is surprising that the visual differences in the distribution of values are not statistically significant. 

The distribution of values in 4G was large and that is why there is no statistically-significant difference – we were also surprised at this result. We did all statistics with a statistician and this included rigorous criteria for significance.

Figure 4H The images seem to show a difference, whereas the quantification does not. I suggest choosing more representative images. 

Figure 4H has been updated to include a more representative image of axon patterning with CK666 treatment.

Figure 6A The text states that axon damage in the control and isotonic condition is comparable, yet in the image, it appears that the damage in the isotonic treatment at 0 hpw is more distal. 

This is a good observation that we consistently see in isotonic-treated fish after burn. Axon damage localizes more proximally in isotonic-treated samples because the keratinocytes distal to the notochord are likely dead, and the axons innervating those cells are likely immediately destroyed upon burn application. As a result, the distal axons are not present to express GCaMP. We believe isotonic treatment allows keratinocytes to live slightly longer, so axon damage is therefore prevented for longer. This is also the focus of continuing work to further understand the burn microenvironment.

Finally, the materials section could mention bias mitigation measures, e.g. withholding the treatment condition from the experimenter in the touch test. 

We minimized bias in experiments whenever possible, and the conservative statistical measures that were applied to our data further reduce the likelihood of false significance.

Reviewer #3 (Recommendations For The Authors): 

- Line numbers would have facilitated reviewer feedback. 

- Supplementary movies were missing in the submission. 

The lack of supplementary movies upon submission was a mistake and the movies have been uploaded along with the revised manuscript.

Introduction: 

- Pg. 3: "In response to tissue damage, sensory neurons undergo rapid and localized axonal degeneration 4,5." Not sure reference 4 (Reyes et al) is appropriate here as this study was not in the context of tissue damage. 

We have revised this section as suggested by the reviewer.

Results: 

- The expected expression pattern/localization of several transgenes was unclear. Please clearly state what cell type(s) each should label. For example, pg. 5 - "We next sought to further investigate sensory neuron function in burned tissue. For this, we assessed wound-induced axonal damage using zebrafish larvae that express the calcium probe GCaMP." Where is GCaMP expressed? 

The manuscript has been updated to include expression patterns for the included transgenes – in this mentioned case, GCaMP is expressed in neurons under the pan-neuronal Elavl3 promoter.

- Introducing the GCaMP labeling could use some clarification. Pg. 5 - "As shown previously by other groups, GCaMP labels degenerating neurons in real time35." This is confusing. Do the authors mean that GCaMP increases immediately prior to Wallerian degeneration as shown by Vargas et al. (PMID: 26558774)? 

Sustained elevated calcium levels are associated with axon damage. Previous work from other labs has shown that calcium influx follows axon injury (Ziv and Spira, EJN 1993, Adalbert et al., Neuroscience 2012). In these experiments, whenever there are CGaMP-positive punctae, this indicates axon damage. We have revised the manuscript to address this critique.

The Elavl3-GCaMP5 transgenic line will label when calcium levels increase in neurons. However, given the parameters used for imaging in our study (20x magnification, 100 ms exposure, and collection speed every 30 seconds for timelapses), we believe that only sufficiently large increases in calcium that are indicative of cell damage, and not physiological function, are being visualized.

- Figure 1E - Are these panels images of the same fish? Please specify in the legend. 

Figure 1E is comprised of one transected and one burned larva each, live-imaged over the course of six hours. The legend has been updated to include this information.

- Figure 1F - How was the damage area measured? Consider doing this measurement over time to match Figure 1E. 

Axon damage area measurements were performed similar to axon density measurements – maximum intensity z-projected confocal images of the caudal fin were generated using FIJI. For all experiments, the caudal fin area posterior to the notochord was outlined using the Polygon tool and measured to obtain a total surface area ROI. Axon fragments inside the outlined area were manually thresholded so all fragments posterior to the notochord were labeled and no saturated pixels were present, and an area measurement of these thresholded pixels was taken. We have added a section describing these measurements in the Methods section under “Axon damage quantification.”

- Pg. 5 - When introducing the ngn1 MO - please state the expected phenotype and cite the appropriate background literature_._ 

The ngn1 morpholino was cited in the Methods section with the appropriate literature (Cornell and Eisen, Development, 2002), from which we got the morpholino sequence. We thank the reviewer for pointing out the need for more introduction and clarification in the main text, so the ngn1 morpholino has been discussed in greater depth and cited in the main text as well using the same citation.

- The two-wound model is an elegant approach but could be more clearly described in the main text. 

An improved explanation of the two-wound experiment has been added to the text.

- For Figure 3, it would be helpful to have a schematic of the anatomy illustrating the relative positions of axons and epidermal cell types. 

- Figure 3C - should an additional control here be transected? Given that the krt4:lifeact transgene labels both layers of the epidermis, how were the superficial and basal keratinocytes separated? Interpretation of this section should be carefully worded. The authors state that "...suggesting that the superficial keratinocytes are being pulled by the motile basal keratinocytes" (pg.7 ) but isn't another possibility that the superficial cells are stationary? 

It is correct that the krt4:lifeact transgene labels both layers of keratinocytes, which together span 20-30 microns. These layers were separated from the same z-stack collected by confocal imaging. The first z-slice and last z-slice of the same stack were separated using FIJI and pseudocolored to appear as different colors. This clarification has been added to the Methods.

Prior observations with the krt4:lifeact and krt4:utrch (figure 3A) transgenic lines reveal that both keratinocyte layers will move distally after burn application.

- Pg. 7 - "The axons of sensory neurons are ensheathed within actin-rich channels running through basal keratinocytes 50,51." ref 51 is a C. elegans paper which does not have basal keratinocytes.

This was in error. The correct reference has replaced reference 51 (O’brien, J Comp. Neurol., 2012), in which electron microscopy is used to document the development of two layers of epithelial cells that also ensheath sensory neurons in a protective manner similar to glial cells in the central nervous system.

- Figures S1E and F - the authors state that RB and DRG soma don't move. However, it was unclear from the figure panels and legend whether the authors imaged neurons that actually innervate the caudal fin (rather than some other region of the animal). Please clarify. For comparison, Fig S1F needs a pre-injury image to be meaningful. 

The imaged cell bodies were those in the posterior trunk region, which are responsible for innervating the posterior sections of the fish including the caudal fin. From our observations, there was no movement of neuronal cell bodies after the burn.

- Figure 5 title - can the authors clarify what aspect of this figure relates to "sustained epidermal damage" 

The figure 5 title has been updated in response to the reviewer comments.

- Figure 6 - is touch sensitivity really "restored" as the authors suggest? Alternatively, sensitivity may never be lost in isotonic treatment. Or the loss may be delayed? 

We have modified the text accordingly by updating our phrasing – “restored” has been replaced with “improved” to indicate benefit over time.

- Can the authors further disentangle the effects of keratinocyte migration, ROS, and isotonic treatment on axon regeneration? For example, would the addition of CK666 to the Isotonic +1 hpw treatment improve axon regeneration? Can the authors directly manipulate ROS signaling (e.g., through exogenous addition of H2O2 or duox1 MO) to alter regeneration outcomes in their wounding assays? 

See the comments above.

- Figure 6 title - consider removing or clarifying the word "excessive" here 

The title has been revised according to the reviewer suggestion.

- hpw vs hpb were used inconsistently throughout the text 

The manuscript has been revised to use “hpw” when referring to the timeframe after injury application.

Methods: 

- Zebrafish transgenics are missing allele names 

References: 

- Many mistakes were noted in this section e.g., journal names missing, wrong authors, typos, DOIs misformatted 

The references section has been corrected to use formatting consistent with APA citation and eLife preferred guidelines.

eLife assessment

This important study identifies a novel link between the early keratinocyte response to wounds and the subsequent regenerative capacity of local sensory neurons. The evidence supporting the claims of the authors is convincing, although inclusion of conditional genetics or cell-autonomy tests would have strengthened the mechanistic aspects. The work will be of interest to cell and developmental biologists interested in tissue regeneration and cell interactions in a broader context.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Fister et. al. investigate how amputational and burn wounds affect sensory axonal damage and regeneration in a zebrafish model system. The authors discovered that burn injury results in increased peripheral axon damage and impaired regeneration. Convincing experiments show altered axonal morphology and increased Ca2+ fluxes as a result of burn damage. Further experimental proof supports that early removal of the burnt tissue by amputation rescues axonal damage. Burn damage was also shown to markedly increase keratinocyte migration and increase localized ROS production as measured by the dye Pfbsf. These responses could be inhibited by Arp 2/3 inhibition and isotonic treatment.

Strengths:

The authors use state-of-the-art methods to study and compare transection and burn-induced tissue damage. Multiple experimental approaches (morphology, Ca2+ fluxing, cell membrane labeling) confirm axonal damage and the impaired regeneration time. Furthermore, the results are also accompanied by functional response tests of touch sensitivity. This is the first study to extend the role of tissue-damage related osmotic exposure beyond wound closure and leukocyte migration to a novel layer of pathology: axonal damage and regeneration.

The authors provide elegant experiments showing that early removal of the burnt tissue can rescue damage-induced axonal damage, which could also be interpreted in an osmotic manner. In the revised version of the paper the authors indeed show that tail fin transections close faster than burn wounds, allowing for lower hypotonic exposure time. However, their new experiments suggest that axonal damage and slow regeneration in tail fin burn wounds are not a direct consequence of the extended exposure time to hypotonic water.

Weaknesses:

The conclusions of the paper claiming a link between burn-induced epithelial cell migration, spatial redox signaling, and sensory axon regeneration are mainly based on correlative observations. Arp 2/3 inhibition impairs cell migration but has no significant effect on axon regeneration and restoration of touch sensitivity.

Genetic approaches have been tested during the revision process to directly prove the role of ROS production by targeting DUOX, however, the combination of DUOX morpholino and burn injury was lethal to the larvae and long-term pharmacological inhibition over 1 hour was also detrimental.

Reviewer #3 (Public Review):

Fister and colleagues use regeneration of the larval zebrafish caudal fin to compare the effects of two modes of tissue damage-transection and burn-on cutaneous sensory axon regeneration. The authors found that restoration of sensory axon density and function is delayed following burn injury compared to transection.

The authors hypothesized that thermal injury triggers signals within the wound microenvironment that impair sensory neuron regeneration. The authors identify differences in the responses of epithelial keratinocytes to the two modes of injury: keratinocytes migrate in response to burn but not transection. Inhibiting keratinocyte migration with a small-molecule inhibitor of Arp2/3 (CK666) resulted in decreased production of reactive oxygen species (ROS) at early, but not late, timepoints. Preventing keratinocyte migration by wounding in isotonic media resulted in increased sensory function 24 hours after burn.

Strengths of the study include the beautiful imaging and rigorous statistical approaches used by the authors. The ability to assess both axon density and axon function during regeneration is quite powerful. The touch assay adds a unique component to the paper and strengthens the argument that burns are more damaging to sensory structures and that different treatments help to ameliorate this.

A weakness of the study is the lack of genetic and cell autonomous manipulations. Additional comparisons between transection and burns, in particular with manipulations that specifically modulate ROS generation or cell migration without potentially confounding effects on other cell types or processes would help to strengthen the manuscript. In terms of framing their results, the authors refer to "sensory neurons" and "sensory axons" throughout the text - it should be made clear what type of neuron(s)/axon(s) are being visualized/assayed. Along these lines, a broader discussion of how burn injuries affect sensory function in other systems-and how the authors' results might inform our understanding of these injury responses-would be beneficial to the reader.

In summary, the authors have established a tractable vertebrate system to investigate different sensory axon wound healing outcomes in vivo that may ultimately allow for the identification of improved treatment strategies for human burn patients. Although the study implicates differences in keratinocyte migration and associated ROS production in sensory axon wound healing outcomes, the links between these processes could be more rigorously established.

Points forts de la vidéo "Décrypter la recherche - ep#2 "Les formes de soutien à la vie associative : du national au local"" avec des timestamps de 0h00min à 0h59min

0h00min - 0h05min : Introduction et contexte de la recherche

• Présentation de l'épisode et de l'invitée Mathilde Rininassi, chargée de recherche.
• Contexte de la recherche : questionner l'action publique de soutien à la vie associative en France.
• Objectifs de l'étude : comprendre les formes de soutien à la vie associative au niveau national et les interactions entre les acteurs publics et associatifs.

0h05min - 0h15min : Illisibilité et diversité des dispositifs de soutien

• Difficulté pour les associations de naviguer dans la multitude de dispositifs de soutien.
• Existence de dispositifs portés par différents ministères et organismes d'État.
• Manque de coordination et de lisibilité des politiques publiques de soutien à la vie associative.

0h15min - 0h25min : Méthodologie de la recherche

• Étude qualitative basée sur 42 entretiens semi-directifs.
• Rencontre avec des agents de 15 ministères ou organismes d'État.
• Interrogation de 19 têtes de réseau ou associations.

0h25min - 0h35min : Formes de soutien à la vie associative

• Typologie des formes de soutien :
  • Soutien financier: subventions, appels à projets, etc.
  • Soutien organisationnel: accompagnement à la structuration, formation, etc.
  • Soutien informationnel: mise à disposition de ressources, communication, etc.
• Variation des formes de soutien selon les ministères et les types d'associations.

0h35min - 0h45min : Rôle des têtes de réseau et des fédérations

• Implication des têtes de réseau dans la mise en œuvre des politiques publiques.
• Coordination et structuration du secteur associatif par les fédérations.
• Dialogue entre les acteurs publics et associatifs.

0h45min - 0h55min : Constats et perspectives

• Absence d'une politique publique unifiée de soutien à la vie associative.
• Nécessité d'une meilleure coordination entre les acteurs publics.
• Simplification des procédures administratives et des financements.
• Renforcement du dialogue entre les acteurs publics et associatifs.

0h55min - 0h59min : Conclusion et remerciements

• Remerciements aux participants et aux intervenants.
• Invitation à l'épisode suivant.

Note: Il est important de noter que les points forts ne sont pas exhaustifs et que la vidéo contient d'autres informations riches. Je vous encourage à regarder la vidéo dans son intégralité pour une meilleure compréhension du sujet.

Points forts de la vidéo [Webinaire] Renforcement du monde associatif - Ep.1 "Face aux attaques des libertés associatives" Introduction

Le contexte actuel est marqué par des attaques croissantes contre les libertés associatives. Le RNMA et le Collectif des associations citoyennes organisent un cycle de webinaires pour explorer les risques et construire des réponses collectives. Ce premier épisode se concentre sur la question de la réponse collective face aux menaces qui pèsent sur les libertés associatives. Les libertés associatives en France en 2024

Définition des libertés associatives Etat des lieux des menaces et des attaques contre les libertés associatives Acteurs et enjeux de la défense des libertés associatives Illustration des menaces par deux organisations

Le Planning familial et son expérience face aux attaques anti-avortement. La Fédération d'associations de protection de la nature et les difficultés de financement. Stratégies de réponse collective

Mobilisation et solidarité entre associations. Travail interassociatif et création de coalitions. Outillage juridique des associations. Développement de contentieux stratégiques. Construction d'une jurisprudence favorable aux associations. Conclusion

Importance de la réflexion et de l'action collective pour défendre les libertés associatives. Le RNMA propose un cycle de webinaires pour approfondir la réflexion et construire des stratégies communes. Ressources

Lien vers la production de Vox Public sur "Anticiper et oui des raps entre guillemets". Livre de Jean-Baptiste Jobart sur les libertés associatives. Fiche réflex sur les stratégies de défense des libertés associatives. Adresse mail du RNMA pour recevoir les ressources et les dates des prochains webinaires. Moments clés

00:00 - Introduction et présentation des intervenants 05:30 - Définition des libertés associatives et contexte actuel 10:30 - Exposé de Jean-Baptiste Jobart sur les menaces aux libertés associatives 25:00 - Témoignage de Karine sur l'expérience du Planning familial 32:30 - Témoignage de Stéphane Giroud sur les difficultés des associations de protection de la nature 42:00 - Discussion sur les stratégies de réponse collective 57:00 - Conclusion et présentation des prochains webinaires

本文（15章など）で、axが表している plot のことを プロット と書いているので、ここは プロット の方が良いと思います。 たとえば、plt.subplots()で作成したプロット（ax）の中に複数の折れ線グラフを含めることもできるので、axのことを「グラフ」と呼ぶとちょっとややこしく感じます。

例: ひとつのプロット（ax）に複数の折れ線グラフがあるケース https://matplotlib.org/stable/gallery/lines_bars_and_markers/errorbar_limits_simple.html#sphx-glr-gallery-lines-bars-and-markers-errorbar-limits-simple-py

他の箇所も同様。

プロット

原文が separate plot で、ここでのプロットはAxesオブジェクトのことなので

別々のプロット

の方が適切だと思います。

原文:

You can include as many individual graphs in one figure as you want.

individual の意味が抜けています。このindividualは「1つのプロット（Axesオブジェクト）内に複数のグラフを描く」のではなく、「Axesオブジェクト自体を分けて、別々にグラフを描く」という意味が強調されていると思うので、

1つの図に、独立したグラフをいくつでも含めることができます。

の方が適切だと思います。

プロット

[技術的な正確性]

ここは1つのFigureオブジェクトに複数のAxesオブジェクトを作る例なので、

1つの図に複数のプロット

が正しい表現かと思います。

描画

描画

本文では datetime object を 「datetime オブジェクト」と訳しています（コード表記の有無であえて訳を使い分けてる？）

日付

本文や Working with dates and times　では 日付 と訳されているのですが、一部の箇所で 日 になっています。日本語としては 日付 の方が自然だと思います。

日付

代案:

また、facecolor で塗りつぶしに使う色を、オプション引数のalpha で色の透明度を受け取ります。

元の原稿だと、文前半と末尾の表現が揃っておらず違和感があります。

の　が続くので、シンプルに データセット間　の方が良いかと思います

の　が続くので、シンプルに データセット間　の方が良いかと思います

描画

描画

名詞の プロット とややこしいので

typo 「の」

原文:

When you make multiple plots, you can emphasize relationships in the data

原文の plots を プロット と訳しているのですが、この表現だとAxisオブジェクトを複数作っているように受け取れてややこしいので、ここは グラフ などの表現の方が技術的には正確だと思います。

「複数のプロット」と言われると 下記のような複数のAxesオブジェクトを作るケースを想像します。

fig, axes = plt.subplots(2)

サンプルコードを見たところ、ここはAxesオブジェクト自体を複数作るのではなく「Axesオブジェクトを1つ作って、その中に複数のグラフを含める」パターンなので、plots = グラフ の方が適切かと。

代案:

1つの図にいくつもデータを描画できます。

動詞の plot を「プロット」と訳すと、名詞の plot （主にAxesオブジェクト）とややこしいので...

原文は Multiple plots なのですが、サンプルコードを見ると「1つのFigureオブジェクトに複数のAxesオブジェクト（プロット）がある」のではなく「1つのAxesオブジェクト（プロット）に複数のグラフがある」という内容になっているので、ここは

複数のグラフ

の方が良いと思いました。

全体的に原文が

• Figureオブジェクト（図/描画オブジェクト）
• Axesオブジェクト（プロット/サブプロット）
• Axesオブジェクト内に描画されるデータ（グラフ？）

をあまり区別せずにひとくくりで plot と呼んでいるようで悩ましいのですが、実際に指しているものが何かを意識して用語を使い分けた方が混乱しないと思います。

別コメントで「変数ax を指している plot の訳は プロット の方が良い」的なことを書いているのですが、ここの原文が指している plot は 図全体のことを指しているので、ここは「グラフ」のまま、あるいは 「図」で良いと思います。

別コメントで「変数ax を指している plot の訳は プロット の方が良い」的なことを書いているのですが、ここの原文が指している plot は fig の方なので、ここは「グラフ」のまま、あるいは 「図」で良いと思います。

好みかもしれませんが、やや違和感ありました （カスタマイズ（名詞）と完全に削除できます（動詞）が並列になってるから？

以下はどうでしょうか

軸をカスタマイズしたり、完全に削除したりすることも可能です。

原文:

You can plot as much data as you want on one plot. H

前半の plot が変数 ax のことを指していると思うので

1つのプロットに必要なだけ大量のデータを描画できます。

とかですかね。

やや冗長でわかりにくいので

各点の色を決めるための値

はどうでしょうか

「軸のスケールを追加」が日本語として変なので

タイトル、ラベルの追加と、軸のスケーリング

あるいは

タイトル、ラベルの追加と、軸のスケール変更

（原文が Adding titles and labels, and scaling axes なので、 adding と scaling が並列の関係になります）

[技術的な正確性]

This convention is used even when there's only one plot in the figure.

現在の原稿だとfigureもplotも「図」になっているのですが、

• figure は描画オブジェクト（変数fig）
• plot はプロット（変数ax）

のことで、明確にオブジェクトの種類が違うので、用語を分けた方が良いと思います。

代案:

この慣例は、図の中にプロットが1つしかない場合でも使われます。

（ここでの convention が「axは図（描画オブジェクトfig）の中のプロットを表す」ということであれば、慣例というよりライブラリのルールなので、 慣例　 より 決まり / ルール の方がしっくりくる気もしますが...

プロット（理由は別コメントと同様）

[日本語の文法]

代案:

簡単な可視化を始める上で役に立ちます。

Faits marquants de la vidéo [Webinaire] Renforcement du monde associatif - Ep.2 "Face à la marchandisation des associations" avec des timestamps:

00:00:00 - Introduction et présentation des intervenants * Le webinaire fait partie d'un cycle sur le renforcement du monde associatif, suite à l'ouvrage "Quel monde associatif de demain?". * Cet épisode se concentre sur la question de la marchandisation des associations. * Les intervenants sont Marianne Langlais (Observatoire citoyen de la marchandisation des associations), Frédéric Bildé (CP Sud-Ouest et secrétaire de la LASS), Thomas Lowers (Réseau national des maisons des associations) et Léa Boirois (chargé de mission au RNMA).

