see they were not made for the south

oh thats insane

ok girl stop laughing it aint that funny

yeah he couldnt survive long

girl calm down

:)

aw i'm proud of you

ITS CORONATION DAY YIPPIE

JON IS MY SON IDCC

ravens...

he loves his siblings so much omg

:(( happy birthday kid

yall better fix it up SOON

flat earther theories SORRY

so?? they should just get better

YES SHE WAS

URHGH LET THEM REUNITE PLSS

NOOOO

:(

nu uh

NOO HES GONNA GET NED KILLED

rat boyfriend

never trust anyone

awww

norse mythology

who?

nooo

oh nahh

oh i hate them all

YES

making me sad over a WOLF

YES

bitch

poor girls :((

nooo lady

alicent...

you cant just say perchance

he's so pathetic

i like him more here

:(

aww

disprefer = don't love ?

infelicitous

I don't think it's that specialist of a word... :shrug:

Appropriate word choice in the same way that "liked by some, disliked by others" is appropriate.

Résumé de la vidéo [00:00:02][^1^][1] - [00:48:41][^2^][2]:

Cette vidéo présente le framework Observable pour créer des tableaux de bord, des rapports et des applications web de manière efficace et gratuite. Elle explique comment utiliser Observable pour documenter des fonctionnalités, introduit le concept de Data loader pour rafraîchir les données, et montre comment intégrer des réalisations Observable dans un site web statique.

Points forts: + [00:00:08][^3^][3] Introduction à Observable * Présentation du framework Observable comme générateur de site statique gratuit et open source * Utilisation de Markdown et JavaScript pour la documentation * Hébergement gratuit sur des plateformes comme GitHub Pages + [00:01:36][^4^][4] Spécialisation pour les tableaux de bord * Observable est spécialisé pour les applications nécessitant un rafraîchissement régulier des données * Introduction du concept de Data loader pour une mise à jour périodique des données * Création de sites web statiques capables de rafraîchir leurs données efficacement + [00:03:00][^5^][5] Développement JavaScript avec Observable * Observable comme environnement de développement JavaScript unique avec réactivité entre déclarations * Explication de la réactivité et de la dépendance des variables dans Observable * Utilisation de Markdown, LaTeX et JavaScript pour créer des contenus interactifs + [00:10:13][^6^][6] Utilisation de bibliothèques et gestion de versions * Observable permet d'appeler des bibliothèques externes et contient un gestionnaire de versions simplifié * Partage et publication de classeurs pour la collaboration et la réutilisation * Exemples de tutoriels et de cours disponibles sur Observable + [00:24:26][^7^][7] Démarrage avec le framework * Processus de création, d'édition et de prévisualisation d'un site avec Observable * Utilisation de GitHub Actions pour le rafraîchissement automatique des données * Intégration d'animations et de visualisations dans un site web statique + [00:40:15][^8^][8] Exemples d'applications créées avec Observable * Présentation d'applications variées, telles que l'évolution des joueurs d'échecs et un tableau de bord d'hôtel * Conversion d'une application JavaScript existante en une version améliorée avec Observable

Résumé de la vidéo [00:00:00][^1^][1] - [03:28:51][^2^][2]:

Cette vidéo présente une conférence organisée par l'UNICEF France, axée sur l'adolescence et les défis associés. Elle met en lumière les expériences des adolescents, les perspectives des experts et les initiatives de soutien.

Points forts: + [00:00:00][^3^][3] Introduction de la conférence * Présentation par l'administrateur de l'IUF, Olivier Houdet * Discussion sur le rôle de l'IUF et son engagement envers l'Europe + [00:50:24][^4^][4] Budget et structure d'Horizon Europe * Explication des différents piliers et de la répartition budgétaire * Importance de l'intégration des pays périphériques dans la recherche européenne + [01:16:41][^5^][5] L'importance de la persévérance dans la recherche * Témoignages sur l'expérience de soumission de projets et l'apprentissage par l'échec * Encouragement à resoumettre les projets pour augmenter les chances de succès + [02:11:06][^6^][6] Financement et soutien des projets * Discussion sur les attentes spécifiques des projets et l'importance du soutien institutionnel * L'impact du financement public sur la compétitivité des appels à projets + [03:12:47][^7^][7] Choisir le bon moment pour soumettre un projet * Partage d'expériences personnelles sur la recherche de la bonne idée et du timing approprié * L'importance de l'innovation et de l'adaptation aux évolutions du domaine de recherche

Clara, pra ser sincera achei a introdução dela meio fraca... Muito bem, ela explica a comissão da Anistia, situando o leitor. Mas acho que falta um pequeno fechamento, dizendo do que se trata o artigo (na 1a frase da introdução isso aparece) e a importância de publicá-lo ou republicá-lo neste momento.

Acho que esta imagem, tendo em vista a boa imagem que Luiz arrumou para a abertura do artigo, deve ser colocada dentro do caso brasileiro, subindo com a imagem '300 Carlos' para este local.

Atenção gente. Há negritos por demais neste texto! Havia começado a marcar de baixo para cima, assinalar 'grifo da autora'. Mas são tantos, que me parece inoperante repetir a cada vez. Uma ou duas vezes ela própria inseriu 'grifo nosso'. Talvez o melhor seja pegar o primeiro deles e fazer uma nota dizendo que todos os grifos neste texto advêm da pluma da autora ou são de autoria de Eneá...

assinalar se é grifo da autora. Abaixo, em 'punidos', me parece que ela mesma assinalou (grifo nosso)

Assinalar o grifo dela

Assinalar, se grifo da autora

Se for grifo dela, é preciso assinalar

Não vamos usar negrito nas bibliografias, pois grita muito dentro do blog. Luiz usou itálico para alguns títulos de obras. Talvez seja melhor continuar e padronizar todos os textos da mesma forma. Também é preciso ter um padrão entre uma referência e outra. Está faltando a bio dela, com a foto.

Isso aqui faz parte da bio dela e não deve entrar no resumo do texto

Não vamos por palavras chave, sobretudo em inglês, pois não colocamos em outros textos do Coletivo

observational pov reveals speedy roles and movements endorsed by the economy

terrain imagery creates a sense of high economy

reminds me of people leaving stuff in the streets for the world of nyc to take for itself.

i look at this as an interpretation of how NYC is full of people from many different backgrounds and cultures and how everyone is very unique from an outside POV but a anemone gives the idea of only certain things fit in

large surroundings high freelance creations

Tropical environment

Perhaps it could be referring to New Yorkers as similar to bees almost like there's a hive mind where people share similar ideals, beliefs, or living situations.

this remind me of the phase " nyc is the city that never sleeps" because no matter what time of the night nyc is always full of life

this talk about how the city is not owned by the people but by a few.

nyc is so busy its not unlikely to hear traffic

this immediately brought my mind to Hudson yards, one of the biggest tourist attractions in nyc but when you look into these buildings they are sky high and empty. its like they are looking down at us with their eyes closed.

This can be interpreted as NYC is similar to a sea anemone because only the proper fish can use this as a habitat and achieve symbiosis. Could also compare New Yorkers as symbiotic with the environment. This could also be interpreted as New York is meant for those who belong?

this makes me think of how many different buildings there are all around NYC

in my opinion this term connects to NYC perfectly, a bunch of people look around and see so many different things and people, and the "keen-eyed" or very perceptive people can see one thing about someone and pick out what makes them unique in this huge city.

this makes me think how many different cultures are found in New York.

What deeper meanings may this hold?

this makes me think of the "NYC Dream" and how ive woken up from the said dream.

Introduction to the Perkins Brailler - Statewide Vision Resource Centre

https://en.wikipedia.org/wiki/Perkins_Brailler

Probably important justify the inaccurate nature of AI, that motivates this study of trust and verification

For our study, might also need to use a mixed design approach to mitigate learning effect.