05:30:00 - Définition de la marchandisation des associations * La marchandisation des associations se caractérise par un recul des subventions de fonctionnement et une augmentation des financements par des commandes publiques et des ressources marchandes. * Cela entraîne une transformation des modes de fonctionnement des associations, avec une pression pour la performance et la quantification des résultats.

10:00:00 - Impacts de la marchandisation des associations * La marchandisation des associations peut avoir des impacts négatifs sur leur autonomie, leurs valeurs et leurs pratiques. * Elle peut conduire à une perte de pouvoir des bénévoles et à une instrumentalisation des associations par les pouvoirs publics ou les entreprises.

15:00:00 - Témoignages de terrain * Des intervenants partagent leurs expériences de la marchandisation des associations dans différents secteurs d'activité. * Ils soulignent les difficultés rencontrées pour maintenir des pratiques associatives authentiques et solidaires.

25:00:00 - Pistes de démarchandisation * Les intervenants discutent de différentes stratégies pour lutter contre la marchandisation des associations. * Il s'agit de renforcer le pouvoir des associations, de promouvoir des financements alternatifs et de repenser les relations avec les pouvoirs publics.

40:00:00 - Questions-réponses * Les participants au webinaire posent des questions aux intervenants sur la marchandisation des associations et les pistes de démarchandisation.

55:00:00 - Conclusion et annonce du prochain épisode * Les intervenants résument les points clés du webinaire et invitent les participants à suivre le prochain épisode sur la managérialisation des associations.

1:59:46 - Fin du webinaire

This is still unclear to me

eLife assessment

This important study reports the discovery of a novel nucleotide ubiquitylation activity by the DTX3L E3 ligase. Solid evidence is presented for ubiquitin attachment to single-stranded oligonucleotides. This very interesting biochemical finding can be used as a starting point for studies to establish relevance in a physiological setting.

Reviewer #1 (Public Review):

In the article by Dearlove et al., the authors present evidence in strong support of nucleotide ubiquitylation by DTX3L, suggesting it is a promiscuous E3 ligase with capacity to ubiquitylate ADP ribose and nucleotides. The authors include data to identify the likely site of attachment and the requirements for nucleotide modification.

While this discovery potentially reveals a whole new mechanism by which nucleotide function can be regulated in cells, there are some weaknesses that should be considered. Is there any evidence of nucleotide ubiquitylation occurring cells? It seems possible, but evidence in support of this would strengthen the manuscript. The NMR data could also be strengthened as the binding interface is not reported or mapped onto the structure/model, this seems of considerable interest given that highly related proteins do have the same activity.

The paper is for the most part well well-written and is potentially highly significant, but it could be strengthened as follows:

(1) The authors start out by showing DTX3L binding to nucleotides and ubiquitylation of ssRNA/DNA. While ubiquitylation is subsequently dissected and ascribed to the RD domains, the binding data is not followed up. Does the RD protein alone bind to the nucleotides? Further analysis of nucleotide binding is also relevant to the Discussion where the role of the KH domains is considered, but the binding properties of these alone have not been analysed.<br /> (2) With regard to the E3 ligase activity, can the authors account for the apparent decreased ubiquitylation activity of the 232-C protein in Figure 1/S1 compared to FL and RD?<br /> (3) Was it possible to positively identify the link between Ub and ssDNA/RNA using mass spectrometry? This would overcome issues associated with labels blocking binding rather than modification.<br /> (4) Furthermore, can a targeted MS approach be used to show that nucleotides are ubiquitylated in cells?<br /> (5) Do the authors have the assignments (even partial?) for DTX3L RD? In Figure 4 it would be helpful to identify the peaks that correspond to the residues at the proposed binding site. Also do the shifts map to a defined surface or do they suggest an extended site, particularly for the ssDNA.<br /> (6) Does sequence analysis help explain the specificity of activity for the family of proteins?<br /> (7) While including a summary mechanism (Figure 5I) is helpful, the schematic included does not necessarily make it easier for the reader to appreciate the key findings of the manuscript or to account for the specificity of activity observed. While this figure could be modified, it might also be helpful to highlight the range of substrates that DTX3L can modify - nucleotide, ADPr, ADPr on nucleotides etc.

Reviewer #2 (Public Review):

Summary:

The manuscript by Dearlove et al. entitled "DTX3L ubiquitin ligase ubiquitinates single-stranded nucleic acids" reports a novel activity of a DELTEX E3 ligase family member, DTX3L, which can conjugate ubiquitin to the 3' hydroxyl of single-stranded oligonucleotides via an ester linkage. The findings that unmodified oligonucleotides can act as substrates for direct ubiquitylation and the identification of DTX3 as the enzyme capable of performing such oligonucleotide modification are novel, intriguing, and impactful because they represent a significant expansion of our view of the ubiquitin biology. The authors perform a detailed and diligent biochemical characterization of this novel activity, and key claims made in the article are well supported by experimental data. However, the studies leave room for some healthy skepticism about the physiological significance of the unique activity of DTX3 and DTX3L described by the authors because DTX3/DTX3L can also robustly attach ubiquitin to the ADP ribose moiety of NAD or ADP-ribosylated substrates. The study could be strengthened by a more direct and quantitative comparison between ubiquitylation of unmodified oligonucleotides by DTX3/DTX3L with the ubiquitylation of ADP-ribose, the activity that DTX3 and DTX3L share with the other members of the DELTEX family.

Strengths:

The manuscript reports a novel and exciting observation that ubiquitin can be directly attached to the 3' hydroxyl of unmodified, single-stranded oligonucleotides by DTX3L. The study builds on the extensive expertise and the impactful previous studies by the Huang laboratory of the DELTEX family of E3 ubiquitin ligases. The authors perform a detailed and diligent biochemical characterization of this novel activity, and all claims made in the article are well supported by experimental data. The manuscript is clearly written and easy to read, which further elevates the overall quality of submitted work. The findings are impactful and will help illuminate multiple avenues for future follow-up investigations that may help establish how this novel biochemical activity observed in vitro may contribute to the biological function of DTX3L. The authors demonstrate that the activity is unique to the DTX3/DTX3L members of the DELTEX family and show that the enzyme requires at least two single-stranded nucleotides at the 3' end of the oligonucleotide substrate and that the adenine nucleotide is preferred in the 3' position. Most notably, the authors describe a chimeric construct containing RING domain of DTX3L fused to the DTC domain DTX2, which displays robust NAD ubiquitylation, but lacks the ability to ubiquitylate unmodified oligonucleotides. This construct will be invaluable in the future cell-based studies of DTX3L biology that may help establish the physiological relevance of 3' ubiquitylation of nucleic acids.

Weaknesses:

The main weakness of the study is in the lack of direct evidence that the ubiquitylation of unmodified oligonucleotides reported by the authors plays any role in the biological function of DTX3L. The study leaves plenty of room for natural skepticism regarding the physiological relevance of the reported activity, because, akin to other DELTEX family members, DTX3 and DTX3L can also catalyze attachment of ubiquitin to NAD, ADP ribose and ADP-ribosylated substrates. Unfortunately, the study does not offer any quantitative comparison of the two distinct activities of the enzyme, which leaves plenty of room for doubt. One is left wondering, whether ubiquitylation of unmodified oligonucleotides is just a minor and artifactual side activity owing to the high concentration of the oligonucleotide substrates and E2~Ub conjugates present in the in-vitro conditions and the somewhat lower specificity of the DTX3 and DTX3L DTC domains (compared to DTX2 and other DELTEX family members) for ADP ribose over other adenine-containing substrates such as unmodified oligonucleotides, ADP/ATP/dADP/dATP, etc. The intriguing coincidence that DTX3L, which is the only DTX protein capable of ubiquitylating unmodified oligonucleotides, is also the only family member that contains nucleic acid interacting domains in the N-terminus, is suggestive but not compelling. A recently published DTX3L study by a competing laboratory (PMID: 38000390), which is not cited in the manuscript, suggests that ADP-ribose-modified nucleic acids could be the physiologically relevant substrates of DTX3L. That competing hypothesis appears more convincing than ubiquitylation of unmodified oligonucleotides because experiments in that study demonstrate that ubiquitylation of ADP-ribosylated oligos is quite robust in comparison to ubiquitylation of unmodified oligos, which is undetectable. It is possible that the unmodified oligonucleotides in the competing study did not have adenine in the 3' position, which may explain the apparent discrepancy between the two studies. In summary, a quantitative comparison of ubiquitylation of ADP ribose vs. unmodified oligonucleotides could strengthen the study.

Author response:

Public Reviews: 

Reviewer #1 (Public Review): 

In the article by Dearlove et al., the authors present evidence in strong support of nucleotide ubiquitylation by DTX3L, suggesting it is a promiscuous E3 ligase with capacity to ubiquitylate ADP ribose and nucleotides. The authors include data to identify the likely site of attachment and the requirements for nucleotide modification. 

While this discovery potentially reveals a whole new mechanism by which nucleotide function can be regulated in cells, there are some weaknesses that should be considered. Is there any evidence of nucleotide ubiquitylation occurring cells? It seems possible, but evidence in support of this would strengthen the manuscript. The NMR data could also be strengthened as the binding interface is not reported or mapped onto the structure/model, this seems of considerable interest given that highly related proteins do have the same activity. 

The paper is for the most part well well-written and is potentially highly significant, but it could be strengthened as follows: 

(1) The authors start out by showing DTX3L binding to nucleotides and ubiquitylation of ssRNA/DNA. While ubiquitylation is subsequently dissected and ascribed to the RD domains, the binding data is not followed up. Does the RD protein alone bind to the nucleotides? Further analysis of nucleotide binding is also relevant to the Discussion where the role of the KH domains is considered, but the binding properties of these alone have not been analysed. 

We thank the reviewer for the suggestion. We have tested DTX3L RD for ssDNA binding using NMR (see Figure 4A and Figure S2), which showed that DTX3L RD binds ssDNA. We also tested the DTX3L KH domains for RNA/ssDNA binding using an FP experiment. However, the FP experiment did not show significant changes upon titrating RNA/ssDNA. It seems that the KH domains alone are not sufficient to bind RNA/ssDNA and both KH and RD domains are required for binding. Understanding how DTX3L binds RNA/ssDNA is an ongoing research in the lab. We will revise the Discussion on the KH domains.

(2) With regard to the E3 ligase activity, can the authors account for the apparent decreased ubiquitylation activity of the 232-C protein in Figure 1/S1 compared to FL and RD? 

We will address this question in the revision.

(3) Was it possible to positively identify the link between Ub and ssDNA/RNA using mass spectrometry? This would overcome issues associated with labels blocking binding rather than modification. 

We have tried to use mass spectrometry to detect the linkage between Ub and ssDNA/RNA, but was unable to do so. We suspect that the oxyester linkage might be labile, posing a challenge for mass spectrometry techniques. Similarly, a recent preprint from Ahel lab, which utilises LC-MS, detects the Ub-NMP product rather than the linkage (https://www.biorxiv.org/content/10.1101/2024.04.19.590267v1.full.pdf).

(4) Furthermore, can a targeted MS approach be used to show that nucleotides are ubiquitylated in cells? 

This will require future development and improvement of the MS approach, specifically the isolation of labile oxyester-linked products from cells and the optimisation of the MS detection method.

(5) Do the authors have the assignments (even partial?) for DTX3L RD? In Figure 4 it would be helpful to identify the peaks that correspond to the residues at the proposed binding site. Also do the shifts map to a defined surface or do they suggest an extended site, particularly for the ssDNA.

We only collected HSQC spectra which was insufficient for assignments. We have performed a competition experiment using ADPr and labelled ssDNA, showing that ADPr competes against the ubiquitination of ssDNA (Figure 4D). We will provide an additional experiment showing that ssDNA with a blocked 3’-OH can compete against ubiquitination of ADPr. These data, together with our NMR analysis, will further strengthen the evidence that ssDNA and ADPr compete the same binding pocket in DTX3L RD. Understanding how DTX3L RD binds ssDNA/RNA is an ongoing research in the lab.

(6) Does sequence analysis help explain the specificity of activity for the family of proteins? 

We will performed sequence alignment of DTX proteins RD domains and discuss this point in the revision.

(7) While including a summary mechanism (Figure 5I) is helpful, the schematic included does not necessarily make it easier for the reader to appreciate the key findings of the manuscript or to account for the specificity of activity observed. While this figure could be modified, it might also be helpful to highlight the range of substrates that DTX3L can modify - nucleotide, ADPr, ADPr on nucleotides etc. 

We will modify this Figure as suggested.

Reviewer #2 (Public Review): 

Summary: 

The manuscript by Dearlove et al. entitled "DTX3L ubiquitin ligase ubiquitinates single-stranded nucleic acids" reports a novel activity of a DELTEX E3 ligase family member, DTX3L, which can conjugate ubiquitin to the 3' hydroxyl of single-stranded oligonucleotides via an ester linkage. The findings that unmodified oligonucleotides can act as substrates for direct ubiquitylation and the identification of DTX3 as the enzyme capable of performing such oligonucleotide modification are novel, intriguing, and impactful because they represent a significant expansion of our view of the ubiquitin biology. The authors perform a detailed and diligent biochemical characterization of this novel activity, and key claims made in the article are well supported by experimental data. However, the studies leave room for some healthy skepticism about the physiological significance of the unique activity of DTX3 and DTX3L described by the authors because DTX3/DTX3L can also robustly attach ubiquitin to the ADP ribose moiety of NAD or ADP-ribosylated substrates. The study could be strengthened by a more direct and quantitative comparison between ubiquitylation of unmodified oligonucleotides by DTX3/DTX3L with the ubiquitylation of ADP-ribose, the activity that DTX3 and DTX3L share with the other members of the DELTEX family. 

Strengths: 

The manuscript reports a novel and exciting observation that ubiquitin can be directly attached to the 3' hydroxyl of unmodified, single-stranded oligonucleotides by DTX3L. The study builds on the extensive expertise and the impactful previous studies by the Huang laboratory of the DELTEX family of E3 ubiquitin ligases. The authors perform a detailed and diligent biochemical characterization of this novel activity, and all claims made in the article are well supported by experimental data. The manuscript is clearly written and easy to read, which further elevates the overall quality of submitted work. The findings are impactful and will help illuminate multiple avenues for future follow-up investigations that may help establish how this novel biochemical activity observed in vitro may contribute to the biological function of DTX3L. The authors demonstrate that the activity is unique to the DTX3/DTX3L members of the DELTEX family and show that the enzyme requires at least two single-stranded nucleotides at the 3' end of the oligonucleotide substrate and that the adenine nucleotide is preferred in the 3' position. Most notably, the authors describe a chimeric construct containing RING domain of DTX3L fused to the DTC domain DTX2, which displays robust NAD ubiquitylation, but lacks the ability to ubiquitylate unmodified oligonucleotides. This construct will be invaluable in the future cell-based studies of DTX3L biology that may help establish the physiological relevance of 3' ubiquitylation of nucleic acids. 

Weaknesses: 

The main weakness of the study is in the lack of direct evidence that the ubiquitylation of unmodified oligonucleotides reported by the authors plays any role in the biological function of DTX3L. The study leaves plenty of room for natural skepticism regarding the physiological relevance of the reported activity, because, akin to other DELTEX family members, DTX3 and DTX3L can also catalyze attachment of ubiquitin to NAD, ADP ribose and ADP-ribosylated substrates. Unfortunately, the study does not offer any quantitative comparison of the two distinct activities of the enzyme, which leaves plenty of room for doubt. One is left wondering, whether ubiquitylation of unmodified oligonucleotides is just a minor and artifactual side activity owing to the high concentration of the oligonucleotide substrates and E2~Ub conjugates present in the in-vitro conditions and the somewhat lower specificity of the DTX3 and DTX3L DTC domains (compared to DTX2 and other DELTEX family members) for ADP ribose over other adenine-containing substrates such as unmodified oligonucleotides, ADP/ATP/dADP/dATP, etc. The intriguing coincidence that DTX3L, which is the only DTX protein capable of ubiquitylating unmodified oligonucleotides, is also the only family member that contains nucleic acid interacting domains in the N-terminus, is suggestive but not compelling. A recently published DTX3L study by a competing laboratory (PMID: 38000390), which is not cited in the manuscript, suggests that ADP-ribose-modified nucleic acids could be the physiologically relevant substrates of DTX3L. That competing hypothesis appears more convincing than ubiquitylation of unmodified oligonucleotides because experiments in that study demonstrate that ubiquitylation of ADP-ribosylated oligos is quite robust in comparison to ubiquitylation of unmodified oligos, which is undetectable. It is possible that the unmodified oligonucleotides in the competing study did not have adenine in the 3' position, which may explain the apparent discrepancy between the two studies. In summary, a quantitative comparison of ubiquitylation of ADP ribose vs. unmodified oligonucleotides could strengthen the study. 

We thank the reviewer for the constructive feedback. We agree that evidence for the biological function is lacking. While we have tried to detect Ub-ssDNA/RNA from cells, we found that Isolating and detecting labile oxyester-linked Ub-ssDNA/RNA products remain challenging due to (1) low levels of Ub-ssDNA/RNA products, (2) the presence of DUBs and nucleases that rapidly remove the products during the experiments, and (3) our lack of a suitable MS approach to detect the product. For these reasons, we feel that discovering the biological function will require future effort and expertise and is beyond the scope of our current manuscript.

In the manuscript (PMID: 38000390), the authors used PARP10 to catalyse ADP-ribosylation onto 5’-phosphorylated ssDNA/RNA. They used the following sequences which lacks 3’-adenosine, which could explain the lack of ubiquitination.

E15_5′P_RNA [Phos]GUGGCGCGGAGACUU

E15_5′P_DNA [Phos]GTGGCGCGGAGACTT

We will perform the experiment using this sequence to verify this. We have cited this manuscript but for some reasons, Pubmed has updated its published date from mid 2023 to Jan 2024. We will update the Endnote in the revised manuscript.

We agree that it is crucial to compare ubiquitination of oligonucleotides and ADPr by DTX3L to find its preferred substrate. We have challenged oligonucleotide ubiquitination by adding excess ADPr and found that ADPr efficiently competes with oligonucleotide (Figure 4D). We will perform more thorough competition experiments by titrating with increasing molar excess of either ADPr or ssDNA to examine the effect on the ubiquitination of ssDNA and ADPr, respectively.

Reviewer #1 (Public Review):

The authors characterized a new non-coding RNA, which they named as PITAR. They first showed that the PITAR expression levels are higher in glioblastoma, and then demonstrated that knockdown of PITAR in glioblastoma cells decreased cell growth, induced G0/G1 arrest and apoptosis. They further identified the E3 ubiquitin ligase TRIM28 is the target of PITAR, and showed that PITAR bound to the TRIM28 mRNA and regulated the stability and expression of the latter. Since TRIM28 has been reported to be an E3 ubiquitin ligase for the tumor suppressor p53, the authors tried to link the PITAR function to p53 regulation. They showed that one PITAR siRNA increased the levels of p53 and p21, and the stability of p53, and these effects could be diminished by overexpression of TRIM28. They also showed that PITAR overexpression decreased the levels of adriamycin-induced p53/p21 expression and reversed DNA damage-induced G2/M arrest. Lastly, the authors showed that PITAR siRNA decreased the growth of glioblastoma, while PITAR overexpression increased glioblastoma growth and counteracted temozolomide for its anti-glioblastoma activity.

Overall, the manuscript has provided evidence supporting the important role of PITAR in the regulation of the growth of glioblastoma. The results supporting the regulation of PITAR on TRIM28 appear to be convincing. However, some weaknesses are also noted.

(1) More than one siRNA/shRNA should be used in critical experiments. For example, Fig 7A-E are important experiments demonstrating PITAR suppresses tumor growth. It is compelling that the siPITAR tumors disappeared at the end of the experiment. While this might be due to apoptosis, using another siRNA to confirm the results would be necessary. The authors may also need to use this model to test their hypothesis that PITAR regulates tumor growth through p53. They can check p53, p21, apoptosis levels in tumor sections.

(2) The data supporting that PITAR downregulates p53 stability and activity can be strengthened. The half-life of endogenous p53 protein is generally 20-30 min, and thus the cycloheximide chase experiments (Fig 5E) need to use shorter treatment time. The ubiquitinated p53 bands are not clear (Fig 5F), and the data suggesting that PITAR regulates p53 ubiquitination are not convincing. While the p53 protein level was largely altered by PITAR/TRIM28, the mRNA levels of its target genes, including p21 and MDM2 only marginally changed (Fig S6D). Other p53 targets, particularly proapoptotic genes, may need to be examined.

(3) The model depicting the role of PITAR in the cellular response to DNA-damaging agents is confusing. If DNA damaging agents like TMZ induce PITAR to inactivate p53, PITAR overexpression would confer TMZ resistance. However, Fig 7G did not support this. While the experimental design is quite problematic given that U87 cells already express a high level of PITAR, PITAR-overexpressing cells were still sensitive to TMZ treatment (this is apparent when checking the images in Fig 7F, although the large error bars shown in Fig 7G may lead to a "not significant" conclusion). The authors may need to test whether PITAR downregulation, which would increase p53 activity, has any effects on TMZ-insensitive tumors. Such results are more therapeutically relevant. It would also be helpful if the authors test whether PITAR is overexpressed in TMZ-resistant clinical samples.

eLife assessment

This important study reports, with convincing evidence, that a long non-coding RNA disrupts the activity of the tumor suppressor p53 to contribute to the growth and therapeutic response of glioblastoma. The work will be relevant to scientists working on non-coding RNAs and brain tumors.