Because it's about degree (on a continuum), it would usually be clearer (and therefore preferred) to specify whether, for instance, you mean "less preferred" or "least preferred". "dispreferred" is ambiguous in that regard: I had assumed it meant (was using it to mean) less preferred ( not the most preferred), but apparently others (https://languagelog.ldc.upenn.edu/nll/?p=2186) read it and see "least preferred".

Because it's neither a new word, nor an ugly word, any more than "distaste", "dislike" is an ugly word.

Referring to how "dis-" might imply it's the opposite.

I can see their point,which I think is that "To favor or prefer (something) less than the alternatives." simply makes it not your maximum preference (so in that sense, it would merely be the absence of the state of being the maximum), not necessarily your minimum (least favorite) rated/preferred choice.

But I think it can actually mean the opposite of preferring. To me, to disprefer something is nearly the same as if you show a distaste for something.

Accompagner les élèves empêchés d'apprendre avec Emmanuelle Piquet Le contenu de la page que vous consultez aborde le sujet délicat du suicide chez les adolescents et les répercussions sur les familles. Voici un résumé des points clés :

• Impact sur les familles : Le suicide d'un adolescent entraîne une profonde détresse chez les proches, qui doivent faire face à la perte et à ses conséquences émotionnelles complexes.
• Rôle des premiers répondants : Les interactions avec les premiers répondants, tels que les policiers et les pompiers, peuvent avoir un impact significatif sur l'expérience des familles lors de telles tragédies.
• Chercher un sens : Les familles cherchent souvent à comprendre les raisons derrière le suicide, bien qu'il soit difficile d'obtenir des réponses complètes.
• Soutien et guérison : Le soutien professionnel et familial est crucial pour la guérison. Les familles explorent différentes façons de se souvenir et d'honorer l'adolescent, tout en trouvant un sens à leur propre expérience.

Ces points soulignent l'importance d'un soutien empathique et d'une communication ouverte pour aider les familles à naviguer dans le processus de deuil.

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

Learn more at Review Commons

---

Reply to the reviewers

__Reviewer #1 (Evidence, reproducibility and clarity (Required)):_ _ __ In this manuscript, Jones et al. report on a potential role for fam83fa in zebrafish hatching, radiation response and autophagy. The authors are commended for generating multiple KO lines and maternal-zygotic embryos for analysis. However, important controls are lacking and the data is circumstantial throughout with very little mechanistic insight into the precise roles, if any, of fam83f in these processes.

We thank the reviewer for recognizing the strengths of our manuscript, and highlighting areas we might improve. Please see the specific comments below addressing the points raised. In respect of mechanistic insight, while we agree that our manuscript does not provide this, it was not intended to. Rather, we aim to communicate our descriptive findings on the role of Fam83fa in vivo, providing data for follow-up studies by other researchers into the mechanistic role of Fam83fa.

1. Validation of the KO phenotypes (hatching, IR sensitivity) requires rescue with WT fam83fa WT mRNA, but not 1-500 or fam83fb mRNA.

We thank the reviewer for raising the issue of rescue experiments. Such experiments are frequently used in knock-down experiments, where non-specificity may be a problem, but they are used more rarely in genetic knock-outs, where the gene defect is well defined. In the case of Fam83fa, a particular difficulty is that overexpression of fam83fa itself causes a p53-mediated DNA damage response (DDR) (Salama et al., 2019). Moreover, we have shown by both qRT-PCR and western blotting that injection of fam83fa mRNA into zebrafish embryos (the traditional technique by which rescue experiments are performed) induces a p53-mediated DDR. As a result, it would be very difficult to interpret the results of any rescue experiment, because one would have to be absolutely certain that levels of fam83fa re-expression recapitulate and do not exceed endogenous levels. As a tool for specificity, we therefore used more than one fam83fa-/- mutant line, carrying a different genomic mutation, and validated that the same phenotype was present in both. We are happy to provide the qRT-PCR and western blot data confirming the results of fam83fa mRNA injection, if required. We have included an additional section into the manuscript detailing this issue. 2.