Reviewer #2 (Public Review):

This study established an alternate way of p53 inactivation and proposed PITAR as a potential therapeutic target, so the impact is high. In addition, this manuscript has apparent strengths, including a logically designed research strategy, in vitro and in vivo study, and well-designed control.

This manuscript identified a long noncoding RNA, PITAR (p53 Inactivating TRIM28 associated RNA), as an inhibitor of p53. PITAR is highly expressed in glioblastoma (GBM) and glioma stem-like cells (GSC). The authors found that TRIM28 mRNA, which encodes a p53-specific E3 ubiquitin ligase, is a direct target of PITAR. PITAR interaction with TRIM28 RNA stabilized TRIM28 mRNA, which resulted in increased TRIM28 protein levels, enhanced p53 ubiquitination, and attenuated DNA damage response. While PITAR silencing inhibited the growth of WT p53 containing GSCs in vitro and reduced glioma tumor growth in vivo, its overexpression enhanced the tumor growth and promoted resistance to Temozolomide. DNA damage also activated PITAR, in addition to p53, thus creating an incoherent feedforward loop. Together, this study established an alternate way of p53 inactivation and proposed PITAR as a potential therapeutic target.

P53 is a well-established tumor suppressor gene contributing to cancer progression in many human cancers. It plays a vital role in preserving genome integrity and inhibiting malignant transformation. p53 is mutated in more than 50% of human cancers. In cancers that do not carry mutations in p53, the inactivation occurs through other genetic or epigenetic alterations. Therefore, further study of the mechanism of regulation of wt-p53 remains vital in cancer research. This study identified a novel LncRNA PITAR, which is highly expressed in glioblastoma (GBM) and glioma stem-like cells (GSCs) and interacts with and stabilizes TRIM28 mRNA, which encodes a p53-specific E3 ubiquitin ligase. TRIM28 can inhibit p53 through HDAC1-mediated deacetylation and direct ubiquitination in an MDM2-dependent manner. Thus, the overall impact of this study is high because of the identification of a novel mechanism in regulating wt-p53.

The other significant strengths of this manuscript included an apparent research strategy design and a clearly outlined and logically organized research approach. They provided both the in vitro and in vivo studies to evaluate the effect of PITAR. They offered reasonable control of the study by validating the results in cells with mutant p53. They also performed a rescue experiment to confirm the PITAR and TRIM28 relationship regulating p53. The conclusions were all supported by solid results. The overall data presentation is clear and convincing.

Author response:

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

Reviewer #1 (Public Review):

(1) Only one PITAR siRNA was tested in majority of the experiments, which compromises the validity of the results.

We thank the reviewer for this comment. We have now used two siRNAs to demonstrate PITAR functions in various assays. In the revised manuscript, we carried out additional experiments with two siRNAs, and the results are presented in Figures 2C, D, F, G, H, I, and J; Figures 5A, B, Supplementary Figure 2B, C, D, E, and F.

(2) Some results are inconsistent. For example, Fig 2G indicates that PITAR siRNA caused G1 arrest. However, PITAR overexpression in the same cell line did not show any effect on cell cycle progression in Fig 5I.

The reason for the fact that PITAR silencing showed a robust G1 arrest, unlike PITAR overexpression, is as follows. Since glioma cells overexpress PITAR (which keeps the p53 suppressed), silencing PITAR (which will elevate p53 levels) in glioma cells shows a robust phenotype in cell cycle profile (in the form of increased G1 arrest). In contrast, the overexpression of PITAR in glioma cells fails to show robust changes in the cell cycle profile because glioma cells already have high levels of PITAR.

(3) The conclusion that PITAR inactivates p53 through regulating TRIM28, which is highlighted in the title of the manuscript, is not supported by convincing results. Although the authors showed that a PITAR siRNA increased while PITAR overexpression decreased p53 level, the siRNA only marginally increased the stability of p53 (Fig 5E). The p53 ubiquitination level was barely affected by PITAR overexpression in Fig 5F.

We disagree with the fact that PITAR silencing only marginally increased the stability of p53. In the cycloheximide experiment in Figure 5E, the half-life of p53 is increased by 60 % (50 mins to 120 mins), which is quite significant in altering the DNA damage response by p53. Further, we also want to point out that the other arm of p53 degradation by Mdm2 remains intact under these conditions. We also provide an improved p53 ubiquitination western blot in the revised version (Figure 5F). 

(4) To convincingly demonstrate that PITAR regulates p53 through TRIM28, the authors need to show that this regulation is impaired/compromised in TRIM28-knockout conditions. The authors only showed that TRIM28 overexpression suppressed PITAR siRNA-induced increase of p53, which is not sufficient.

We thank the reviewer. In the revised manuscript, we demonstrate that PITAR overexpression fails to inhibit p53 in TRIM28 silenced cells (Supplementary Figure 5G; Figure 5K, L, M, N).

(5) Note that only one cell line was investigated in Fig 5.

In revised manuscript, the impact of PITAR silencing and PITAR overexpression on p53 functions are demontsrared for one more glioma cell line (Supplemenatry Figure 5B, C, D, and E).

(6) Another major weakness of this manuscript is that the authors did not provide any evidence indicating that the glioblastoma-promoting activities of PITAR were mediated by its regulation of p53 or TRIM28 (Fig 6 and Fig 7). Thus, the regulation of glioblastoma growth and the regulation of TRIM28/p53 appear to be disconnected.

We would like to respectfully disagree with the reviewer on this particular point.  We have indeed provided the following evidence in the first version of the manuscript: glioblastoma-promoting activities of PITAR were mediated by its regulation of p53 or TRIM28.

(1) To show the importance of p53:

We show that PITAR silencing failed to inhibit the colony growth of p53-silenced U87 glioma cells (U87/shp53#1). We also show that while PITAR silencing decreased TRIM28 RNA levels in U87/shNT and U87/shp53#1 glioma cells, it failed to increase CDKN1A and MDM2 (p53 targets) at the RNA level in U87/shp53#1 cells unlike in U87/siNT cells (Supplementary Figure 6 Panels A, B, C, and D). 

(2) To show the importance of TRIM28 and p53:

The importance of p53 is also demonstrated in the context of patient-derived GSC lines. We demonstrate that PITAR silencing-induced reduction in the neurosphere growth (WT p53 containing patient-derived GSC line) is accompanied by a reduction in TRIM28 RNA and an increase in the CDKN1A RNA without a change in p53 RNA levels (Supplementary Figure 7 Panels A, B, C, D, and E). We also demonstrate that PITAR overexpression-induced neurosphere growth is accompanied by an increase in the TRIM28 RNA, and a decrease in CDKN1A RNA without a change in p53 RNA levels (Supplementary Figure 7 Panels F, G, H, and I). However, PITAR silencing failed to decrease neurosphere growth in mutant p53 containing GSC line (MGG8) (Supplementary Figure 7 Panels J, K, L, M, N, and F).

(3) We show that the TRIM28 protein level is drastically reduced in small tumors formed by U87/siPITAR cells (Supplementary Figure 7 Panel E).

(4) We show that glioma tumors formed by U87/PITAR OE cells express high levels of TRIM28 protein but reduced levels of p21 protein (Supplementary Figure 7 Panel B).

Further, we did additional experiments to prove the importance of TRIM28.

In the revised manuscript, we have carried out an additional experiment to prove the requirement of TRIM28 for tumor-promoting functions of PITAR overexpression. Earlier, we have shown that exogenous overexpression of PITAR promotes glioma tumor growth and imparts resistance to Temozolomide chemotherapy (Figure 7F and G; Supplementary Figure 9A and B). In the revised manuscript, we show that the tumor growth-promoting function of PITAR overexpression requires TRIM28. U87-Luc/PITAR OE cells formed a larger tumor compared to U87-Luc/VC cells (Figure 7H, and I; compare red line with blue line). U87-Luc/shTRIM28 cells formed very small-sized tumors (Figure 7H, and I; compare green line with blue line). Further, PITAR overexpression (U87-Luc/PITAR OE) was less efficient in promoting glioma tumor growth in TRIM28 silenced cells (Figure 7H, and I; compare pink line with red line). Thus, we prove that, as a whole, TRIM28 mediates the tumor growth-promoting functions of PITAR.

(7) It is not clear what kind of message the authors tried to deliver in Fig 7F/G. Based on the authors' hypothesis, DNA-damaging agents like TMZ would induce PITAR to inactivate p53, which would compromise TMZ's anti-cancer activity. However, the data show that TMZ was very effective in the inhibition of U87 growth. The authors may need to test whether PITAR downregulation, which would increase p53 activity, have any effects on TMZ-insensitive tumors. Such results are more therapeutically relevant.

Reviewer #1 rightly pointed out that TMZ induces PITAR expression, which should compromise TMZ's anti-cancer activity.

We demonstrate the same as below:

Figure 7F&G demonstrates the following two facts:1. PITAR overexpression increases the glioma-tumor growth (Figure 7G, compare red line with the blue line), 2. PITAR overexpressing glioma tumors are resistant to TMZ chemotherapy (Figure 7G, compare the pink line with the green line).

In addition, Figure 7 F and G also demonstrate that TMZ treatment of tumors formed by U87/VC glioma cells inhibited the growth but not eliminated the tumor growth completely (compare pink line with blue line). We believe that the inability of TMZ to eliminate the tumor growth completely is because of the chemoresistance imparted by the DNA damage induced PITAR.

Further, in Figure 2I, we indeed show that PITAR-silenced cells are more sensitive to TMZ and Adriamycin chemotherapy.

(8) Lastly, the model presented in Fig 7H is confusing. It is not clear what the exact role of PITAR in the DNA damage response based on this model. If DNA damage would induce PITAR expression, this would lead to inactivation of p53 as revealed by this manuscript. However, DNA damage is known to activate p53. Do the authors want to imply that PITAR induction by DNA damage would help to bring down the p53 level at the end of DNA damage response? The presented data do not support this role unfortunately.

We respect the views and questions raised by the reviewer.

We would like explain as below the importance of our model.

Yes, it is true that DNA damage induces p53. We show here that DNA damage also induces PITAR in a p53-independent manner, which, in turn, inhibits p53. Here is our explanation. Even though DNA damage activates p53, there exists an autoregulatory negative feedback loop that controls the extent and duration of p53 response to DNA damage (Wu et al., 1993; Haupt et al., 1997; Kubbutat, Jones and Vousden, 1997; Zhang et al., 2009).  It is proposed that the p53-Mdm2 feedback loop generates a “digital clock” that releases well-timed quanta of p53 until the damage is repaired or the cell dies (Lahave et al., 2004). In addition, it has also been shown that TRIM28, through its association with Mdm2, also contributes to p53 inactivation (Wang et al., 2005b; Czerwińska, Mazurek, and Wiznerowicz, 2017).

Based on the above reports and our current work, we propose that DNA damage-induced PITAR, through its ability to increase the TRIM28 levels, contributes to the control of the DNA damage response of p53 along with Mdm-2. The difference is as follows: Since Mdm-2 is also a transcriptional target of p53, the p53-Mdm-2 axis is an autoregulatory negative feedback loop to control the DNA damage response by p53. In contrast, PITAR is not a transcriptional target of p53, and DNA damage-induced activation of PITAR is p53-independent. Hence, the PITAR-TRIM28 axis in controlling the DNA damage response of p53 creates an Incoherent feedforward regulatory network.  The experimental evidence provided in the revised manuscript is as follows: 1) We have already (the first version of the manuscript) shown that exogenous overexpression of PITAR significantly inhibits DNA damage-induced p53 (Figures 6A, B, C, and D). 2) In the revised manuscript, we show that the DNA damage response of p53 (duration and extent of p53 activation after a pulse of ionizing radiation) in PITAR-silenced cells follows similar kinetics in terms of duration, but the extent of p53 activation was much stronger (Supplementary figures 8H, I, J, and K).  This is because the TRIM28 component in TRIM28/Mdm-2 axis is compromised as PITAR silencing reduces the TRIM28 levels. 3) We also demonstrate that DNA damage-induced TRIM28 is dependent on PITAR (Figure 6K; Supplementary Figure 5G)

Reviewer #1(Recommendations For The Authors):

(1) Fig 7A, what is the explanation for the observation that tumors disappeared in most of the mice in the siPITAR group? Did the authors check if apoptosis was induced here?

We agree to the point that the lack of tumor growth in the siPITAR group is likely due to the induction of apoptosis. We would like to point out that in vitro experiments indeed demonstrate that PITAR silencing induces apoptosis in Figure 2H and Supplementary Figure 2F.

(2) The authors need to explain why Fig 6 used a cell line different from other experiments. It would be better to check other cell lines.

The purpose of RG5 and MGG8 is as follows. 1) We wanted to establish the growth-promoting functions of PITAR in patient-derived GSC lines. 2) We also wanted to show the importance of WT p53 for the growth-promoting functions of PITAR.

However, in the revised manuscript we moved this portion under the subsection “PITAR inhibits p53 protein levels by its association with TRIM28 mRNA“.

Further,the experiments related to DNA damage induced activation of PITAR in p53-independent manner and its impact on DNA damage response by p53 is moved to a new section entitled “PITAR is induced by DNA damage in a p53-independent manner, which in turn diminishes the DNA damage response by p53”

(3) It would be more convincing if the authors could test more p53 target genes in addition to p21.

We thank the reviewer for this comment and the specific suggestions for checking additional p53 targets. In the revised manuscript, we have checked the MDM2 transcript levels in Supplementary Figure 6D. 

Reviewer #2 (Recommendations For The Authors):

(1) In the text, they mentioned " Figure 4J". There is no Figure 4J in Figure 4. It may be Figure 4K.

We thank reviewer #2. We corrected this information in the revised manuscript.

(2) The molecular weight markers in Western blots were missed in several Figure panels, including Figure 4J, Figure 5K, and Supple. Figure 3B, Supple. Figure 5G, H, Supple. Figures 6A and 7A.

We thank reviewer #2, and we have included the molecular weight markers in all the mentioned figures.

Reviewer #1 (Public Review):

The authors characterized a new non-coding RNA, which they named as PITAR. They first showed that the PITAR expression levels are higher in glioblastoma, and then demonstrated that knockdown of PITAR in glioblastoma cells decreased cell growth, induced G0/G1 arrest and apoptosis. They further identified the E3 ubiquitin ligase TRIM28 is the target of PITAR, and showed that PITAR bound to the TRIM28 mRNA and regulated the stability and expression of the latter. Since TRIM28 has been reported to be an E3 ubiquitin ligase for the tumor suppressor p53, the authors tried to link the PITAR function to p53 regulation. They showed that one PITAR siRNA increased the levels of p53 and p21, and the stability of p53, and these effects could be diminished by overexpression of TRIM28. They also showed that PITAR overexpression decreased the levels of adriamycin-induced p53/p21 expression and reversed DNA damage-induced G2/M arrest. Lastly, the authors showed that PITAR siRNA decreased the growth of glioblastoma, while PITAR overexpression increased glioblastoma growth and counteracted temozolomide for its anti-glioblastoma activity.

Overall, the manuscript has provided preliminary evidence supporting the important role of PITAR in the regulation of the growth and drug resistance of glioblastoma. The results supporting the regulation of PITAR on TRIM28 appear to be convincing. However, the study suffers significant weaknesses summarized as below.

(1) Only one PITAR siRNA was tested in majority of the experiments, which compromises the validity of the results. Some results are inconsistent. For example, Fig 2G indicates that PITAR siRNA caused G1 arrest. However, PITAR overexpression in the same cell line did not show any effect on cell cycle progression in Fig 5I.

(2) The conclusion that PITAR inactivates p53 through regulating TRIM28, which is highlighted in the title of the manuscript, is not supported by convincing results. Although the authors showed that a PITAR siRNA increased while PITAR overexpression decreased p53 level, the siRNA only marginally increased the stability of p53 (Fig 5E). The p53 ubiquitination level was barely affected by PITAR overexpression in Fig 5F. To convincingly demonstrate that PITAR regulates p53 through TRIM28, the authors need to show that this regulation is impaired/compromised in TRIM28-knockout conditions. The authors only showed that TRIM28 overexpression suppressed PITAR siRNA-induced increase of p53, which is not sufficient. Note that only one cell line was investigated in Fig 5.

(3) Another major weakness of this manuscript is that the authors did not provide any evidence indicating that the glioblastoma-promoting activities of PITAR were mediated by its regulation of p53 or TRIM28 (Fig 6 and Fig 7). Thus, the regulation of glioblastoma growth and the regulation of TRIM28/p53 appear to be disconnected.

(4) It is not clear what kind of message the authors tried to deliver in Fig 7F/G. Based on the authors' hypothesis, DNA damaging agents like TMZ would induce PITAR to inactivate p53, which would compromise TMZ's anti-cancer activity. However, the data show that TMZ was very effective in the inhibition of U87 growth. The authors may need to test whether PITAR downregulation, which would increase p53 activity, have any effects on TMZ-insensitive tumors. Such results are more therapeutically relevant.

(5) Lastly, the model presented in Fig 7H is confusing. It is not clear what the exact role of PITAR in the DNA damage response based on this model. If DNA damage would induce PITAR expression, this would lead to inactivation of p53 as revealed by this manuscript. However, DNA damage is known to activate p53. Do the authors want to imply that PITAR induction by DNA damage would help to bring down the p53 level at the end of DNA damage response? The presented data do not support this role unfortunately.

we are fucked on all fronts. now what? suicide or killing spree? matter of taste ...

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Reply to the reviewers

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Reply to the Reviewers

We want to thank the three reviewers for their invaluable and constructive feedback. We respond to each comment individually, describing how we plan to address them in our revised manuscript.

Reviewer #1

1. Given the emphasis on super-resolution imaging deep inside a sample, we were surprised to see no mention of other forms of structured illumination that allow super-resolution imaging in samples thicker than a single cell. These include the 'spot-scanning' implementations of SIM that offer better imaging at depth by virtue of pinholes, and include MSIM, iSIM, and rescan confocal technologies. The two-photon / AO implementation of iSIM seems particularly germane, e.g. https://pubmed.ncbi.nlm.nih.gov/28628128/ Please consider citing these works, as they help place the existing work into context.

Response:

We want to thank reviewer #1 for the good point. To address this comment, we plan to add to the discussion section a description of these super resolution techniques, together with other SIM methods, explaining how they compare to our approach.

1. As we're sure the authors appreciate, besides aberrations, a major additional obstacle to 3D SIM in thick tissues is the presence of out-of-focus background. Indeed, this point was mentioned by Gustafsson in his classic 2008 paper on 3D SIM (https://pubmed.ncbi.nlm.nih.gov/18326650/): 'The application area of three-dimensional structured illumination microscopy overlaps with that of confocal microscopy, but the two techniques have different and complementary strengths. Structured illumination microscopy offers higher effective lateral resolution, because it concentrates much of the excitation light at the very highest illumination angles, which are most effective for encoding high-resolution information into the observed data, whereas confocal microscopy spreads out its illumination light more or-less uniformly over all available angles to form a focused beam. For very thick and compactly fluorescent samples, however, confocal microscopy has an advantage in that its pinhole removes out-of focus light physically. Structured illumination microscopy is quite effective at removing out-of-focus light computationally, because it is not subject to the missing-cone problem, but computational removal leaves behind the associated shot noise. Therefore confocal microscopy may be preferable on very thick and dense samples, for which the in-focus information in a conventional microscope image would be overwhelmed by out-of-focus light, whereas structured illumination microscopy may be superior in a regime of thinner or sparser samples.' This point is not mentioned at all in the manuscript, yet we are certain it is at least partially responsible for the residual image artifacts the authors mention. Please discuss the problem of out of focus light on 3D samples, particularly with an eye to the 'spot-scanning' papers mentioned above.

Response:

We appreciate this significant obstacle and we want to thank Reviewer #1 for emphasising its importance. To address the comment, we plan to add a discussion of the significance of out-of-focus light to SIM imaging to the introduction, results, and discussion sections of the manuscript.

1. The authors use a water dipping lens, yet they image into samples that are mounted on coverslips, i.e. they use a dipping lens to image through a coverslip:

This almost certainly introduces spherical aberration, which the authors seem to observe: see attached pdf for reference

We find this troubling, as it seems that in the process of building their setup, the authors have made a choice of objective lens that introduces aberrations - that they later correct. At the very least, this point needs to be acknowledged in the manuscript (or please correct us if we're wrong) - as it renders the data in Figs. 3-4 somewhat less compelling than if the authors used an objective lens that allowed correction through a coverglass, e.g. a water dipping lens with a correction collar. In other words, in the process of building their AO setup, the authors have introduced system aberrations that render the comparison with 3D SIM somewhat unfair. Ideally the authors would show a comparison with an objective lens that can image through a glass coverslip.

Response:

We want to thank Reviewer #1 for raising this point, which we did not describe clearly enough, leading to confusion. We should have made it clearer that we used a water dipping/immersion objective lens with a correction collar which extends from no coverslip (dipping) up to well beyond a standard #1.5 (170 um thick) coverslip. We adjusted this collar before each image acquisition session, to ensure that the system is optimised for each experiment individually and that the spherical aberrations are minimal before any DM-based correction. We plan to elaborate and emphasise this point in several places in the revised manuscript, including in the figure legends, materials and methods and results sections, to avoid any ambiguity and confusion about the use of the correction collar and this particular water immersion/dipping objective lens.

1. The authors tend to include numbers for resolution without statistics. This renders the comparisons meaningless in my opinion; ideally every number would have a mean and error bar associated with it. We have included specific examples in the minor comments below.

Response:

This is a good point, which we address below, in three minor comments. In summary, to address this comment, we plan to include statistical information in the revised manuscript.

1. In Fig. 5, after the 'multipoint AO SIM', the SNR in some regions seems to decrease after AO: see attached pdf for reference

Please comment on this issue.

Response:

We want to thank Reviewer #1 for the insightful comment. There are multiple phenomena in effect here, which cause the drop in intensity. The most prominent one is photobleaching, as the AO image stack (right) was acquired after the bypass one (left). To address this comment, we plan to add additional data and to include a brief discussion about this issue and other related points.

1. Please provide timing costs for the indirect AO methods used in the paper, so the reader understands how this time compares to the time required for taking a 3D SIM stack. In a similar vein, the authors in Lines 213-215, mention a 'disproportionate measurement time' when referring to the time required for AO correction at each plane - providing numbers here would be very useful to a reader, so they can judge for themselves what this means. What is the measurement time, why is it so long, and how does it compare to the time for 3D SIM? It would also be useful to provide a comparison between the time needed for AO correction at each (or two) planes without remote focusing (RF) vs. with RF, so the reader understands the relative temporal contributions of each part of the method. We would suggest, for the data shown in Fig. 5, to report a) the time to acquire the whole stack without AO (3D SIM only); b) the time to acquire the data as shown; c) the time to acquire the AO stack without RF. This would help bolster the case for remote focusing in general; as is we are not sure we buy that this is a capability worth having, at least for the data shown in this paper.

Response:

We agree that the timing (and other) costs can be an important consideration, and we want to thank Reviewer #1 for bringing up this good point. To address this issue, we plan to expand our description of the AO methods, also including numbers for the time it takes to perform the different parts. In terms of comparisons, the RF makes no contribution to the timing costs of the aberration correction, a point that we want to make clearer in the results and the methods and materials sections, as the two are independent processes in our approach. Instead, the RF can be compared to standard focusing with a piezo stage, a point which we discuss in the supplementary material. We plan to make this point clearer in the discussion section of the main manuscript, and to emphasise better the advantages of the RF in terms of imaging speed.

1. Some further discussion on possibly extending the remote focusing range would be helpful. We gather that limitations arose from an older model of the DM being used, due to creep effects. We also gather from the SI that edge effects at the periphery of the DM was also problematic. Are these limitations likely non-issues with modern DMs, and how much range could one reasonably expect to achieve as a result? We are wondering if the 10 um range is a fundamental practical limitation or if in principle it could be extended with commercial DMs.

Response:

Regrettably, we were not able to try other DMs on the Deep3DSIM system. However, Jiahe and colleagues show in [1] that similar DM-based remote focusing, even with the same model deformable mirror, can be pushed to 120 um (Strehl ratio >0.8) with a 0.42 NA dry lens (20 mm WD) and close-loop wavefront compensation operation. While this is not directly translatable to high NA 3D-SIM imaging, we expect that with a stable version of the same DM the useable RF range could be easily increased twice or even more. We thank Reviewer #1 for the good comment, which we plan to address by revising the text to make the limitations clearer and by citing relevant studies.

[1] Cui, J., Turcotte, R., Emptage, N. J., & Booth, M. J. (2021). Extended range and aberration-free autofocusing via remote focusing and sequence-dependent learning. Optics Express, 29(22), 36660-36674.

Minor comments:

1. The paper mentions Ephys multiple times, even putting micromanipulators into Fig. 1 - although it is not actually used in this paper. If including in Figure 1, please make it clear that these additional components are aspirational and not actually used in the paper.

Response:

Although not shown in the context of this paper, the Deep3DSIM system was built specifically around experiments such as electrophysiology, which can benefit from the upright configuration and the water-dipping-capable objective lens. To address this comment, we plan to clarify the role of the micromanipulators and to update Figure 1 accordingly.

1. The abstract mentions '3D SIM microscopes', 'microscopes' redundant as the 'm' in 'SIM' stands for 'microscope'.

Response:

We accept that “3D SIM microscopes” sounds repetitious and we plan to revise the wording of the abstract to “3D SIM system”.

1. 'fast optical sectioning', line 42, how can optical sectioning be 'fast'? Do they mean rapid imaging with optical sectinong?

Response:

Yes, we meant rapid imaging with optical sectioning. We plan to change the wording to make it less ambiguous.

1. line 59, 'effective imaging depth may be increased to some extent using silicone immersion objectives', what about water immersion objectives? We would guess these could also be used.

Response:

Yes, water immersion objective lenses also fall in the same category and we plan to rephrase this part to state it explicitly.

1. line 65 - evidence for 'water-dipping objectives are more sensitive to aberrations' ? Please provide citation or remove. They are certainly more prone to aberrations if used with a coverslip as done here.

Response:

The refractive index (RI) of cells and tissues [1] is closer to the RI of silicone oil (~1.4) than it is to water (~1.33). Therefore, because of the larger difference in RI, imaging with a water-dipping objective lens is more prone to aberrations from RI mismatch. We plan to rephrase this argument to make it clearer.

[1] Jacques, S. L. (2013). Optical properties of biological tissues: a review. Physics in Medicine & Biology, 58(11), R37.

1. 'fast z stacks' is mentioned in line 103. How fast is fast?

Response:

The speed would depend on the way Z-stacks are being acquired. For example, acquisitions with two channels would be at least twice as fast, because of the ability to do simultaneous imaging on the Deep3DSIM system. Likewise, experiments that can benefit from the remote focusing can be several times faster than using a Z piezo stage, and this point is discussed in the supplementary material (section “Step response”). Finally, thanks to the electronic design of the imaging system, orchestrating everything via digital logic (e.g. TTL) signals, and thanks to the elaborate control software, we can ensure that all image acquisitions are carried out as quickly as possible, operating near the limit of the underlying hardware devices. We plan to explain these points in a clear way in the discussion section, and we plan to provide more numbers in the supplementary material.

1. line 116 'we imaged 100 nm diameter green fluorescent beads'. Deposited on glass? Given that this paper is about imaging deep this detail seems worth specifying in the main text.

Response:

Yes, in this case the beads were deposited on glass. We plan to include this detail in the description of the experiment.

1. lines 127-130, when describing changes in the bead shape with numbers for the FWHM, please provide statistics - quoting single numbers for comparison is almost useless and we cannot conclude that there is a meaningful improvement without statistics.

Response:

We agree with this comment. We plan to include statistical information for all the FWHM numbers.

1. In the same vein, how can we understand that remote focus actually improves the axial FWHM of the widefield bead? Is this result repeatable, or it just noise?

Response:

The lower axial FWHM with remote focusing is likely caused by data fitting or quantification error. Together with the inclusion of statistical information, we plan to review all the resolution values and to ensure that they are accurate and sensible.

1. line 155, 'Because of the high spatial information...' -> 'Because of the high resolution spatial information...'

Response:

We agree with this comment. To address it, we plan to rephase this part.

1. When quoting estimated resolution #s from microtubules (lines 158-163) similarly please provide statistics as for beads.

Response:

We agree with this comment. To address it, we plan to include statistical information for the resolution values from microtubules.

1. It seems worth mentioning the mechanism of AO correction (i.e. indirect sensing) in the main body of the text, not just the methods.

Response:

We agree with this comment. To address it, we plan to describe briefly the aberration correction method in the introduction or the results section.

1. How long do the AO corrections take for the datasets in the paper?

Response:

The duration of the aberration correction routines is directly proportional to the number of Zernike modes, the number of iterations, the exposure time of the camera, and other parameters. In our experiments, it was usually in the order of tens of seconds. To address this comment, and in line with the sixth major comment, we plan to include more details about the timing of the different parts of the AO methods.

1. Were the datasets in Fig. 2-4 acquired with remote focusing, or in conventional z stack mode? Please clarify this point in the main text and the figure captions.

Response:

The only data acquired with RF in Fig. 2-4 are one bead in Fig. 2A and another bead in Fig. 2B, both labelled accordingly. We plan to make it clearer in the text that the rest of Figure 2, as well as Figures 3 and 4, were acquired with the piezo Z stage.

1. It would be helpful when showing z projections in Figs. 3-5 to indicate the direction of increasing depth (we assume this is 'down' due to the upright setup, but this would be good to clarify)

Response:

The direction is indicated by the arrows labelled with ‘Z’. We plan to clarify this in the figure captions.

1. line 174, 'showed significant improvements in both intensity and contrast after reconstruction' - we see the improvements in contrast and resolution, it is harder to appreciate improvements in intensity. Perhaps if the authors showed some line profiles or otherwise quantified intensity this would be easier to appreciate.

Response:

We agree with this comment. To address it, we plan to change Figure 3 to illustrate the improvement in intensity, likely with line profiles, as suggested by the reviewer.

1. line 195 'reduced artefacts' due to AO. We would agree with this statement - the benefit from AO is obvious, and yet there are still artefacts. If the authors could clarify what these (residual) artefacts are, and their cause (out of focus light, uncorrected residual aberrations, etc) this would be helpful for a reader that is not used to looking at 3D SIM images.

Response:

We agree with this comment. To address it, we plan to explain this point in both the results and the discussion sections.

1. Line 197, 'expected overall structure', please clarify what is expected about the structure and why.

Response:

We agree with this comment. To address it, we plan to describe better the Canoe (Cno) protein, including an explanation of its expression pattern, which is the honeycomb-like structure observed in the images.

1. Line 199, what is a 'pseudo structure'?

Response:

We used this expression to refer to unclear (e.g. dim, fuzzy) structures. We plan to improve the wording of that part of the results section.

1. Fig. 4B, 'a resolution of ~200 nm is retained at depth', please clarify how this estimate was obtained, ideally with statistics.

Response:

We agree with this comment. To address it, we plan to clarify this point in the results section, including statistical information.

1. Fig. 4D, please comment on the unphysical negative valued intensities in Fig. 4D, ideally explaining their presence in the caption. It would also be helpful to highlight where in the figure these plots arise, so the reader can visually follow along.

Response:

We agree with this comment. To address it, we plan to explain how negative intensities arise in SIM reconstruction, often a result of spherical aberrations, and we plan to indicate where the line profile in Figure 4D comes from.

1. Line 245, 'rapid mitosis'. What does rapid mean, i.e. please provide the expected timescale for mitosis.

Response:

The mitotic cycles at this developmental stage are short, e.g. 5 minutes per mitosis, compared to those of somatic cells where it takes several hours. We plan to include this information in the main text.

1. For the data in Fig. 6, was remote refocusing necessary?

Response:

Yes, it was necessary because the point of Figure 6 is to demonstrate the combination of remote focusing and SIM super-resolution in live samples. Drosophila embryos are a very good sample for this kind of demonstration, because they are often subject to micromanipulation (e.g. injection and electrophysiology), and these are the kind of experiments that can benefit greatly from the optical axial scanning of the remote focusing, where the sample can remain stationary. However, there is nothing preventing the imaging of this kind of sample with a piezo Z stage or with some other kind of mechanical actuator. In this sense, the remote focusing is not strictly necessary but still much more convenient in some applications. We plan to make this point clearer in the discussion section.

1. What is the evidence for 'reduced residual aberrations', was a comparative stack taken without AO? In general we feel that the results shown in Fig. 6 would be stronger if there were comparative results shown without AO (or remote focusing).

Response:

We agree with this comment. In general, it is difficult to make direct comparisons (e.g. as in Figures 3-5) with live samples, because of the dynamic character of the samples, where it is often impossible to capture the same scene more than once. To address this comment, we plan to revise the wording of the relevant part of the results section, to ensure that the data in Figure 6 is properly described.

1. Line 350, 'incorporation of denoising algorithms' - citations would be helpful here.

Response:

We agree with this comment. To address it, we plan to add references to the relevant statement, showing examples of denoising in 3D-SIM imaging and reconstruction.

1. Line 411, 'All three were further developed and improved' - vague, how so?

Response:

A detailed breakdown of all the changes is available on the respective software repositories. We also plan to add a summary in the supplementary material.

1. Sensorless AO description; how many Zernike modes were corrected?

Response:

We usually corrected 8 modes: Z5 to Z11 and Z22, using Noll indexing. We plan to add a table to the supplementary material, describing which modes were corrected for each dataset.

1. Multi-position aberration correction. Was the assumption of linearity in the Zernike correction verified or met? Why is this a reasonable assumption?

Response:

By their very definition, some aberrations, such as defocus and spherical aberrations, change linearly with depth. Others are also proportional to the imaging depth, and first-order approximation (i.e. straight line) is the most sensible for just two correction points, as is the case with the dataset presented in Figure 5. We plan to explain this point better in the results section.

1. Fig. S1B is not useful; if the idea is to give a visual impression of the setup, we would recommend providing more photos with approximate distances indicated so that the reader has a sense of the scale of the setup. As is - it looks like a photograph of some generic optical setup.

Response:

We agree with this comment. To address it, we plan on including more photos in the supplementary material, to give a better sense of the scale.

1. SI pattern generation - 'the maximum achievable reconstruction resolution was only slightly reduced to about 95% of the theoretical maximum'. We don't understand this sentence, as the resolution obtained on the 100 nm beads is considerably worse than 95% of the theoretical maximum. Or do the authors mean 95% of the theoretical maximum given their pitch size of 317 nm for green and 367 nm for red?

Response:

Limiting the stripe width to about 90% of what is achievable leads to a reduction of the theoretical maximum resolution to 95% of what it could be. We plan to rephrase this part to make it clearer.

1. SI Deformable mirror calibration 'spanning the range [0.1, 0.9]' - what are the units here?

Response:

These are normalised control amplitudes, i.e. [10%, 90%], which means that they are unitless. We plan to explain this in a clearer way.

1. What are the units in Fig. S5C, S5D?

Response:

Errors are in radians, defined by the calibration interferometric wavefront sensor. We plan on updating the figure to include this information.

1. It would be useful to define 'warmup' also in the caption of SI Fig. S6A.

Response:

We agree with this comment. We plan to change the caption of Figure S6A to clarify this point.

1. SI Remote Focusing, 'four offsets, {-5 mm, -2.5 mm, 2.5 mm, 5 mm}...' are the units mm or um?

Response:

The units are supposed to be um (micrometres). We plan on fixing this error.

1. '...whereas that of the 10 beads was...' here, do the authors mean the position of the beads derived from the movement of the piezo stage, as opposed to the remote focusing?

Response:

This is the average standard deviation between the 10 different beads, all from volumes acquired with remote focusing. We plan on rephrasing this part to make it clearer.

1. The authors refer to the 'results from Chapter 3.2'. What are they talking about? Do they mean a supplementary figure, or earlier supplementary results? In general, we found the discussion in this paragraph difficult to follow.

Response:

This is a remnant from an earlier version of the document which used numbered sectioning. Chapter 3.2 is referring to the section titled “Characterisation of drift and temperature effects”. We plan on revising this paragraph to make it clearer.

1. Supplementary Fig. 9 seems to be not referred to anywhere in the text.

Response:

We agree with this comment. To address this issue, we plan on referring to this figure in the main text.

1. Since the paper emphasizes 3D SIM, OTFs along the axial direction would also be useful to show, in addition to the lateral OTFs shown in Fig. 2D.

Response:

We agree with this comment. To address it, we plan on adding orthogonal views of the OTFs to the supplementary material.

1. When the sample is moved by the piezo, the axial phase of the 3D-SIM illumination pattern is stable as the sample is scanned through the illumination pattern. When remote focusing is performed, the sample is always stable so the axial phase of the 3D-SIM illumination pattern is presumably changing with remote focusing. Can the authors clarify if the 3D SIM illumination pattern is scanned when remote focusing is applied, or is the intensity pattern stable in z?

Response:

Yes, the illumination pattern is scanned. We plan on clarifying how the structured illumination works in the case of remote focusing in the supplementary material.

1. In Supplementary Fig. 9, primary spherical is referred to twice, both at index 11 and 22. The latter is presumably secondary spherical?

Response:

Yes, it is supposed to be secondary spherical aberrations. We plan on fixing this error.

1. we do not understand the x axis label, in Fig. S4D, is it really [0, 50, 50, 50] as written?

Response:

The labels of the x-axis are not well formatted. There are three range of [0, 50] where only the first zero is properly displayed. We will revise this part of the figure to make it clear.

Reviewer #2

1. The authors have provided an incomplete description of the structured illumination microscopy (SIM) reconstruction process. It is unclear whether the approach is based on 2D interference SIM configurations or 3D interference patterns. Furthermore, the specific algorithm utilized for image reconstruction has not been elucidated. Elaborating on these aspects is crucial as they significantly influence the interpretation of the resulting data.

Response:

We want to thank Reviewer #2 for bringing our attention to the incomplete description of the reconstruction process. Our approach was based on 3D interference patterns and it was carried out using the Gustafsson’s reconstruction techniques as implemented by the softWoRx software, designed for the OMX 3D-SIM microscopes. To address this comment, we plan to revise the manuscript and to include more details about the 3D-SIM reconstruction techniques in the methods and materials section.

1. The authors have stated that sample-induced aberrations caused by RI inhomogeneities within the specimen is another major reason for causing artifacts generation. Literature has demonstrated that RI inhomogeneities can lead to non-local distortions in the grid pattern, which suggests that applying uniform reconstruction parameters across the entire image may not be viable. Traditional artifact remediation using the classical Wiener method is likely insufficient under these conditions (PMID: 33896197). The existing adaptive optics (AO) approach, which employs a deformable mirror (DM) alongside an sCMOS camera, is inadequate for tackling the issue at hand. Actually the assertion made in the paper that "aberrations change approximately linearly with depth" is seemingly contradicted by simulations referenced in the cited literature (PMID: 33896197). Consequently, it appears that the current methodology might only achieve a partial mitigation of the problems associated with spherical aberration resulting from RI mismatches. It is advisable, therefore, that the authors explicitly acknowledge this limitation in their manuscript to prevent any potential misinterpretation by readers.

Response:

We are thankful for the thoughtful comment by Reviewer #2. The focus of our work was not the use of advanced 3D-SIM reconstruction and aberration correction methods; instead, we used standard ones which are not able to deal perfectly with anisoplanitism, i.e. when the aberrations vary laterally. As such, our approach provides an average reconstruction and correction across the field of view. In our particular setup this anisoplanitism was not very significant, but we agree that it could be an issue for optical systems with very wide field of view. To address this good point, we plan on clarifying these potential issues in the results and the discussion sections.

1. In Figure 2, the use of COS-7 cells, which are known for their relatively thin axial dimension, for the experiments raises an eyebrow. Notably, there are ample instances in existing research where both 2D-SIM and 3D-SIM, without the integration of adaptive optics, have yielded high-quality super-resolution images of structures such as tubulin and the endoplasmic reticulum. In addition, the authors did not present a direct comparison between BP-SIM and AO-SIM here. Without this comparative analysis, it remains ambiguous whether the enhancements in resolution and contrast and the reduction in artifacts can genuinely be attributed to the mitigation of spherical aberration. To clarify this, it would be beneficial for the authors to include side-by-side comparisons of these modalities to demonstrate the specific improvements attributed to AO-SIM.

Response:

We are grateful to Reviewer #2 for this helpful comment. In Figure 2, we demonstrate the performance we get out of 3D-SIM in terms of optical resolution. We do not make any statements about the impact of the aberration correction on image quality. Nevertheless, to address this comment, we plan to revise the figure to explain more clearly and explicitly this point.

1. In Figures 3 and 4, the authors have illustrated the enhancements achieved through the application of AO. However, there is a discernible presence of hammer-stroke and honeycomb artifacts in pre-AO imaged data, which seem to originate from the amplification of the incorrectly moved out-of-focal background in the frequency domain. Various strategies have been previously suggested to address these specific artifacts, encompassing methods like subtracting background noise in the raw images or employing selective frequency spectrum attenuation techniques, such as Notch filtering and High-Fidelity SIM. To facilitate a more comprehensive understanding, I would recommend that the authors incorporate into their study a comparison that includes BP-SIM data that has undergone either background subtraction or frequency spectrum attenuation. This added data would enable a more complete evaluation and comparison regarding the merits and impact of their AO approach.

Response:

We thank the reviewer for this excellent suggestion and we agree that a pre-processing step, such as background subtraction or frequency spectrum attenuation, can help with the reduction of artefacts. To address this comment, we will re-analyse our data and apply these techniques, and we will add the data to the manuscript, with an appropriate revision to the text.

Reviewer #3

1. There is an overall reference in the manuscript of the novelty possible range of applications of using an upright microscope configuration. Examples mentioned are tissue-based imaging, access to whole-mount specimens for manipulation and electrophysiology. However, authors fail to present any such applications. There is not a single example presented which could not have been obtained with an inverted microscope. Could the authors provide an example where a water-dipping is used. Expanded samples could be one case, since the thickness of the gel makes it difficult to image with an inverted microscope. Another possible example would be to label the extracellular space and do shadow imaging of the tissue (SUSHI PMID: 29474910). ExM might be simpler to do as part of revising the manuscript than SUSHI.