While the hatching phenotype (Fig 3) is convincing, there is no data on HG development in the null embryos. Does the HG develop normally in the absence of fam83fb? If so, this would support the authors conclusions that the role of fam83fb is functional rather than developmental (indirect effect). In situs as in Fig.1 might be helpful here.

Thank you to the reviewer for this helpful suggestion. We agree that we did not investigate whether the hatching gland develops normally in the MZ-fam83fa-/- mutant embryos. No gross morphological differences were observed that led us to investigate this, although we agree it is an interesting question for a future project. In terms of functional vs developmental effects, we are confident that MZ-fam83fa-/- mutant embryos develop at a normal temporal rate, as evidenced by the machine learning based classifier used to assess temporal developmental trajectory (Figure S3 and Jones et al., 2022, 2024). This strongly suggests that the effect of fam83fa KO is functional rather than indirect and caused by (for example) developmental delay.

While the IR sensitivity phenotype (Fig S4) is convincing, IR-induced cell death/apoptosis was not analyzed. There is a large literature describing straightforward assays for cell death/apoptosis detection in zebrafish with assays such as acridine orange or TUNEL labeling, or active casp3 whole-mount IF. Is IR-induced cell death enhanced in fam83fa KOs?

We thank the reviewer for their positive comments and agree that investigating the nature of the cell death occurring following IR would be very interesting. We did make use of both acridine orange and TUNEL labeling following injection of fam83fa mRNA (see 1 above), and whilst the assays themselves were relatively straightforward, due to technical issues the quantification of fluorescence intensity was not. Similarly, we suspect that a significant degree of necrosis is also occurring, which further complicates the issue of data interpretation from both these approaches. We do, however, think this is an important avenue of questioning, and hope that other researchers will explore the mechanism of IR induced cell death in the MZ-fam83fa-/- mutants in the future,

Similarly, there are multiple tools to assay autophagy in zebrafish (e.g., Moss et al., Histochem Cell Biol 2020, PMC7609422; Mathai et al., Cells 2017, PMC5617967). Is autophagy affected in the KOs, with or without IR? These experiments might directly implicate fam83fa in autophagy.

We agree that there are exciting tools with which to assay autophagy in zebrafish, and although we considered some of these, including caudal fin regeneration, we deemed these experiments to be beyond the descriptive scope of this paper, given the time and resources available to us. We hope that other researchers will use our data as a basis for investigating the role of Fam83fa in autophagy further, using assays such as these suggested by the reviewer.

Figure 4: Isn't there a slight reduction in p53 induction at 10 hours?

Although the western blot in Figure 4A gives this impression, this is probably due to loading variability (see the anti-β-actin loading control band). Moreover, over three independent experiments (Figure 4B), this apparent difference is not statistically significant. Taken together with other evidence that the p53-mediated DNA damage response is not affected in MZ-fam83fa-/- mutants, we are confident there is no detectable change in the level of stabilized p53 in the MZ-fam83fa-/- mutants compared to WT.

Given the widely documented, dominant role of p53 in zebrafish IR-sensitivity, the authors should test if the IR sensitivity of fam83fa KO animals is p53-dependent, ideally via a cross into p53 null, but at least via injection of p53 morpholinos.

We agree that p53 is widely documented as playing an essential role in the IR induced DNA damage response in zebrafish. All our experiments suggest there is no difference between the levels of p53 (protein or mRNA) or any of the p53-induced downstream effectors (that we tested) in MZ-fam83fa-/- mutants compared to WT embryos. This was true whether or not the embryos were subjected to genotoxic stressors, including IR treatment. We therefore conclude that the increased sensitivity phenotype we observe as a result of loss of Fam83fa is not caused by a change in p53 activity, at least not as part of the DNA damage response.

Do autophagy inhibitors phenocopy the hatching and IR-sensitivity defects of fam83fa embryos? Do the inhibitors exacerbate the mutant phenotypes or synergize with M or Z mutant phenotypes? (I may have missed this but do M and Z fam83fa null embryos have any phenotype? Or do the phenotypes only manifest in MZ embryos?)