Response:

We are thankful to Reviewer #3 for these interesting comments. To address this comment, we will emphasise more clearly that Figure 6 of our manuscript shows a sample that is often part of live imaging experiments that require microinjection and even electrophysiology. Our aim was to show the proof of principle and the potential of such experiments, rather than to carry out real and complex experiments using electrophysiology or microinjection. Regarding providing an example where water-dipping is used, this is already present in the same Figure 6, which we will describe more explicitly and fully in the revised manuscript. The reviewer’s comments on expansion microscopy and SUSHI are interesting, but the primary purpose of our microscope system is to facilitate super resolution live cell imaging experiments. Nevertheless, to address this comment, we will add an explanation of the relevance of our approach to improving deep super resolution imaging of expanded specimens.

1. On the main text it is described a 5-fold volumetric resolution, which is confusing since authors only mention lateral and axial resolutions. Their measurements correspond to a ~1.6-fold lateral improvement and ~1.7-fold axial improvement. These are however not the 95% of the achievable resolution theoretical maximum, as stated in p7 SI (2 fold increase of 282nm), but only the 80-85%. This point should be rephrased in the manuscript.

Response:

We want to thank Reviewer #3 for bringing up this important point. To address it, we plan to make changes to the text, both in the main manuscript and in the supplementary material, to make it clearer what are the resolution improvement that we achieve and what are the limitations to our approach.

1. [OPTIONAL] p4 and related to figure 2, it would be important to report also measurements of beads with SIM but without AO, just as done for WF. Is there an improvement of using AO on SIM? This is reported for the fixed cells but not for the beads.

Response:

We found no significant improvement in resolution when AO was applied to SIM. To address this comment, we plan to add the extra data to Figure 2, demonstrating this point.

1. Figure 2, it is odd the comparison between WF+/- AO and SIM +/- AO are done using different cellular structures. Since wavelengths used are not the same it is difficult to interpret if there is any improvement of using AO on SIM compared to SIM without AO. Same questions arise as above, Is there an improvement of using AO on SIM?

Response:

We agree that the data in Figure 2C and 2D is presented in unusual way. Our intention was not to make a comparison between bypass and AO, but instead to characterise the super-resolution capabilities of the system. We use different channels because doing -/+ AO consecutively leads to noticeable intensity drop due to photobleaching. We are grateful to Reviewer #3 for the valuable comment, which we plan to address by revising Figure 2.

1. "A significant benefit and uniqueness of the Deep3DSIM design is its upright configuration, whereas commercial SIM systems are built around inverted microscopes and are usually restricted to imaging thin samples, such as cultured cells." (p5) is not correct. The commercial DeepSIM module from CREST Optics can be mounted on an inverted microscope as well as image deep into tissue (seehttps://crestoptics.com/deepsim/ and application notes therein) and be used with essentially any objective. This point should be rephrased in the text.

Response:

We want to thank Reviewer #3 for bringing our attention to this error. Of course, we meant commercial 3D-SIM systems, such as GE Healthcare DeltaVision OMX and Nikon N-SIM. To address this issue, we plan to rephrase this part of the results section. Regarding the commercial DeepSIM module from CREST Optics, as far as we can tell, it uses a different method – 2D lattice multi-spot SIM – which comes at the cost of signal loss when sample-induced aberrations are strong. This is very different from our method, which uses a deformable mirror to manipulate the phase information of both the excitation and the emission light at the back-pupil plane of the objective lens, which can theoretically provide 2× resolution enhancement with no signal lost.

1. Fig 3 reports the improvements of AO on SIM for imaging over 10um in tissue. What are the Zernike modes measured? Or how does the pupil look like before and after correction? It would be also good to report the Fourier amplitudes as done in Fig 2C as a quantitative measure of improvement. It would be good to point out the artifacts observed on the BP SIM image reconstruction (labelled with 3x, fringes are noticeable).

Response:

We thank Reviewer #3 for the good suggestions. We plan to add information about the measured Zernike modes to the results section, as well as to add a brief discussion about the noticeable reconstruction artefacts. In terms of pupil and Fourier amplitudes, we plan to change Figure 3 to include all this information or, alternatively, to include it in the supplementary material.

1. Many key details relating to image acquisition and AO correction are missing for all figures. How is the AO optimization implemented? Is it implemented via a genetic algorithm (progressive optimization of parameters) or using more clever strategies? Not clear if the optimization is implemented using images obtained with flat illumination or after SIM imaging/processing of a given dataset. How long does the AO optimization take? How sensitive to noise is the process? What metric do they use to estimate the sensorless AO correction? On pag12, they say "Fourier domain image metric" for measurements with fine details; otherwise, ISOsense when not high frequencies are present. Could the authors report the formula used to calculate the first metric? What do they consider to be low and high frequencies in this case? Is there a reason why ISOsense is not always used, or is there an automatic way to choose between the two? How many images were acquired for AO correction? Which samples were corrected with ISOsense and which ones with Fourier domain image metric? (see for example the detailed experimental reporting in the Supp Mat from Lin et al Nat Commun 2021).

Response:

We are grateful to Reviewer #3 for the extensive list of questions. The optimisation is done via non-linear least square, it uses widefield images, and it is performed before the actual image acquisition, i.e. well before any SIM reconstruction takes place. The methods used for aberration correction are described in the Methods and materials section, and further in the cited literature, e.g. Antonello et al 2020 and Hall et al 2020. ISOsense needs to be manually chosen over the Fourier image metric, and this should be done when large mode biases lead to small changes in the metric value, which is likely to happen when there are little or no sharp features in the images. One of the disadvantages of our implementation of ISOsense is that the structured illumination pattern is continuously exposed over the sample, which leads to photobleaching and phototoxicity. None of the datasets shown in the manuscript use ISOsense. To address all of the questions from this comment, we plan to significantly expand our descriptions of the AO methods, both in the main text and in the supplementary material.

1. Fig 4. Data presented for larval brain tissue is a very clear example of adding AO to image deep into tissue as the effect at ~130 cannot be understated. Here too, it would be also good to report the Fourier amplitudes as done in Fig 2C as a quantitative measure of improvement and possibly the SNR of reconstructed images. Having a way to quantitatively describe how much better are those images would be great. Also, what are the aberrations corrected? Can the wavefront or Zernike amplitude of the modes be reported? Same as for Fig 3, details about AO correction are missing.

Response:

We are grateful to Reviewer #3 for the helpful comment. We will address it by adding the Fourier amplitudes to Figure 4, as suggested, and by reporting the Zernike mode amplitudes of the aberration corrections.

1. [OPTIONAL] "It is worth noting that aberrations can differ across larger fields, and therefore, after applying an average correction, residual aberrations can still be observed in some regions of the AO-corrected bead images. However, the overall PSF shapes were still dramatically improved with AO compared to the equivalent without AO." This point is very interesting although not result either in the main text or in the SI is presented.

Response:

The residual aberrations are present in the right image of Figure 4B, although we did not highlight them specifically. We are thankful to Reviewer #3 for the good suggestion and we plan to implement it by changing Figure 4 to show a few of the beads with residual aberrations.

1. "As we found that the aberrations change approximately linearly in depth, we could measure the aberration in two planes and calculate the corrections in intermediate planes by interpolation, an approach which we termed "multi-position AO"." This is, personally, one of the major contributions of this work to the community. Unfortunately, it is not reported in detail. Not only for SIM but for imaging with WF or confocal, such linear change for aberrations with depth is not well known. Again, here the details of AO correction and image metrics are missing. To establish that for most thick biological structures 'aberrations change approximately linearly in depth' would be foundational to the widespread use of AO within standard imaging. Would it be possible for the authors to elaborate on this point and present detailed results? What is the error from measuring and correcting more than 2 planes? What is the error from just measuring and AO correcting at the deeper plane, i.e. from a single measurement? Authors could also show a case in which a linear assumption works nicely (or how well it works). For example, comparing an intermediate plane (or a plane beyond) imaged after AO optimization or after coefficient interpolation of the Zernike modes and compare it against correcting directly that plane.

Response:

Some aberrations, such as defocus and spherical aberrations, are mathematically defined as varying linearly with depth. The change in other aberrations with depth can also be estimated with a linear model, which is a standard first-order approximation in the case of two datapoints, such as corrections done in Figure 5. It is not possible to do regression analysis with just a single point, so it is impossible to apply our multi-position AO at a single plane. We are grateful to Reviewer #3 for the constructive comment. To address the questions in this comment, we plan to provide a more detailed description of the correction estimation methods to the results section, as well as a discussion on the accuracy of the linear model in the discussion section.

1. The image of the cos-7 cell in metaphase, for Fig 5 is, however, very disappointing. See Fig 1 of Novak et al Nat Commun 2018 for an example of a single z-plane of a cell in metaphase. Having the possibility to correct for the entire 3D volume, I would expect amazing 3D volumes (movies and/or projections) associated with this imaging which are not presented.

Response:

We thank Reviewer #3 for the interesting comment. The example in Novak et al 2018 was acquired with STED microscopy, which is an entirely different imaging method and thus produces different results. Nevertheless, we will revise the discussion of Figure 5 to ensure that the right expectations are set.

1. In Figure 6, they use AO in remote configuration mode to allow imaging of live specimens. It needs to be clarified if this is an a priori characterization that is then kept fixed while recording in time. The last acquired volume of fig 6A and B have a higher amount of artifacts with respect to time 00:00. Are those artifacts due to lower SNR (maybe due to sample bleaching) or due to some change in the aberrations of the specimen?

Response:

We want to thank Reviewer #3 for the valuable comment. We assume that by change in artefacts, Reviewer #3 is referring to the overall green fluorescent structure. Indeed, this last volume shows the anaphase to telophase transition where the mitotic spindle is being reorganised and disassembled. As such, the structure is much less well-defined than in the first volume. The changes in aberrations over time are not particularly significant in this case, and the photobleaching is not that impactful in such an experiment where relatively thin volumes are acquired with substantial time delay between them. To address this comment, we plan to revise the discussion of the figure and to ensure that the scene observed in the last volume is clearer.

1. "These results demonstrate that the remote focusing functionality of the system can be successfully applied for live 3D-SIM experiments, allowing four-dimensional acquisition while keeping the specimen stationary, thus avoiding the usual agitation and perturbations associated with mechanical actuation." Generally, this statement is true, but for the specific example shown of drosophila embryogenesis is it relevant? If they use piezo-driven Z-stack imaging with AO, does that lead to incorrect reconstructions or motion-induced artifacts? Related to the results shown in Fig 6, the fair comparison would be AO SIM vs SIM (without AO), not AO SIM vs AO WF.

Response:

We are grateful to Reviewer #3 for the insightful comment. Drosophila embryos are quite robust to perturbations due to their shape and size, and the restrictions imposed by SIM experiments (e.g. small Z steps and Z levels held for long periods of time) make motion-induced artefacts not very impactful. Regarding the results, the point of Figure 6 is not to demonstrate the advantages of aberration correction, which we do not claim in the caption or in the relevant part of the discussion, but to demonstrate that remote focusing works well with 3D-SIM reconstruction, which is known to have stringent requirements about the image quality. To address this comment, we plan to revise the figure and its relevant part of the results section.

1. When performing remote focusing, is the effective NA of the imaged plane changing with respect to the NA of the objective used at its focal plane?

Response:

We thank Reviewer #3 for the good question. The effective NA is not altered by the remote focusing. We plan to mention this detail in the results section.

1. [OPTIONAL] Did the authors run calculations to explore whether a commercial upright microscope could be used instead of their design? Are there any fundamental flaws that would make impossible using a commercial base? If not, could an AO SIM module be designed such that it adds on a commercial base? It would be important to discuss this point.

Response:

We thank Reviewer #3 for bringing up this interesting point. A lot of considerations, calculations, and modelling were done in the design of the Deep3DSIM system. Of course, the use of a commercial upright microscope stand was part of the deliberation. One of the obvious limitations is the difficult access to the pupil-conjugated plane. On the other hand, a commercial microscope stand is not well compatible with many of the key parts of the system, which were designed around specific biological applications, such as dual camera system for fast live simultaneous imaging and the heavy-duty Z stage intended to support two heavy micromanipulators. To address this comment, we plan to add a discussion of the compatibility of Deep3DSIM with commercial microscope stands to the discussion section and the supplementary material.

Minor comments:

1. Fig 2 lacks a color bar for D panels, which is in log scale. Authors should also show the Fourier transform along the z direction.

Response:

The colour mapping in Figure 2 uses the lookup tables called Cyan Hot and Orange Hot, as indicated in the caption, which come from the ImageJ software. To address this comment, we plan to improve the caption to reflect the fact that the plots are in log scale. We also want to include Fourier transforms along Z, either in the figure itself or in the supplementary material.

1. p4, "Such minor aberrations tend to be insignificant in conventional microscopy modalities such as widefield and confocal (Wang and Zhang, 2021). Therefore..." If optical aberrations are insignificant for single cells in widefield and confocal why do experiments here? These sentences should be rephrased to motivate better the experiments performed.

Response:

We agree with this comment. To address it, we plan to rephrase this part of the results section to motivate better the experiments.

1. Imaged microtubules look abnormal, 'dotty' (figure 2) in both WF and SIM. See https://sim.hms.harvard.edu/portfolio/microtubules/ or Fig 1 of Wegel, et al Dobbie Sci Rep 2016, for better examples of continuous microtubule structures as imaged with SIM.

Response:

The dottiness of the microtubule structures is not related to the SIM reconstruction, because the same dottiness is seen in the respective WF data, too. It is a product of the sample preparation and it has only aesthetic significance. Nevertheless, to address this comment we plan to mention the dottiness in the results section.

1. Is also the remote focusing performed via optimization of metrics similar to the one used for compensating aberrations?

Response:

Yes, as mentioned in the Methods and materials (p. 13), the calibration of the remote focusing involved sensorless aberration correction of several Zernike modes, such as defocus and spherical aberrations.

1. Figure 2, the order of names on the top right of the panel should match the order of curves presented.

Response:

We agree with this comment. To address it, we plan to reorder the curves in Figure 2.

1. I value the efforts to improve open-source tools for system and AO control and GUI. And those tools seemed to have been modified for this work, although those modifications are not described. Would it be possible for the authors to describe those modifications?

Response:

A detailed breakdown is publicly available at the respective software repositories. To address this comment, we plan to add a summary of software changes to the supplementary material.

1. Reported average values of the FWHM of imaged beads in 3D (p4) require also to report errors associated with those measurements.

Response:

We agree with this comment. To address it, we plan to add statistical information to the FWHM values on page 4.

1. Page 13, second paragraph states that "The results from chapter 3.2..." I believe that was a copy/paste from a thesis but should be corrected for a peer-reviewed publication, as there is no chapter 3.2.

Response:

This is a leftover from an older version of the document which used numbered sectioning. In this case “chapter 3.2” refers to subsection “Characterisation of drift and temperature effects”. We plan on fixing this mistake in the revised manuscript.

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Referee #3

Evidence, reproducibility and clarity

Summary

The work of Wang et al entitled "Deep super-resolution imaging of thick tissue using structured illumination with adaptive optics" presents the use of a deformable mirror to simultaneously perform adaptive optics 'AO' and remote focusing 'RF' on a custom-designed upright microscope configuration. The work is novel and represents a timely application of this type of technology to imaging biological specimens. AO enables the correction of refractive index mismatch and sample-induced aberrations while remote focusing allows focusing through the sample without moving the specimen or the objective. The use of AO improves the final image reconstructed using a traditional SIM processing strategy. I greatly value the idea presented (SI pattern generation p7 Supp Inf) about maximizing the contrast of the projected structured illumination. This could be an excellent way to improve SIM imaging since image reconstructions suffer from artifacts when signal to noise ratio is low. However, since it is only one of the factors considered for reducing the stripe width it is unclear how it compares to imaging with the width that maximizes resolution.

Authors do also a very good job describing, characterizing and designing experiments to deal with instabilities exhibited by the deformable mirror.

One of the key aspects of the paper, that could be stressed more, is that including AO gives access to better-quality raw images that are then be used for standard reconstruction pipeline SIM processing. When aberrations are compensated, the illumination pattern closely matches what is expected by the SIM image formation model. Since these raw recorded images are sharper and closer to the actual assumption behind the SIM image reconstruction model, they will have a major positive impact in reducing artifacts that the inversion algorithm is returning. This is particularly evident in Figure 4.

Major comments:

• There is an overall reference in the manuscript of the novelty possible range of applications of using an upright microscope configuration. Examples mentioned are tissue-based imaging, access to whole-mount specimens for manipulation and electrophysiology. However, authors fail to present any such applications. There is not a single example presented which could not have been obtained with an inverted microscope. Could the authors provide an example where a water-dipping is used. Expanded samples could be one case, since the thickness of the gel makes it difficult to image with an inverted microscope. Another possible example would be to label the extracellular space and do shadow imaging of the tissue (SUSHI PMID: 29474910). ExM might be simpler to do as part of revising the manuscript than SUSHI.
• On the main text it is described a 5-fold volumetric resolution, which is confusing since authors only mention lateral and axial resolutions. Their measurements correspond to a ~1.6-fold lateral improvement and ~1.7-fold axial improvement. These are however not the 95% of the achievable resolution theoretical maximum, as stated in p7 SI (2 fold increase of 282nm), but only the 80-85%. This point should be rephrased in the manuscript.
• [OPTIONAL] p4 and related to figure 2, it would be important to report also measurements of beads with SIM but without AO, just as done for WF. Is there an improvement of using AO on SIM? This is reported for the fixed cells but not for the beads.
• Figure 2, it is odd the comparison between WF+/- AO and SIM +/- AO are done using different cellular structures. Since wavelengths used are not the same it is difficult to interpret if there is any improvement of using AO on SIM compared to SIM without AO. Same questions arise as above, Is there an improvement of using AO on SIM?
• "A significant benefit and uniqueness of the Deep3DSIM design is its upright configuration, whereas commercial SIM systems are built around inverted microscopes and are usually restricted to imaging thin samples, such as cultured cells." (p5) is not correct. The commercial DeepSIM module from CREST Optics can be mounted on an inverted microscope as well as image deep into tissue (see https://crestoptics.com/deepsim/ and application notes therein) and be used with essentially any objective. This point should be rephrased in the text.
• Fig 3 reports the improvements of AO on SIM for imaging over 10um in tissue. What are the Zernike modes measured? Or how does the pupil look like before and after correction? It would be also good to report the Fourier amplitudes as done in Fig 2C as a quantitative measure of improvement. It would be good to point out the artifacts observed on the BP SIM image reconstruction (labelled with 3x, fringes are noticeable).
• Many key details relating to image acquisition and AO correction are missing for all figures. How is the AO optimization implemented? Is it implemented via a genetic algorithm (progressive optimization of parameters) or using more clever strategies? Not clear if the optimization is implemented using images obtained with flat illumination or after SIM imaging/processing of a given dataset. How long does the AO optimization take? How sensitive to noise is the process? What metric do they use to estimate the sensorless AO correction? On pag12, they say "Fourier domain image metric" for measurements with fine details; otherwise, ISOsense when not high frequencies are present. Could the authors report the formula used to calculate the first metric? What do they consider to be low and high frequencies in this case? Is there a reason why ISOsense is not always used, or is there an automatic way to choose between the two? How many images were acquired for AO correction? Which samples were corrected with ISOsense and which ones with Fourier domain image metric? (see for example the detailed experimental reporting in the Supp Mat from Lin et al Nat Commun 2021).
• Fig 4. Data presented for larval brain tissue is a very clear example of adding AO to image deep into tissue as the effect at ~130 cannot be understated. Here too, it would be also good to report the Fourier amplitudes as done in Fig 2C as a quantitative measure of improvement and possibly the SNR of reconstructed images. Having a way to quantitatively describe how much better are those images would be great. Also, what are the aberrations corrected? Can the wavefront or Zernike amplitude of the modes be reported? Same as for Fig 3, details about AO correction are missing.
• [OPTIONAL] "It is worth noting that aberrations can differ across larger fields, and therefore, after applying an average correction, residual aberrations can still be observed in some regions of the AO-corrected bead images. However, the overall PSF shapes were still dramatically improved with AO compared to the equivalent without AO." This point is very interesting although not result either in the main text or in the SI is presented.
• "As we found that the aberrations change approximately linearly in depth, we could measure the aberration in two planes and calculate the corrections in intermediate planes by interpolation, an approach which we termed "multi-position AO"." This is, personally, one of the major contributions of this work to the community. Unfortunately, it is not reported in detail. Not only for SIM but for imaging with WF or confocal, such linear change for aberrations with depth is not well known. Again, here the details of AO correction and image metrics are missing. To establish that for most thick biological structures 'aberrations change approximately linearly in depth' would be foundational to the widespread use of AO within standard imaging. Would it be possible for the authors to elaborate on this point and present detailed results? What is the error from measuring and correcting more than 2 planes? What is the error from just measuring and AO correcting at the deeper plane, i.e. from a single measurement? Authors could also show a case in which a linear assumption works nicely (or how well it works). For example, comparing an intermediate plane (or a plane beyond) imaged after AO optimization or after coefficient interpolation of the Zernike modes and compare it against correcting directly that plane.
• The image of the cos-7 cell in metaphase, for Fig 5 is, however, very disappointing. See Fig 1 of Novak et al Nat Commun 2018 for an example of a single z-plane of a cell in metaphase. Having the possibility to correct for the entire 3D volume, I would expect amazing 3D volumes (movies and/or projections) associated with this imaging which are not presented.
• In Figure 6, they use AO in remote configuration mode to allow imaging of live specimens. It needs to be clarified if this is an a priori characterization that is then kept fixed while recording in time. The last acquired volume of fig 6A and B have a higher amount of artifacts with respect to time 00:00. Are those artifacts due to lower SNR (maybe due to sample bleaching) or due to some change in the aberrations of the specimen?
• "These results demonstrate that the remote focusing functionality of the system can be successfully applied for live 3D-SIM experiments, allowing four-dimensional acquisition while keeping the specimen stationary, thus avoiding the usual agitation and perturbations associated with mechanical actuation." Generally, this statement is true, but for the specific example shown of drosophila embryogenesis is it relevant? If they use piezo-driven Z-stack imaging with AO, does that lead to incorrect reconstructions or motion-induced artifacts? Related to the results shown in Fig 6, the fair comparison would be AO SIM vs SIM (without AO), not AO SIM vs AO WF.
• When performing remote focusing, is the effective NA of the imaged plane changing with respect to the NA of the objective used at its focal plane?
• [OPTIONAL] Did the authors run calculations to explore whether a commercial upright microscope could be used instead of their design? Are there any fundamental flaws that would make impossible using a commercial base? If not, could an AO SIM module be designed such that it adds on a commercial base? It would be important to discuss this point.

Minor comments

• Fig 2 lacks a color bar for D panels, which is in log scale. Authors should also show the Fourier transform along the z direction.
• p4, "Such minor aberrations tend to be insignificant in conventional microscopy modalities such as widefield and confocal (Wang and Zhang, 2021). Therefore..." If optical aberrations are insignificant for single cells in widefield and confocal why do experiments here? These sentences should be rephrased to motivate better the experiments performed.
• Imaged microtubules look abnormal, 'dotty' (figure 2) in both WF and SIM. See https://sim.hms.harvard.edu/portfolio/microtubules/ or Fig 1 of Wegel, et al Dobbie Sci Rep 2016, for better examples of continuous microtubule structures as imaged with SIM.
• Is also the remote focusing performed via optimization of metrics similar to the one used for compensating aberrations?
• Figure 2, the order of names on the top right of the panel should match the order of curves presented.
• I value the efforts to improve open-source tools for system and AO control and GUI. And those tools seemed to have been modified for this work, although those modifications are not described. Would it be possible for the authors to describe those modifications?
• Reported average values of the FWHM of imaged beads in 3D (p4) require also to report errors associated with those measurements.
• Page 13, second paragraph states that "The results from chapter 3.2..." I believe that was a copy/paste from a thesis but should be corrected for a peer-reviewed publication, as there is no chapter 3.2.

Referee Cross-Commenting

The other two reviewers raise relevant and important points that would contribute to the overall improvement of the work. I think that authors should try to address most, if not all, of the comments as long as they don't require more than 3-6 months to get done.

Significance

General assessment:

Although a very good and timely idea is presented the overall the manuscript still needs a lot of work. There is a lack of many key details of AO correction, all applications chosen could have been done in an inverted scope and some of the example images reported are suboptimal (Fig 2 and 5) that need further experimental work. Details and metrics, one example of the advantage of using an upright microscope and overall better examples of imaged cells could be provided.

This work builds upon recent work of implementing AO for 3D SIM (Lin et al Nat Commun 2021) to propose to use a deformable mirror to perfrom AO as well as remote focusing in an upright microscope configuration.

Audience: this work will be of interest for a specialized group of researchers, but it will contribute to the goal of adding AO tools to every microscope that will greatly impact the whole imaging community.

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Referee #2

Evidence, reproducibility and clarity

The authors want to develop a structured illumination microscopy (SIM) system for deep tissue superresolution imaging. Here they have developed a SIM system based on the upright configration, and use deformable mirror to compensate for the relection index (RI) mismatch and improve the resolution and contrast in deep tissues. They also showed examples of SR imaging of COS-7 cells and Drosophila larval brains and embryos.

However, I do have some concerns regarding the paper.

1. The authors have provided an incomplete description of the structured illumination microscopy (SIM) reconstruction process. It is unclear whether the approach is based on 2D interference SIM configurations or 3D interference patterns. Furthermore, the specific algorithm utilized for image reconstruction has not been elucidated. Elaborating on these aspects is crucial as they significantly influence the interpretation of the resulting data.
2. The authors have stated that sample-induced aberrations caused by RI inhomogeneities within the specimen is another major reason for causing artifacts generation. Literature has demonstrated that RI inhomogeneities can lead to non-local distortions in the grid pattern, which suggests that applying uniform reconstruction parameters across the entire image may not be viable. Traditional artifact remediation using the classical Wiener method is likely insufficient under these conditions (PMID: 33896197). The existing adaptive optics (AO) approach, which employs a deformable mirror (DM) alongside an sCMOS camera, is inadequate for tackling the issue at hand. Actually the assertion made in the paper that "aberrations change approximately linearly with depth" is seemingly contradicted by simulations referenced in the cited literature (PMID: 33896197). Consequently, it appears that the current methodology might only achieve a partial mitigation of the problems associated with spherical aberration resulting from RI mismatches. It is advisable, therefore, that the authors explicitly acknowledge this limitation in their manuscript to prevent any potential misinterpretation by readers.
3. In Figure 2, the use of COS-7 cells, which are known for their relatively thin axial dimension, for the experiments raises an eyebrow. Notably, there are ample instances in existing research where both 2D-SIM and 3D-SIM, without the integration of adaptive optics, have yielded high-quality super-resolution images of structures such as tubulin and the endoplasmic reticulum. In addition, the authors did not present a direct comparison between BP-SIM and AO-SIM here. Without this comparative analysis, it remains ambiguous whether the enhancements in resolution and contrast and the reduction in artifacts can genuinely be attributed to the mitigation of spherical aberration. To clarify this, it would be beneficial for the authors to include side-by-side comparisons of these modalities to demonstrate the specific improvements attributed to AO-SIM.
4. In Figures 3 and 4, the authors have illustrated the enhancements achieved through the application of AO. However, there is a discernible presence of hammer-stroke and honeycomb artifacts in pre-AO imaged data, which seem to originate from the amplification of the incorrectly moved out-of-focal background in the frequency domain. Various strategies have been previously suggested to address these specific artifacts, encompassing methods like subtracting background noise in the raw images or employing selective frequency spectrum attenuation techniques, such as Notch filtering and High-Fidelity SIM. To facilitate a more comprehensive understanding, I would recommend that the authors incorporate into their study a comparison that includes BP-SIM data that has undergone either background subtraction or frequency spectrum attenuation. This added data would enable a more complete evaluation and comparison regarding the merits and impact of their AO approach.

Significance

The authors want to develop a structured illumination microscopy (SIM) system for deep tissue superresolution imaging. Here they have developed a SIM system based on the upright configration, and use deformable mirror to compensate for the relection index (RI) mismatch and improve the resolution and contrast in deep tissues.

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Referee #1

Evidence, reproducibility and clarity

Review, 3D SIM + AO, Wang and coworkers

In this manuscript, Wang and coworkers report an upright 3D SIM system with adaptive optics (AO) correction. They demonstrate that AO improves imaging into thick 3D samples, including Drosophila larval brain. They also explore the use of remote focusing with their setup. The authors clearly demonstrate a gain with AO, and we are convinced that the microscope they build offers some utility over existing state of the art, particularly in samples thicker than a single cell. That said, we have concerns with the manuscript that we would like to see addressed before recommending publication:

• Given the emphasis on super-resolution imaging deep inside a sample, we were surprised to see no mention of other forms of structured illumination that allow super-resolution imaging in samples thicker than a single cell. These include the 'spot-scanning' implementations of SIM that offer better imaging at depth by virtue of pinholes, and include MSIM, iSIM, and rescan confocal technologies. The two-photon / AO implementation of iSIM seems particularly germane, e.g. https://pubmed.ncbi.nlm.nih.gov/28628128/ Please consider citing these works, as they help place the existing work into context.
• As we're sure the authors appreciate, besides aberrations, a major additional obstacle to 3D SIM in thick tissues is the presence of out-of-focus background. Indeed, this point was mentioned by Gustafsson in his classic 2008 paper on 3D SIM (https://pubmed.ncbi.nlm.nih.gov/18326650/): 'The application area of three-dimensional structured illumination microscopy overlaps with that of confocal microscopy, but the two techniques have different and complementary strengths. Structured illumination microscopy offers higher effective lateral resolution, because it concentrates much of the excitation light at the very highest illumination angles, which are most effective for encoding high-resolution information into the observed data, whereas confocal microscopy spreads out its illumination light more or-less uniformly over all available angles to form a focused beam. For very thick and compactly fluorescent samples, however, confocal microscopy has an advantage in that its pinhole removes out-of focus light physically. Structured illumination microscopy is quite effective at removing out-of-focus light computationally, because it is not subject to the missing-cone problem, but computational removal leaves behind the associated shot noise. Therefore confocal microscopy may be preferable on very thick and dense samples, for which the in-focus information in a conventional microscope image would be overwhelmed by out-of-focus light, whereas structured illumination microscopy may be superior in a regime of thinner or sparser samples.' This point is not mentioned at all in the manuscript, yet we are certain it is at least partially responsible for the residual image artifacts the authors mention. Please discuss the problem of out of focus light on 3D samples, particularly with an eye to the 'spot-scanning' papers mentioned above.
• The authors use a water dipping lens, yet they image into samples that are mounted on coverslips, i.e. they use a dipping lens to image through a coverslip: see attached pdf for reference

This almost certainly introduces spherical aberration, which the authors seem to observe: see attached pdf for reference

We find this troubling, as it seems that in the process of building their setup, the authors have made a choice of objective lens that introduces aberrations - that they later correct. At the very least, this point needs to be acknowledged in the manuscript (or please correct us if we're wrong) - as it renders the data in Figs. 3-4 somewhat less compelling than if the authors used an objective lens that allowed correction through a coverglass, e.g. a water dipping lens with a correction collar. In other words, in the process of building their AO setup, the authors have introduced system aberrations that render the comparison with 3D SIM somewhat unfair. Ideally the authors would show a comparison with an objective lens that can image through a glass coverslip. - The authors tend to include numbers for resolution without statistics. This renders the comparisons meaningless in my opinion; ideally every number would have a mean and error bar associated with it. We have included specific examples in the minor comments below. - In Fig. 5, after the 'multipoint AO SIM', the SNR in some regions seems to decrease after AO: see attached pdf for reference

Please comment on this issue.

• Please provide timing costs for the indirect AO methods used in the paper, so the reader understands how this time compares to the time required for taking a 3D SIM stack. In a similar vein, the authors in Lines 213-215, mention a 'disproportionate measurement time' when referring to the time required for AO correction at each plane - providing numbers here would be very useful to a reader, so they can judge for themselves what this means. What is the measurement time, why is it so long, and how does it compare to the time for 3D SIM? It would also be useful to provide a comparison between the time needed for AO correction at each (or two) planes without remote focusing (RF) vs. with RF, so the reader understands the relative temporal contributions of each part of the method. We would suggest, for the data shown in Fig. 5, to report a) the time to acquire the whole stack without AO (3D SIM only); b) the time to acquire the data as shown; c) the time to acquire the AO stack without RF. This would help bolster the case for remote focusing in general; as is we are not sure we buy that this is a capability worth having, at least for the data shown in this paper.
• Some further discussion on possibly extending the remote focusing range would be helpful. We gather that limitations arose from an older model of the DM being used, due to creep effects. We also gather from the SI that edge effects at the periphery of the DM was also problematic. Are these limitations likely non-issues with modern DMs, and how much range could one reasonably expect to achieve as a result? We are wondering if the 10 um range is a fundamental practical limitation or if in principle it could be extended with commercial DMs.

Minor comments

• The paper mentions Ephys multiple times, even putting micromanipulators into Fig. 1 - although it is not actually used in this paper. If including in Figure 1, please make it clear that these additional components are aspirational and not actually used in the paper.
• The abstract mentions '3D SIM microscopes', 'microscopes' redundant as the 'm' in 'SIM' stands for 'microscope'.
• 'fast optical sectioning', line 42, how can optical sectioning be 'fast'? Do they mean rapid imaging with optical sectinong?
• line 59, 'effective imaging depth may be increased to some extent using silicone immersion objectives', what about water immersion objectives? We would guess these could also be used.
• line 65 - evidence for 'water-dipping objectives are more sensitive to aberrations' ? Please provide citation or remove. They are certainly more prone to aberrations if used with a coverslip as done here.
• 'fast z stacks' is mentioned in line 103. How fast is fast?
• line 116 'we imaged 100 nm diameter green fluorescent beads'. Deposited on glass? Given that this paper is about imaging deep this detail seems worth specifying in the main text.
• lines 127-130, when describing changes in the bead shape with numbers for the FWHM, please provide statistics - quoting single numbers for comparison is almost useless and we cannot conclude that there is a meaningful improvement without statistics.
• In the same vein, how can we understand that remote focus actually improves the axial FWHM of the widefield bead? Is this result repeatable, or it just noise?
• line 155, 'Because of the high spatial information...' -> 'Because of the high resolution spatial information...'
• When quoting estimated resolution #s from microtubules (lines 158-163) similarly please provide statistics as for beads.
• It seems worth mentioning the mechanism of AO correction (i.e. indirect sensing) in the main body of the text, not just the methods.
• How long do the AO corrections take for the datasets in the paper?
• Were the datasets in Fig. 2-4 acquired with remote focusing, or in conventional z stack mode? Please clarify this point in the main text and the figure captions.
• It would be helpful when showing z projections in Figs. 3-5 to indicate the direction of increasing depth (we assume this is 'down' due to the upright setup, but this would be good to clarify)
• line 174, 'showed significant improvements in both intensity and contrast after reconstruction' - we see the improvements in contrast and resolution, it is harder to appreciate improvements in intensity. Perhaps if the authors showed some line profiles or otherwise quantified intensity this would be easier to appreciate.
• line 195 'reduced artefacts' due to AO. We would agree with this statement - the benefit from AO is obvious, and yet there are still artefacts. If the authors could clarify what these (residual) artefacts are, and their cause (out of focus light, uncorrected residual aberrations, etc) this would be helpful for a reader that is not used to looking at 3D SIM images.
• Line 197, 'expected overall structure', please clarify what is expected about the structure and why.
• Line 199, what is a 'pseudo structure'?
• Fig. 4B, 'a resolution of ~200 nm is retained at depth', please clarify how this estimate was obtained, ideally with statistics.
• Fig. 4D, please comment on the unphysical negative valued intensities in Fig. 4D, ideally explaining their presence in the caption. It would also be helpful to highlight where in the figure these plots arise, so the reader can visually follow along.
• Line 245, 'rapid mitosis'. What does rapid mean, i.e. please provide the expected timescale for mitosis.
• For the data in Fig. 6, was remote refocusing necessary?
• What is the evidence for 'reduced residual aberrations', was a comparative stack taken without AO? In general we feel that the results shown in Fig. 6 would be stronger if there were comparative results shown without AO (or remote focusing).
• Line 350, 'incorporation of denoising algorithms' - citations would be helpful here.
• Line 411, 'All three were further developed and improved' - vague, how so?
• Sensorless AO description; how many Zernike modes were corrected?
• Multi-position aberration correction. Was the assumption of linearity in the Zernike correction verified or met? Why is this a reasonable assumption?
• Fig. S1B is not useful; if the idea is to give a visual impression of the setup, we would recommend providing more photos with approximate distances indicated so that the reader has a sense of the scale of the setup. As is - it looks like a photograph of some generic optical setup.
• SI pattern generation - 'the maximum achievable reconstruction resolution was only slightly reduced to about 95% of the theoretical maximum'. We don't understand this sentence, as the resolution obtained on the 100 nm beads is considerably worse than 95% of the theoretical maximum. Or do the authors mean 95% of the theoretical maximum given their pitch size of 317 nm for green and 367 nm for red? SI Deformable mirror calibration

'spanning the range [0.1, 0.9]' - what are the units here?

What are the units in Fig. S5C, S5D?

It would be useful to define 'warmup' also in the caption of SI Fig. S6A. SI Remote Focusing, 'four offsets, {-5 mm, -2.5 mm, 2.5 mm, 5 mm}...' are the units mm or um? '...whereas that of the 10 beads was...' here, do the authors mean the position of the beads derived from the movement of the piezo stage, as opposed to the remote focusing? The authors refer to the 'results from Chapter 3.2'. What are they talking about? Do they mean a supplementary figure, or earlier supplementary results? In general, we found the discussion in this paragraph difficult to follow. Supplementary Fig. 9 seems to be not referred to anywhere in the text. - Since the paper emphasizes 3D SIM, OTFs along the axial direction would also be useful to show, in addition to the lateral OTFs shown in Fig. 2D. - When the sample is moved by the piezo, the axial phase of the 3D-SIM illumination pattern is stable as the sample is scanned through the illumination pattern. When remote focusing is performed, the sample is always stable so the axial phase of the 3D-SIM illumination pattern is presumably changing with remote focusing. Can the authors clarify if the 3D SIM illumination pattern is scanned when remote focusing is applied, or is the intensity pattern stable in z? - In Supplementary Fig. 9, primary spherical is referred to twice, both at index 11 and 22. The latter is presumably secondary spherical? - we do not understand the x axis label, in Fig. S4D, is it really [0, 50, 50, 50] as written? see attached pdf for reference

Referee Cross-Commenting

I don't have much to add; the other reviewers raise good points and I think it would be good if the authors could respond to their feedback in a revised manuscript.

Significance

Nearly all fluorescence images deteriorate as a function of depth. Methods to ameliorate this depth-dependent degradation are thus of great practical value, as they improve the information content of images and thus (hopefully) biological insight. In this work, the authors develop a method to improve super-resolution imaging (3D SIM) at depth, by combining it with adaptive optics.

Résumé de la vidéo [00:00:00][^1^][1] - [01:54:14][^2^][2]:

Cette vidéo présente une réunion de l'Institut Bertrand Schwartz, axée sur la participation des jeunes et l'implication des élus locaux dans les missions locales. Les intervenants discutent des bénéfices et des risques de la proximité avec les administrés, ainsi que des changements nécessaires dans les pratiques des élus pour favoriser la participation citoyenne.

Points forts : + [00:00:00][^3^][3] Introduction et déroulé de la réunion * Présentation des intervenants * Objectifs de la réunion * Importance de la participation des jeunes + [00:02:00][^4^][4] Rappel de la démarche et des principes * Implication des élus locaux * Contribution des jeunes aux politiques * Importance de la décentralisation + [00:05:00][^5^][5] Recherche-action pour la participation des jeunes * Trois types d'acteurs : jeunes, professionnels, élus * Objectifs des webinaires * Changement de posture des élus + [00:10:00][^6^][6] Outil de mesure de la participation * Types de participation : consultants, collaborateurs, pilotes * Importance de la non-participation assumée * Risques de fausse participation + [00:31:20][^7^][7] Discussion sur l'implication des élus * Importance des échanges directs avec les jeunes * Changement de contexte avec la garantie jeune * Rôle des missions locales comme médiateurs

Est-ce que cela répond à votre demande ?

Great.

Childhood engagement with fantasy can influence adults' susceptibility to false narratives, highlighting the importance of protecting individuals from misinformation, especially in an era where fake news is pervasive and challenging to evade.

essai annotation es ins

MIT Sensemaking

Uncertainity

...

怎么缩容和扩容是一样的标识

明白了都是要再hash

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

Learn more at Review Commons

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Reply to the reviewers

Reviewer #1:

This study provides negative in vivo evidence for the use of two PERK inhibitors and of TUDCA for the treatment of Sli1-related Marinesco-Sjögren syndrome (MSS).

Overall, the manuscript reports a substantial amount of work and the study could be published in its present format. The experiments are well described in terms of methodology and appropriate analysis has been applied. Claims are proportionate and not overstated

I would have only minor comments related to some clarifications that the authors could make in the present manuscript and a suggestion for experiments that could improve the manuscript.

First, although this is not my expertise, the in vitro analysis of CHOP luciferase assays suggests that very high concentrations, in particular of TUDCA, are needed to observe an effect. The authors may wish to clarify their opinion and whether this could be the reason why in vivo they have been unable to obtain any inhibition of the PERK pathway.

The reviewer is correct in pointing out that high concentrations of trazodone, DBM and TUDCA were required to inhibit the PERK pathway in the CHOP::luciferase reporter cell lines. However, as we state in the Discussion, we do not think that their lack of effect in vivo was due to insufficient drug levels, since woozy mice were treated with trazodone, DBM or TUDCA according to dose regimens and administration routes that have proved effective in other neurodegenerative disease mouse models. Moreover, our analysis did not find major differences in drug bioavailability between mice with the woozy genetic background (CXB5/ByJ) and C57BL/6J mice in which these drugs had shown neuroprotective effects (see also the response to the next point).

Second, it seems to me that when measuring the Trazodone metabolism there is a difference between acute and chronic treatment. It would be worth discussing what the authors make of that and what is more relevant (I assume chronic) to the disease model outcome.

We realized that the nomenclature used in Figures 6 and 7 was confusing, leading the reader to think there were differences in trazodone levels between chronically and acutely treated mice.

The experiment shown in Figure 6 was designed to test whether there were differences in trazodone pharmacokinetics and metabolism between mice of the woozy strain, which have the CXB5/ByJ genetic background, and C57BL/6J mice in which trazodone had shown neuroprotective effects in previous studies. In contrast, Figure 7 illustrates the levels of trazodone and m-CPP in control and woozy mice (both of which have the CXB5/ByJ genetic background) that had been chronically treated with trazodone for 5 weeks. These are the same animals as in Figure 3, as we state in Figure 7 legend. Therefore one should compare the levels of trazodone and m-CPP in Figure 7 with those of the "woozy" group (CXB5/ByJ genetic background) in Figure 6. This comparison shows that trazodone and m-CPP levels are comparable after chronic and acute (6h) treatment.