This is an excellent question, and indeed one we attempted to address. We tried to optimize several autophagy inhibitors including bafilomycin A1, chloroquine and wortmannin, as well as the proteasomal inhibitor MG132. In addition, we tried to optimize the autophagy promoters Torin1 and rapamycin. Unfortunately, we regularly saw global effects in zebrafish embryos that were difficult to characterize and control by dosage. At the same time, we were also working to confirm the specific effects of these drugs on autophagy using p62 and LC3-I and LC3-II western blots, which themselves were difficult to optimize. We attempted to optimize these experiments for 6 months before the COVID lockdown occurred, at which point they were abandoned. We would be delighted for future researchers to continue these experiments, as we are now unable to pursue this further due to closure of the Smith lab, but we agree that these are very pertinent questions. We hope the descriptive data provided in our paper will prompt other researchers in the autophagy field to further explore the role of Fam83fa in autophagy. In response to the zygotic phenotype question, this was something we did not investigate. As there was no immediately apparent phenotype in the zygotic generation, for ease of screening larger numbers of embryos we proceeded immediately to the maternal-zygotic (MZ) generation.

Reviewer #1 (Significance (Required)):

The role of Fam83f is not known. This study in zebrafish might be the first to clarify the function of this protein in vivo.

We thank the reviewer for this positive insight, and we agree that our work is the first do so in vivo.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Fam83f is one of the proteins about which little is known. The authors Jones et al., tried to shed light on Fam83f function by knocking out the gene in zebrafish. Here they found that fam83 is expressed in the hatching gland and that larvae without Fam83f hatch significantly earlier than wild-type animals. The authors furthermore investigated the response of fam83f knock-out animals to DNA damage and found increased sensitivity to ionizing radiation and MMS. In order to find out more about Fam83f function in the DNA damage response, the authors performed RNA-seq after employing DNA damage and here they saw upregulation of several autophagy/lysosome-associated proteins and downregulation of some phosphatidylinositol-3-phosphate binding proteins, among others. Finally, the authors found that Fam83f is targeted to the lysosome. The manuscript is overall well written and clear in its general statement.

We thank the reviewer for their encouraging comments.

In the manuscript, the authors describe the investigation of several aspects of Fam83f function and particularly the role in hatching seems to be important for Fam83f as the gene is strongly expressed in the hatching gland and its absence leads to a clear and considerable earlier hatching. Unfortunately, all aspects of Fam83f function that are described in the manuscript are investigated very superficially, the conclusions are not supported by data and important controls are lacking. As such, the RNA-seq results are not confirmed by qRT-PCR, the role of the Fam83f LIR domain is not confirmed by co-IPs and it has not been investigated whether the presence of Fam83f in lysosomes is due to its degradation or whether it has a function in this cellular compartment.

We thank the reviewer for their input and will address each point raised below: -

• All aspects of Fam83f function are investigated superficially.

We agree that we have not provided an in-depth analysis of the mechanistic role of Fam83fa. It was because there were so many roles that we decided to make this paper rather descriptive in nature, hoping that the observations will prove useful to other researchers who may wish to define the mechanistic roles of Fam83fa more deeply. Even without in-depth investigation, our findings are previously unreported and the phenotypes we report are clear. We have amended our manuscript to make it apparent that this paper is intended to be descriptive in nature, and we hope this addresses this issue.

• Important controls are lacking - RNA-seq results are not confirmed by qRT-PCR

We thank the reviewer for their comment. We did not include qRT-PCR data as a control for the RNA-seq data because 1) each RNA-seq experiment was repeated on three biological replicates across three independent experiments and 2) we conducted RNA-seq on two different MZ-fam83fa-/- mutant lines and only considered genes that were mis-regulated in both mutants. Taken together, we considered this to be sufficient validation for the manuscript. However, we also performed confirmatory qRT-PCR for several of the differentially expressed genes identified, including the three main PI(3)P binding genes. We have now included these data in the supplementary information as an additional control - see Figure S6G which is now also referred to in the main text, and additional primer sequences have been added to Table S1.

• The role of the Fam83f LIR domain is not confirmed by co-Ips

We agree with the reviewer that this is an important experiment, and we worked closely with Dr Brian Ludwig and Dr Karen Vousden (The Francis Crick Institute) to test this. We tried to express zebrafish Atg8 and Gabarap (the two main ATG8 proteins that bind to LIR domains) but were unable to express sufficient levels of protein to perform the co-Ips. The text in the manuscript has now been amended to reflect that this experiment is required to confirm the role of the putative LIR domain in Fam83fa.

• *it has not been investigated whether the presence of Fam83f in lysosomes is due to its degradation or whether it has a function in this cellular compartment *

Whilst we agree with the reviewer that this is an important question, we did not intend this paper to expand beyond a descriptive role of the observations we made following the loss of Fam83fa in vivo. These are important questions to follow up on to determine the mechanism of action of Fam83fa, and we hope that other researchers will pursue these avenues of investigation following the publication of our observations.

Also, there is no leading concept in the manuscript. Starting from a role in hatching, the authors go to the DNA damage response and finally to the presence of Fam83f in lysosomes. How are these different aspects linked? Is the presence of Fam83f in lysosomes important for the suppression of hatching and how does Fam83f delays this process? (One would have wished that the authors would not have been that broad and were more focused on a particular aspect which then could have been investigated in depth.)

We agree with the reviewer that the paper gives a broad overview of our observations and does not examine the underlying mechanisms in detail. However, we believe that descriptive papers such as this, where observations following genetic perturbation are reported, are equally important, providing as they do important foundational data for other researchers to take forward. We do postulate on the links between the hatching, DNA damage and lysosomal phenotypes we observe in the discussion section, and we have expanded on this following the reviewers' comments, to make our hypothesized link between these phenomena clearer.

Specific comments: - All materials should be described in material and methods including the antibodies that have been used

The antibodies used together with concentrations and catalog numbers are now in Materials and Methods

• Abbreviations should be explained

The manuscript has been revised to ensure all abbreviations are explained. We thank the reviewer for bringing this oversight to our attention.

• Figure 4A: Levels of p53 should also be shown for untreated fam83f -/-KO1 and KO2 animals

The authors thank the reviewers for raising this point. Extracts from untreated MZ-fam83fa-/- KO1 and KO2 embryos were not included on this particular blot, as p53 was observed to be undetectable in all embryos, across all our experiments (WT and both mutants) unless genotoxic stress was applied. No quantification could therefore be performed as the expression level was essentially zero. However, we have now included an example p53 western blot in Supplemental Figure 5A, which shows WT, MZ-fam83fa-/- KO1 and MZ-fam83fa-/- KO2 untreated blots for p53 (all undetectable) alongside treated embryos (detected).

• Some references are missing (e.g. page 17, lane 320/321: As this group of cells arises....)

This citation and reference have now been added; thank you to the reviewer for highlighting this omission.

• Lane 369: The authors write about 4 KO lines but only two are shown in the figure.

We thank the reviewer for this observation. In Figure 2B only KO1 and KO2 schematic diagrams are shown for simplicity (as these are the lines taken forward for further investigation). We have now amended the manuscript text to make this clear.

• Lane 374/375: The NMD is not proven

Absolutely - we have now revised the text to change this sentence accordingly and thank the reviewer for noting this.

• Lane 380: how can RNA levels of fam83fa be upregulated when the gene has been knocked out? Why are these genes only upregulated in KO1? How relevant is this?

This was a typographical error, and we are very grateful to the reviewer for picking up on this. It should have read 'fam83fb'. As nonsense-mediated decay and associated transcriptional adaptation have been previously reported in zebrafish, this finding may be of considerable interest to the community. It is a side observation, and not necessarily directly related to the role of Fam83fa in vivo, but we felt it important to include. Indeed, as a result of this observation we have recently shared our MZ-fam83fa-/- lines with another group who are planning to investigate precisely this question - why are fam83fb and fam83g only upregulated in KO1?