To avoid confusion, we have changed the mouse nomenclature. We have renamed the control group of mice as "CT" (previously "WT") throughout the text and figures. In Figure 6, we have used CXB5/ByJ instead of "woozy" to emphasize the comparison between the different genetic backgrounds (CXB5/ByJ vs C57BL/6J). Finally, we have replaced the colors of symbols in Figure 7 in order to match those of Figure 3. We have also made the description and discussion of these results clearer in the revised manuscript.

With respect to the experiments a simple and informative addition would be the evaluation of the PERK pathway in mice treated with TUDCA, as this is missing.

The effect of TUDCA treatment on the PERK pathway is shown in Figure 5, where we measured CHOP mRNA levels in Purkinje cells microdissected from mice treated with 0.4% TUDCA in the chow, and in Figure 9C and D, where we measured the percentage of CHOP-immunopositive Purkinje cells in the cerebellum of same groups of mice by immunohistochemistry.

Figure 10 illustrates the results of an additional experiment in which woozy mice were treated with 500 mg/kg TUDCA intraperitoneally (ip), to test whether this alternative dosing regimen was any better. Like the treatment per os, TUDCA ip had no beneficial effect on motor dysfunction. Therefore we deemed it unnecessary to check the effect on PERK pathway inhibition in this group of mice.

A more difficult but potentially more interesting line of investigation is that of searching for potential actions of Trazodone that are PERK independent and might be responsible for the partial rescue observed in the beam walking test, which is much more sensitive and specific than rotarod, so worth considering. Assuming authors want to go down this route and add significance to their study my suggestion would be an unbiased RNA seq from the brain samples they already have. However, this is a suggestion to steer the study towards a more positive outcome and it is not necessary to support their current conclusions.

We agree with the reviewer that it would be interesting to investigate the mechanism by which trazodone slightly ameliorated the motor performance of woozy mice in the beam walking test. In the Discussion, we speculated that this could be due to an effect of trazodone on cerebellar serotonergic neurotransmission, which would require electrophysiological investigations to demonstrate. Of course, other mechanisms may also be operative, which RNA seq may help identify, as the reviewer suggests. However, this would be a complex and lengthy investigation, the results of which would not change the main conclusions of the present paper. We plan to explore this line of investigation in a future study.

Reviewer #2:

Lavigna et al. described the effect of Trazodone in Marinesco-Sjögren syndrome model mice. Although the results are somewhat disappointing, this research has provided fundamental evidence for the future development of MSS therapeutics. There are few minor comments to further improve the manuscript

Major comment<br /> P14<br /> "Trazodone metabolism to m-CPP was slightly impaired in woozy mice compared to C57BL/6J mice. This was evident from the m-CPP/trazodone ratio, calculated on the AUC0-t in the plasma, which was 0.34 in woozy and 0.67 in C57BL/6J mice."

Why was the concentration different between WT and woozy mice? Which organ mainly contributes to the metabolism of trazodone? Is the function of this target organ different between WT and woozy mice?<br /> Similar to trazodone, m-CPP clearance from plasma was slightly faster in woozy than in C57BL/6J mice.<br /> Is m-CPP eliminated via the kidney? Or liver? Why is there a difference? Does SIL1 functions in liver or kidney? Needs discussion. This is the same for brain m-CPP levels.

As explained in the response to the second comment of reviewer #1, "woozy" in Figure 6 referred to mice with the CXB5/ByJ genetic background, and in this experiment we compared trazodone pharmacokinetics and metabolism between CXB5/ByJ and C57BL/6J mice. We have modified the nomenclature of Figure 6 and the Results to make this clear.

Trazodone undergoes extensive hepatic metabolism, and only a small percentage is excreted unchanged in the urine. Metabolism involves hydroxylation, oxidation and dealkylation reactions, forming in particular the 5HT-active metabolite m-CPP (by CYP3A4). This and other metabolites are mainly excreted in urine, as conjugates [1-3]. The slight differences in trazodone pharmacokinetics and metabolism between the CXB5/ByJ and C57BL6/J mice shown in Figure 6 is not attributable to loss of SIL1 function, since both groups of mice carried wild-type Sil1 alleles, but is most likely due to subtle differences between the two strains, for example in the binding to plasma proteins, metabolic enzymes, transporters and/or the excretion processes. The available data do not allow to clarify this issue.

The main point, however, is that no major differences were found in the plasma and brain concentrations of trazodone between these two strains of mice, which could have explained the lack of efficacy of trazodone in woozy mice, as we now further stress in the revised Discussion.

Minor comments

P3 L5 mutation should be variant.

This has been changed.

P4 L1 eIF2a-P should be phosphorylated eIF2α (p-eIF2α). The reviewer prefers (p-eIF2α) than (eIF2α-p) throughout the manuscript.

There is no standard rule for indicating phosphorylated proteins, and phosphorylated eIF2α is referred to in various ways in different papers, with the "p" in capital or lowercase, preceding or following the protein name, separated by a dash or not. We would prefer to maintain the current nomenclature for consistency with our previous publications, unless the Editor deems otherwise.

P9 L11 M-CPP should be fully spelled out the first time it appears. m-Chlorophenylpiperazine (m-CPP)

M-CPP is spelled out the first time it appears in the Material and Methods, subheading Drug treatments and bioanalysis.

Please explain the difference between the expected function of trazodone and its metabolite m-CPP. Why m-CPP is not effective.

Based on the observation that mice of the woozy strain had lower brain levels of m-CPP than C57BL6/J mice (Figure 6), we hypothesized that the lack of effect of trazodone in woozy mice could be due to m-CPP mediating the PERK signaling inhibitory activity of trazodone. However, experiments in CHOP::luciferase reporter cells demonstrated that m-CPP does not inhibit PERK signaling (Figure 2D). The precise mechanism by which trazodone inhibits PERK signaling is not known [4], which makes it difficult to speculate why its main metabolite, m-CPP, does not exhibit this activity.

P11 L3 Fig. 3 Fig. 3A and B?

Yes, we specifically refer to panels A and B of Figure 3. We have indicated this in the revised manuscript.

P11 L6 at 7 weeks of age?

We have re-done the statistical analysis by two-way ANOVA and reported the results in the legend to Figure 3. There is a significant difference between vehicle- and trazodone-treated woozy mice in the number of missteps when the two groups are compared globally. No statistically significant difference in the number of missteps is detected at specific time points by post-hoc analysis. There is no statistically significant difference between vehicle- and trazodone-treated woozy mice in the time to traverse the beam. The Results section has been revised accordingly.

P12 L17 ~4 times, 4 times? Please state the exact value.

Done.

Figure 7 Why are brain m-CPP levels higher than plasma levels? Is trazodone metabolized in brain tissue?

Trazodone is extensively metabolized in the liver through Cytochrome P450 (Rotzinger et al., 1999). It is well documented that m-CPP readily passes the blood-brain barrier, much better than the parent compound, explaining its high brain levels [2].

P19 L7 ISRIB; please fully spell out the first time it appears.

Done.

References

1. Rotzinger S, Bourin M, Akimoto Y, Coutts RT, Baker GB (1999) Metabolism of some “second”- and “fourth”-generation antidepressants: iprindole, viloxazine, bupropion, mianserin, maprotiline, trazodone, nefazodone, and venlafaxine. Cell Mol Neurobiol 19:427– 442. https://doi.org/10.1023/a:1006953923305
2. Caccia S, Ballabio M, Samanin R, Zanini MG, Garattini S (1981) (--)-m-Chlorophenyl- piperazine, a central 5-hydroxytryptamine agonist, is a metabolite of trazodone. J Pharm Pharmacol 33:477–478. https://doi.org/10.1111/j.2042-7158.1981.tb13841.x
3. DeVane CL, Boulton DW, Miller LF, Miller RL (1999) Pharmacokinetics of trazodone and its major metabolite m-chlorophenylpiperazine in plasma and brain of rats. Int J Neuropsychopharm 2:17–23. https://doi.org/10.1017/S1461145799001303
4. Halliday M, Radford H, Zents KAM, Molloy C, Moreno JA, Verity NC, Smith E, Ortori CA, Barrett DA, Bushell M, Mallucci GR (2017) Repurposed drugs targeting eIF2alpha-P-mediated translational repression prevent neurodegeneration in mice. Brain 140:1768– 1783. https://doi.org/10.1093/brain/awx074

test

这一段没理解

Let's break down the key points about Session Authentication in Django REST Framework in simple terms:

Session Authentication

Overview

• Session Authentication uses Django's default session backend.
• Ideal for: AJAX clients that operate within the same session context as your website.

Credentials Provided

• If successfully authenticated:
• request.user will be a Django User instance.
• request.auth will be None.

Responses for Unauthenticated Requests

• If a request lacks valid authentication credentials and is denied permission, it will result in an HTTP 403 Forbidden response (instead of a 401 Unauthorized).

Using Session Authentication with AJAX

CSRF Tokens

• CSRF Token Requirement: For "unsafe" HTTP methods (PUT, PATCH, POST, DELETE), you need to include a valid CSRF token.
• Why CSRF Tokens: They help prevent Cross-Site Request Forgery attacks by ensuring that the request is made by an authenticated user.

Login Pages

• Django's Standard Login View: Always use Django's standard login view to ensure proper protection.
• Reason: This ensures that CSRF validation is applied correctly.

CSRF Validation in REST Framework

Differences from Standard Django

• Authenticated Requests: Require CSRF tokens.
• Anonymous Requests: Can be sent without CSRF tokens.
• Login Views: Always need CSRF validation, so they should always have CSRF tokens.

Practical Example

1. Including CSRF Token in AJAX Requests:
2. When making AJAX requests with methods like PUT, PATCH, POST, or DELETE, ensure to include the CSRF token.

3. Example: javascript $.ajax({ type: 'POST', url: '/your-api-endpoint/', headers: { "X-CSRFToken": csrfToken }, data: { yourData }, success: function(response) { console.log(response); } });

4. Using Django's Standard Login View:

5. Always redirect to or render Django's built-in login view for user authentication.

6. Example: ```python from django.contrib.auth.views import LoginView

   class MyLoginView(LoginView): template_name = 'myapp/login.html' ```

Summary

• Session Authentication is useful for AJAX clients within the same session as your website.
• It requires CSRF tokens for "unsafe" HTTP methods.
• Always use Django's standard login view to ensure proper CSRF validation.
• CSRF validation in REST framework differs slightly to support both session and non-session authentication, but authenticated requests always need CSRF tokens, especially on login views.

Here's a simplified breakdown of the information provided regarding unauthorized (401) and forbidden (403) responses in Django REST Framework:

Unauthorized (401) Response

• When: An unauthorized response (401) is used when a request lacks valid authentication credentials.
• Header Requirement: It must include a WWW-Authenticate header, which informs the client how to authenticate for subsequent requests.
• Authentication Scheme: The type of response (401 or 403) depends on the authentication scheme used. For Basic Authentication, it would include: WWW-Authenticate: Basic realm="api"
• Usage: Typically used when a client needs to provide credentials (like username and password) but hasn't yet done so or provided invalid credentials.

Forbidden (403) Response

• When: A forbidden response (403) is used when an authenticated user lacks the necessary permissions to access a resource.
• Header: Does not include a WWW-Authenticate header.
• Authentication Scheme: Even if a request is authenticated but lacks permissions, a 403 response is used.
• Usage: Indicates that the server understood the request but refuses to authorize it, even if credentials are valid.

Apache mod_wsgi Configuration Note

• Context: When deploying Django with Apache using mod_wsgi, ensure that the WSGIPassAuthorization directive is set to 'On' in the appropriate context (server config, virtual host, directory, or .htaccess) if using non-session based authentication like Basic Authentication.
• Purpose: This directive ensures that necessary authorization headers are passed through to the WSGI application, allowing proper authentication handling at the application level.

Practical Considerations for Basic Authentication

• Security: Ensure that Basic Authentication is used only in testing or under controlled environments due to its limitations (e.g., credentials sent as Base64 encoded strings in headers).
• HTTPS: Always enforce HTTPS for Basic Authentication to secure transmission of credentials.
• Client Handling: Clients should be configured to re-request credentials on each login attempt and avoid storing them persistently.

This setup ensures secure and proper handling of authentication and authorization mechanisms within Django REST Framework applications, maintaining robust security practices.

Here's a simplified explanation based on the provided information about authentication in Django REST Framework:

How Authentication Works in Django REST Framework

1. Purpose of Authentication:

2. Authentication identifies the credentials (like username, password, tokens) used in an incoming request. It doesn't decide if the request is allowed or not; that's handled by permissions.

3. Authentication Process:

4. Django REST Framework supports multiple authentication schemes. Each scheme is tried in order until one successfully authenticates the request.

5. If no scheme authenticates the request, request.user will be set to an anonymous user (django.contrib.auth.models.AnonymousUser), and request.auth will be None.

6. Setting Authentication Globally:

7. You can define default authentication schemes for your entire API using DEFAULT_AUTHENTICATION_CLASSES in your settings.

8. Example: python REST_FRAMEWORK = { 'DEFAULT_AUTHENTICATION_CLASSES': [ 'rest_framework.authentication.BasicAuthentication', 'rest_framework.authentication.SessionAuthentication', ] }

9. Setting Authentication Per View:

10. You can also specify authentication schemes on a per-view basis using class-based views or function-based views.

11. Example with class-based views: ```python from rest_framework.authentication import SessionAuthentication, BasicAuthentication from rest_framework.permissions import IsAuthenticated from rest_framework.response import Response from rest_framework.views import APIView

    class ExampleView(APIView): authentication_classes = [SessionAuthentication, BasicAuthentication] permission_classes = [IsAuthenticated]

     def get(self, request, format=None):
         content = {
             'user': str(request.user),  # `django.contrib.auth.User` instance.
             'auth': str(request.auth),  # None
         }
         return Response(content)
    

    - Example with function-based views (`@api_view` decorator):python from rest_framework.decorators import api_view, authentication_classes, permission_classes from rest_framework.authentication import SessionAuthentication, BasicAuthentication from rest_framework.permissions import IsAuthenticated from rest_framework.response import Response

    @api_view(['GET']) @authentication_classes([SessionAuthentication, BasicAuthentication]) @permission_classes([IsAuthenticated]) def example_view(request, format=None): content = { 'user': str(request.user), # django.contrib.auth.User instance. 'auth': str(request.auth), # None } return Response(content) ```

Summary

Authentication in Django REST Framework verifies user credentials in incoming requests. It's defined globally or per view, determines request.user and request.auth, and sets the stage for permission checks that decide if the request is allowed to proceed.

In simple terms, let's break down the concept of pluggable authentication and the key points from the text with examples:

Pluggable Authentication

Pluggable authentication means that the system should be flexible and allow different ways to verify who a user is. Think of it like having different keys for the same door, where each key represents a different method of proving your identity.

Key Points and Examples

1. What is Authentication?
2. Authentication is like checking an ID card at the entrance of a building to ensure the person trying to enter is who they say they are.

3. Example: When you log in to a website, you might enter a username and password. This process verifies your identity.

4. Why Should Auth be Pluggable?

5. Different applications or parts of an application might need different methods to verify identity.

6. Example: One part of your app might use a username and password, while another might use a fingerprint or a token sent to your phone.

7. REST Framework's Role:

8. The REST framework provides various built-in ways to handle authentication, and it allows developers to add custom methods.

9. Example: The REST framework might support OAuth (logging in with Google), token authentication (using a special code), and basic authentication (username and password) out of the box.

10. When Does Authentication Happen?

11. Authentication happens first, before anything else in the request process. This ensures only verified users can access further functionalities.

12. Example: Before checking if a user has permission to view a page or how many times they've accessed it, the system first confirms who the user is.

13. request.user and request.auth Properties:

14. request.user: This property holds the user's details once they've been authenticated.
15. Example: After logging in, request.user might store information like the user's name, email, and roles.
16. request.auth: This property holds any additional authentication information, like tokens.
17. Example: If you log in using a token sent to your email, this token will be stored in request.auth.

Simplified Summary

Authentication needs to be adaptable, allowing different methods to verify user identity. The REST framework supports multiple built-in ways and custom methods for authentication, ensuring it runs first before any other checks. Once authenticated, user details are stored in request.user, and any extra authentication data (like tokens) is stored in request.auth.

Real-life Example

Imagine a school with multiple entrances:

• Main Entrance: Students show their student ID (username and password).
• VIP Entrance: Teachers use a fingerprint scanner (biometric authentication).
• Emergency Entrance: Parents receive a temporary access code (token authentication).

Each entrance verifies identity differently, but all lead into the same school, ensuring only authorized people get in. Similarly, a pluggable authentication system in an application allows different methods to verify users based on the situation.

这个太重要了

без остановки читать или рассказывать

шепнуть на ушко

оказать поддержку

разъяснить

вдуматься

сослаться

поддерживать

подтвердить

требовать

наделять

настойчивый

повторить

докладчик

легендарный

ровно как и

as - не столько сколько

as - настолько, что

like - не такой как

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eLife assessment

This study presents a useful comparison of the dynamic properties of two RNA-binding domains. The data collection and analysis are solid, making excellent use of a suite of NMR experiments and ITC data. Nonetheless, reported evidence was found to only partially support the proposed connection between the backbone dynamics of the tandem domains and their RNA binding activity. This work will be of interest to biophysicists working on RNA-binding proteins.

Reviewer #1 (Public Review):

In the manuscript Chugh and co-workers utilize a suite of NMR relaxation methods to probe the dynamic landscape of the TAR RNA binding protein (TRBP) double-stranded RNA-binding domain 2 (dsRBD2) and compare these to their previously published results on TRBP dsRBD1. The authors show that, unlike dsRBD1, dsRBD2 is a rigid protein with minimal ps-ns or us-ms time scale dynamics in the absence of RNA. They then show that dsRBD2 binds to canonical A-form dsRNA with a higher affinity and with less changes in dynamics compared to dsRBD1.

Strengths:

The authors expertly use a variety of NMR techniques to probe protein motions over six-orders of magnitude in time. Other NMR titration experiments and ITC data support the RNA-binding model.

Weaknesses:

Generally, the data collection and analysis are sound. However, microsecond timescale dynamics for the RNA-bound form of dsRBD2 are inferred from a sample that is only 5% bound. Additionally, the manuscript lacks context with the much broader field of RNA-binding proteins. For example, many studies have shown that RNA recognition motif (RRM) domains have similar dynamic characteristics when binding diverse RNA substrates.

Reviewer #2 (Public Review):

Summary:

Proteins that bind to double-stranded RNA regulate various cellular processes, including gene expression and viral recognition. Such proteins often contain multiple double-stranded RNA-binding domains (dsRBDs) that play an important role in target search and recognition. In this work, Chug and colleagues have characterized the backbone dynamics of one of the dsRBDs of a protein called TRBP2, which carries two tandem dsRBDs. Using solution NMR spectroscopy, the authors characterize the backbone motions of dsRBD2 in the absence and presence of dsRNA and compare these with their previously published results on dsRBD1. The authors show that dsRBD2 is comparatively more rigid than dsRBD1 and claim that these differences in backbone motions are important for target recognition.

Strengths:

The strengths of this study are multiple solution NMR measurements to characterize the backbone motions of dsRBD2. These include 15N-R1, R2, and HetNOE experiments in the absence and presence of RNA and the analysis of these data using an extended-model-free approach; HARD-15N-experiments and their analysis to characterize the kex. The authors also report differences in binding affinities of dsRBD1 and dsRBD2 using ITC and have performed MD simulations to probe the differential flexibility of these two domains.

Weaknesses:

While it may be true that dsRBD2 is more rigid than dsRBD1, the manuscript lacks conclusive and decisive proof that such changes in backbone dynamics are responsible for target search and recognition and for the diffusion of TRBP2 along the RNA molecule.

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

Learn more at Review Commons

---

Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity):

This manuscript compiles LoF variants of M1AP and ZZS proteins (i.e., SHOC1, TEX11 and SPO16) that almost certainly underlie infertility and reports the first case of an infertile man homozygous for a variant in SPO16. The authors validated interactions between human M1AP and ZZS that were found in mice. Analyzing testicular samples from infertile men revealed that those with deficiencies in SHOC1, TEX11 or SPO16 exhibited early meiotic arrest without haploid germ cells, whereas those with M1AP variants displayed a predominant metaphase I arrest with rare haploid germ cells. Further investigations showed that disrupted SHOC1, TEX11 or SPO16 led to defective synapsis and pairing of homologous chromosomes and unpaired DNA DSBs, while M1AP mutations reduced CO events. Importantly, men with LoF variants in M1AP can father healthy children by medically assisted reproduction. Overall, the results are clear and convincing in defining likely causative variants in infertility patients.

Response: We thank reviewer #1 for the appreciation of our work. We already addressed the suggestions raised by reviewer# 1 to improve our manuscript.

I have a few minor comments for improving the manuscript:<br /> • No statistical analyses were performed. The meaning of error bars was not mentioned. It is essential to specify the minimum number of seminiferous tubules counted for each patient.

Response: We added the statistical analysis. We described now in more clarity that all round tubules in a patient's testicular section were counted (l. 646-653).

• Allele frequencies of variants are not provided.

Response: We added the allele frequencies from gnomAD v4.1.0 (SNVs) and gnomAD SVs v2.1 (CNVs) in Table 1.

• Figure 4 should clearly label the representations of each color channel.

Response: Thanks for this suggestion. We labelled each color channel accordingly.

• The authors should clearly label the bands of SPO16 in the right panel of Figure 1B.

Response: We labelled the SPO16 band in Figure 1B more clearly.

• Appendix Figure S1B and S2B, what does "rat" mean in "rat Ins2 Ex3/4/"?

Response: In the minigene assay, an artificial gene was constructed with exon 3 and 4 from the insulin 2 gene of the species rat (Rattus norvegicus). We described this in more detail in the Appendix methods section (l. 119) and in the Figure legend S1B and S2B.

Reviewer #1 (Significance):

Overall, this study significantly contributes to the understanding of some genetic causes of human infertility and offers a potential avenue to treat patients with M1AP variants/ mutants. Since no knock-in animal model was applied to mimic the subtle phenotype variations observed in patients, the functionality of truncated proteins remains unexplored. For example, it is unclear why the germ cells in patient M3260 with the SHOC1 variant can progress to round spermatids (Fig. 2C), while those in Shoc1 KO mice (10.1093/molehr/gaac015) and other patients cannot. However, this is a minor concern.

Response: Thanks for this comment. SHOC1 variant c.1939+2T>C present in M3260 is a predicted splice site variant. In vitro it results in an in-frame exon skipping as shown by the minigene assay (Appendix Figure S2) that is predicted to lead to a loss of only 4% of the protein. We assume that this does not result in a complete loss but only in an impaired protein function enabling significantly reduced progression of spermatogenesis up to the round spermatid stage in few cells (l. 354-360). We addressed this in more detail in the results section (l. 145ff and l. 189ff) and in the Appendix Figure S2 legend. Accordingly, SHOC1 variant c.1939+2T>C is not a LoF variant and we excluded it from the quantification of subsequent analyses. Immunohistological staining of this patients was excluded from Appendix Figure S6, S7, S9, S10, and S11 and incorporated into Appendix Figure S2.

In addition, the recurrent M1AP c.676dup was functionally analysed in our previous work (Wyrwoll et al., 2020, PMID: 32673564). We detected M1AP mRNA in a testicular biopsy from one patient showing that this variant leads not to degradation of the mRNA. Furthermore heterologous expression of the mutant M1AP cDNA in HEK293T cells led to the production of a truncated protein that presumably leads to loss of protein function. We added this information in l. 136. Furthermore, our preliminary experiments of co-immunoprecipitation of truncated M1AP with TEX11 hint to an abolished protein-protein interaction caused by M1AP c.676dup and thus a loss of protein function.

Our field of expertise is gametogenesis and meiosis in mice.

Reviewer #2 (Evidence, reproducibility and clarity):

Summary:<br /> This interesting manuscript provides evidence for the biological and clinical relevance in human males of mutations in genes encoding M1AP and other related proteins. In mice, M1AP, "meiosis 1 associated protein," is known to associate with several proteins (SHOC1, TEX11, and SPO16) in the ZZS complex that promotes DNA recombination and crossover formation during meiosis I prophase. Mutation of these proteins in model organisms disrupts the process of recombination and cause arrest of spermatocytes prior to the first meiotic division. Here the authors took advantage of their MERGE (Male Reproductive Genomics) cohort to screen for human loss-of-function (LoF) mutations in the relevant ZZS complex and M1AP genes and to associate these with human male reproductive phenotypes. They found that men with deficiency of ZZS proteins SHOC1, TEX11 or SPO16 genes were infertile, exhibiting arrest of germ cell development early in meiotic prophase, with aberrations of chromosome synapsis and failure to repair DNA double-strand breaks (DSBs). In interesting contrast, men with M1AP mutations exhibited metaphase arrest, and indeed, in some cases, produced haploid spermatids, which in medically assisted reproduction (ICSI), led to the birth of offspring. Because they demonstrate that M1AP interacts with the other proteins, the authors conclude that M1AP is a "catalyzer," but not essential, for the processes of synapsis, recombination, and formation of haploid gametes.

Major Comments:<br /> The work is clearly presented with detailed methods that should allow adaptation in other laboratories.<br /> Overall, this study is a tour de force with what was no doubt difficult archival samples. The histology is generally of good quality, supporting the conclusions about progress of meiotic prophase in the mutant samples. The images of H&E-stained tissue are particularly striking, especially those in supplemental figures.

Response: We thank Reviewer #2 for the appreciation of our work and the suggestions to improve our manuscript. To provide transparency of our work, we plan to upload each (immuno-) histologically stained testicular section shown in the Main and Appendix Figures in the microscopy image repository OMERO/Open Microscopy Environment (OME).

That said, and with particular reference to Fig. 3A, it is difficult to sub-stage meiotic prophase by immunocytochemistry, even in optimal samples, with only one marker (in this case gH2AX). The staging here is also at odds with the statement in the subsequent section (and Fig. 4B) on absence of pachytene cells in men with mutation of SHOC1, TEX11, or SPO16.

Because precise stages of arrest probably cannot be determined in these samples, the authors would be wiser to use phrases such as "zygotene-like"

Response: We agree with the reviewer that it is indeed difficult to sub-stage meiotic prophase based on IHC for one marker. A precise sub-staging of the meiotic prophase would require identifying the stage of the seminiferous epithelium. The cycle of the human seminiferous epithelium has been subdivided into 12 stages based on the acrosomal development made visible by immunohistochemistry for acrosin. However, in order to properly evaluate the human germ cell associations, only seminiferous tubules showing a well-preserved seminiferous epithelium with no apparent damage to the epithelium and the peritubular wall can be considered. In addition, all the different generations of germ cells have to be present as well as at least six spermatids (Muciaccia et al., 2013, PMID: 23946533). As these requirements cannot be fulfilled in the testicular tissue of men with a meiotic arrest as due to LoF variants in M1AP or the ZZS genes, we followed the reviewer's suggestion and have modified the respective phrases throughout the text, e.g. to 'zygotene-like'.

The authors should also clarify how it was confirmed that the metaphase-like cells were spermatocytes and not spermatogonia (given that gH2AX signal is weak or unclear in some such nuclei). Readers with a focus on the more regularly staged mouse or rat tubules would appreciate a few more guidelines to criteria for staging human tubules.

Response: We thank the reviewer for raising this point. In order to confirm that the metaphase-like cells were indeed spermatocytes we will perform additional IHC staining for γH2AX and MAGEA4 on sequential testis sections (distance 3 µm) on representative samples of the patient cohort as well as controls as the hosts of both antibodies are mice. For a few more guidelines on the criteria for staging human tubules, please refer to the response to the previous point.

Evidence for the birth of a (healthy) child from one individual with M1AP mutation verges on the anecdotal (N=1). It is interesting but raises multiple questions and concerns about both the frequency of chromosomal abnormalities in such individuals and the transmission of the mutant alleles.

Response: We understand very well, that the evidence based on N=1 seems to be sparse. Nevertheless, if it is in principle possible for a man affected by bi-allelic M1AP LoF variants to conceive a child by ICSI then it could be also possible for other couples with a similar genetic condition (M1AP LoF), and thus providing a proof-of-principle (l. 417f). Reviewer #2 is completely right with the concerns regarding chromosomal aberrations and the transmission of the mutant allele. Thus, it is essential for clinicians/geneticists to counsel the affected couple carefully about the small but existed chance to have a biological own child and the accompanied potential but so far unexplored risks as outlined in l. 435ff. Our future research project will address this open and highly relevant question.

The authors conclude that the M1AP protein is an essential "catalyzer" in the meiotic recombination pathway. However, it is not clear from the data presented that M1AP in fact has enzymatic catalysis activity or exactly when and how it participates. Because the word "catalyzer" is not buttressed with hard or convincing evidence, the authors should consider other ways to describe the proposed role of M1AP, perhaps as a "putative component" and/or "modifier" of the recombination pathway.

Response: We appreciate the reviewer's advice, and changed the wording to "functional enhancer".

Minor comments:<br /> Fig. 1A - these are nice illustrations, but overly simplified with respect to timing (synapsis is not completed in zygonema)

Response: We completely agree that Figure 1A is a simplified depiction that could not reflect the temporally and spatially highly complex processes of meiosis. By adding a second dotted box and describing the process in the Figure legend in more detail, we tried to reduce the simplification. Nonetheless, we believe that this simplified schematic help readers, who are less familiar with the progression of meiosis to contextualise the described processess.

Fig. 1B - greater clarity in legend would be appreciated

Response: We described Figure 1B in more detail.

Figs. 2A & 3A - colors in bar graphs are difficult to discriminate

Response: We improved the discrimination of bar graphs accordingly.

Fig. 4A - with full appreciation for the difficulty with this material, the images are of low contrast and require considerable enlargement

Response: We agree with this opinion; and we increased the contrast. In addition, we will improve the way of representation in a revised Figure 4 in the complete revision of the manuscript in accordance with the suggestions of all three Reviewers.

Reviewer #2 (Significance):

This is a very interesting paper, which I evaluated from the perspective of a reproductive geneticist with expertise in meiosis and interest in infertility. I think this report will be of interest to clinicians because it identifies a gene possibly linked to marginal fertility and establishes human protein interactions similar to those previously identified in mice. It reinforces the importance of ZZS genes in humans. The contributions of this report to the field of meiosis confirm previous evidence on M1AP, including mutant phenotypes and protein interactions, extending them to humans. We can thus appreciate the conserved function of the mammalian M1AP protein, but as yet the molecular mechanisms of M1AP are not clarified.

Response: We gratefully thank Reviewer #2 for the thorough evaluation of our work and appreciate the recognition of the significance. Indeed, it was not possible to clarify the molecular mechanisms of M1AP that, hopefully, could be identified as soon as human specific antibodies, which will function in the needed applications, will be available. Additionally, we will perform further experiments as suggested by Reviewer #3 to gain a better understanding of the processess involved. Clarifying the underlying molecular mechanism is not only one of our highest interest but will also be important for the scientific community.

Reviewer #3 (Evidence, reproducibility and clarity):

In this manuscript, Rotte et al. investigate the meiotic molecular function in human of the M1AP protein and of the ZZS complex (SCHOC1, TEX11 and SPO16 proteins). The ZSS complex is a key player of meiotic recombination. It is a sub-complex of the conserved family of the ZMM proteins, essential for the formation of class I crossovers, a proper chromosomes segregation and fertility. Understanding its mode of action, regulation and conservation in human is thus a crucial issue in the fields of meiosis and human reproduction, with potential implications for patients. In that context, the recent identification of the protein M1AP as a partner of the ZSS proteins raise the question of its role, function and conservation. The aim of this study is thus of primary importance.<br /> To perform this molecular characterization, the authors made a cohort (24 total) of men carrying LoF variants in M1AP and ZSS genes. They performed a molecular biology analysis to assess the physical interaction between the human M1AP protein and the three components of the ZSS complex. Their results confirm a previous work performed in mice, mentioned by the authors.<br /> Then, they took advantage of available biopsies from different mutant men to perform a histological and cytological analysis of the impact of the different mutations on meiosis. The main conclusions are that in human, similarly to what is known in different organisms (ranging from yeast to mice), the ZSS complex is essential for crossover formation, synapsis and spermatogenesis, and that defect in the genes is associated with a premature prophase I arrest and no sperm formation. The authors also showed that M1AP protein plays a role in meiotic progression, but to a lesser extend compare to the ZSS proteins, with a metaphase I arrest, an undetectable recombination phenotype, apart of a reduced crossover number and, spermatozoa can form in its absence.

Major points:<br /> The authors investigate the physical interaction between M1AP and the ZSS members through a single approach: Co-IP of tagged proteins after expression in human HEK293T cells. This approach is informative, but to reinforce the conclusions the authors should provide data from independent approaches: yeast two hybrid, expression of recombinant proteins followed by pull down, co-immunostaining (TEX11 antibodies were used in the study and M1AP antibody is present in the literature) are possible non-exclusive approaches to decipher, more in details, the interaction. Moreover, understanding the hierarchy of interactions appears important to understand its rational, regulation and function. What is the meaning of a M1AP interaction with all the members of the complex? Remains an open question.

Response: We thank Reviewer #3 for this comment. In an independent approach we aimed to specify the interaction of M1AP to the ZZS proteins. Thus, we already cloned truncated versions of M1AP to refine the binding site of M1AP to the ZZS proteins (Figure R1). In a preliminary experiment, we co-transfected full-length as well as truncated forms of M1AP with TEX11 and showed via Co-IP that the interaction is only possible with full-length M1AP. Within the full-revision, we plan to finalise these experiments and thus validate the specifity of the interaction between M1AP and TEX11 and thereby gain more insight into the interaction/hierarchy of the interaction of M1AP with the ZZS complex.

Figure R1 Tolerance landscape of M1AP NM_001321739.2 illustrating the respective regions selected for mutagenesis of truncated M1AP constructs. Adapted from MetaDome.

Moreover, in the last couple of years, we spent enormous resources (personnel, time, financial) to get a functional antibody against human M1AP, including testing of different commercial (and already published) antibodies, creating three customised antibodies against different M1AP polypeptides, a nanobody raised against the complete M1AP protein (failed because of the impossibility to purify the protein), and contacting the authors of previously published customised M1AP antibodies (Arango et al., 2013/PMID 23269666 and Li et al. 2023/PMID 36440627). Figure R2 recapitulates some of our attempts. Moreover, we published the initial attempts of establishing an M1AP antibody in Wyrwoll et al., 2020/PMID 32673564. Unfortunately, no human M1AP-specific antibody is available.

Additionally, we tested different TEX11, SHOC1 and SPO16 antibodies in immunohistochemistry and SHOC1 and SPO16 antibodies in immunofluorescence of spermatocyte spreads, which did not result in a specific staining (Figure R3). Due to the lack of a human specific antibody against M1AP as well as antibodies against SHOC1 and SPO16, we are not able to localise these proteins in patient testicular sections to address this highly interesting research question that remains of great interest within our work on M1AP.

Figure R2. Attempts to locate M1AP in the human testis. Previous attempts to identify a commercially available antibody that reliably detects M1AP in the human testis have not been successful (Wyrwoll et al., 2020/ PMID 32673564). Accordingly, we tried to produce a human-specific antibody in cooperation with companies specified in antibody customisation (Eurogentec, Biotem). The last attempt, conducted with Biotem, is exemplarily shown in this figure. A. Human M1AP protein sequence (NP_620159.2) highlighting the antibody epitopes (orange) that were selected so that in men carrying the M1AP LoF variant c.676dup p.Trp226Leufs*4 in a homozygous state, the respective antibody should not be able to bind due to the protein truncation. For rabbit immunisation, both epitopes were pooled. B. HEK293T cells were transfected with DYK-tagged M1AP plasmids, either expressing the wildtype (WT) or the truncated protein (W226L). Sera of day (D) 28 and 42 of the immunised rabbit as well as the purified antibody product, a commercially available anti-M1AP antibody (HPA), and anti-DYK control antibody specificity was confirmed by Western blotting. C. Customised anti-M1AP antibody validation in human testicular control and D. M1AP-deficient tissue did not yield in a reliable staining. Various protocol optimisations were tested (different antigen retrieval, adapted blocking and antibody dilution solution, various primary and secondary antibody concentrations). Date shown represents the best result, respectively. The application of both sera and the purified antibody for spermatocyte spreading was tested in parallel and has not been successful either (data not shown). SC: Sertoli cells, SPC: spermatocytes, M-I: metaphase I cells, RS: round spermatids, ES: elongated spermatids. The scale bar represents 100 µm and 10 µm.

Figure R3. Efforts to identify human-specific antibodies for ZZS localisation. A. Commercially available antibodies for ZZS were tested via Western blotting, aiming to reliably detect SHOC1, SPO16, and TEX11 in human testicular biopsies. HA-tagged wildtype plasmid DNA (WT) was transfected in HEK293T cells and the anti-HA antibody was used as a positive control. Only one antibody detected TEX11 reliable in the purified lysates (anti-TEX11: HPA002950). B.-D. Immunohistochemical staining was performed with all antibodies on human testicular and is representatively shown for anti-SHOC1: #BS155344-R, anti-SPO16: #BS15024-R, and anti-TEX11: HPA002950. Only the anti-TEX11 (#HPA002950) was found to be specific. However, presumably due to the fixation with Bouin's solution, staining could not reliably be repeated in all samples and was not implied in this study. Various protocol optimisations were tested (different antigen retrieval, adapted blocking and antibody dilution solution, various primary and secondary antibody concentrations). Date shown represents the best result, respectively. The application of all antibodies for spermatocyte spreading was tested in parallel and have not been successful (data not shown), except for anti-TEX11 (#HPA002950, Appendix Figure S13). SC: Sertoli cells, SPC: spermatocytes, RS: round spermatids, ES: elongated spermatids. The scale bar represents 100 µm and 10 µm.

The ZZS mutants have a defect in gH2AX pattern, a defect in synapsis and no MLH1 foci, associated to apoptosis and prophase I arrest. M1AP mutation has a minor impact. The characterization of the effect of the different mutations (in particular M1AP) on the recombination process should be addressed further, by cytological means. For example, effect on strand invasion and ssDNA production should be monitored using RPA, DMC1 and RAD51 antibodies. The impact on alternative resolution pathway (e.g. BLOOM dependent) should be tested as well as the effect on other ZMM proteins, in particular MSH4-5, should be investigated. These experiments are essential to characterize, at the molecular level, the function of the different proteins during recombination.

Response: We thank the reviewer for this suggestion and highly appreciate to investigate the different pathways in more depth. We plan to perform additional immunofluorescence staining of spermatocyte spreads of identified patients compared to the control in the planned revision for a better understanding of M1AP within human recombination. We already ordered the antibodies against meiotic marker proteins as suggested by the Reviewer.

We would like to take the opportunity to refer to the extremely limited access to cryopreserved testicular material of the patients presented in this manuscript: for each gene (M1AP, SHOC1, TEX11, SPO16) we were lucky to get one testicular biopsy specimen from one man for only one preparation of spermatocyte spreads. We hope for the Reviewer's understanding that we cannot address each requested staining albeit this would be of highest interest. However, we are very confident that we will provide additional staining added to the yet shown to improve the understanding of M1AP's function on human male meiotic recombination.

In the same line, TEX11 staining in M1AP mutant should be more documented and in particular the different stages shown, as well as the foci counting, to have a quantitative result, that can be compared to MLH1. Moreover, co-immunostaining of different markers with TEX11: RPA, DMC1, MSH and MLH1 are also important to understand how the pathway is perturbed and the recruitment delayed/affected.

Response: In the planned revision, we will include the TEX11 foci counting using the acquired images that will be compared to MLH1 foci quantification. In addition, we plan additional co-immunostaining of TEX11 with different markers dependent on the availability of testicular material. Due to the limited resources of cryopreserved material, we cannot repeat the TEX11 staining in the patients with M1AP LoF variant for documentation of different stages. Slides that have already been stained are unfortunately bleached and cannot be re-analysed.

The published M1AP antibody should be tested to investigate its perturbation in the absence of the ZZS proteins and the hierarchy of event.

Response: As already outlined above, we tried to get any functional M1AP antibody for several years, which was not possible (Figure R2). Thus, we unfortunately cannot address this comment via this approach albeit this research question remains of great interest within our work on M1AP.

OPTIONAL: the obligatory crossover was measured, a comment or calculation of interference would be very interesting, and it seems doable using the MLH1 counting, to test whether thses mutants have an effect on this process.

Response: We thank Reviewer #3 for the suggestion of this interesting question that was not within our focus so far. Due to the limited material and the small number of cells from which we could digitally separate the chromosomes, we believe that the sample size is insufficient to obtain a statistically significant result.

Minor comments<br /> As written, the title is misleading, the paper does not investigate the impact of M1AP in ZSS recombination. Such study implies to study genetic interactions or the genetic dependency between the different proteins, which is not the case here.

Response: Thanks for this comment. We changed the title to "Genotype-specific differences in infertile men due to loss-of-function variants in M1AP or ZZS genes".

Labelled on histological images is not clear. The authors should clearly explain to what marker each staining correspond.

Response: We changed the labelling accordingly.

L67 to 72: the authors should update and use more accurate citations for meiotic recombination.

Response: Thanks for this suggestion. In this section, we have described the fundamental processes of meiosis, which have been repeatedly reviewed by renowned scientists. We have therefore chosen four well-cited expert reviews from different groups as references (PMID: 29385397, 24050176, 27648641, 35613017).

L76: the ZMM are specifically involved in the resolution of class I crossover. Please rephrase.

Response: We rephrased the sentence and changed it throughout the manuscript.

L94: Strictly, the author identified an interaction, they didn't establish how the interaction takes place.

Response: We rephrased the sentence.

FigS13: TEX11 staining should be presented with foci counting as a main figure.

Response: We plan to restructure Figure 4 along with the new meiosis specific markers and will consider this comment.

L255: MLH1 does not quantify homologous recombination but, class I crossovers.

Response: We rephrased the sentence.

L352: The sentence is hard to understand, rephrase please.

Response: We rephrased the sentence.

Reviewer #3 (Significance):

In general, the paper is well written and easy to follow. However, in light of the importance of the questions for the field of meiosis, it currently seems a little superficial, in particular if the authors aim at addressing the molecular function of the different proteins. The role of the ZSS proteins and M1AP in the control of meiotic recombination, at the molecular level is very important to decipher and additional experiments might help to better address this question. In addition, the functional links between M1AP and ZSS remains unclear and to investigate further.<br /> This study gives information for human process, and can be compared to more advanced work done with mice.<br /> This study will be important for the community working on meiosis in mammals, but also for people interested in reproduction.

Response: We thank Reviewer #3 for the thorough evaluation and acknowledgment of the significance of our work. We appreciate the suggestion of performing additional experiments to gain a better and more in depth understanding of the molecular pathways involved. We hope for the Reviewer's understanding that we cannot address all raised comments due to the limited material and the difficulty to get human specific antibodies in a research field that primarily works with highly valuable mouse models.