• Figure 3C is not mentioned in the text and lacks any labelling

Figure 3C is now clearly referred to in the text and a label added to the figure.

• Lane 434/435: all relevant data should be shown (can be done as supplementary figure)

We have now amended this to include an additional supplemental figure (Figure S5A).

• Lane 434: The reference to the figure seems to be incorrect (5A4A)

Amended accordingly - thank you for pointing out this mistake.

• Figure 4C and 4D: what is the difference?

Thank you to the reviewer for noticing this omission. These data are from t1 (+2hrs) and t2 (+10hrs) and have now been labelled accordingly.

• S5C and S5D: why are there 3 clusters?

We thank the reviewer for raising this as it has provided us with an opportunity to present our data more clearly. There are 3 clusters that represent the combination of the two first principal components, which are time and treatment. Therefore, the clusters represent i) untreated at t1, ii) treated at t1 and iii) treated at t2. However, having two plots with different color schemes made this confusing/misleading. We have now replaced the two PCA plots with one that is colored and labelled accordingly with the 3 aforementioned clusters.

• Lane 495 to 505: What does this mean that the GO analysis shows upregulation and downregulation of endopeptidases and why "in contrast"?

We thank the reviewer for this comment, and we agree that this paragraph was misleading/confusing. This has now been rewritten in the main text, clarifying that endopeptidases were consistently upregulated at both timepoints.

Reviewer #2 (Significance (Required)):

The strength of the manuscript is certainly that it provides inside into Fam83f function as there is not much known about Fam83f.

We thank the reviewer for the positive comment, and we agree that very little is known about this highly conserved protein.

These study is probably most interesting for people in the zebrafish and related fields as the authors convincingly show the expression of Fam83f in the hatching gland and also the earlier hatching in the absence of the protein is very clear.

Thank you for the positive feedback.

The weakness of the study is clearly that it does not provide an in-depth analysis. As such, it shows that Fam83f is involved in hatching and can delay the process but it remains elusive how this is achieved. (Likwise, also the investigation into the DNA damage response remains very superficial and does not prove a specific role for Fam83f in the DNA damage response or whether the increased sensitivity is more unspecifically caused by the absence of a gene or eventually even connected to the earlier hatching.

Please refer to responses above (and changes made to the manuscript) clarifying that this study is intended to be descriptive, and provides important foundational data for further in-depth mechanistic studies by other researchers interested in the role of Fam83fa in vivo.

__Reviewer #3 (Evidence, reproducibility and clarity (Required)):_ _ __ In their manuscript "Zebrafish reveal new roles for Fam83f in hatching and the DNA damage-mediated autophagic response", Jones et al. provide an interesting exploration for the function of a poorly studied protein, Fam83f in embryonic development. Using the zebrafish as a model organism, the study combines loss-of-function genetics, phenotypic analysis and RNA-sequencing to characterize and explore the result of Fam83f loss. Upon critical review of the manuscript and the results we offer suggestions to improve the manuscript (see 'minor technical issues'). Additionally, we would like to highlight a weakness of the study in making the connection between Fam83f to the observed phenotype (increased sensitivity to DNA damage), see 'major issues'.

Major issues:

Most of our concern stems from relatively incomplete connection of the loss of fam83f to increased sensitivity to DNA-damage and lysosome function.

Please refer to comments above and changes made to the manuscript to clarify this is a descriptive paper that is not intended to provide in-depth mechanistic insight into the role of Fam83fa.

Is the increased sensitivity in fam83f KO embryos a direct effect to fam83f loss? A rescue experiment (by introduction of Fam83fa mRNA into their KO2 fish line) in the presence of ionizing radiation would help us understand the functional role of this protein in this process. Furthermore, can overexpression of any of the down-regulated genes involved in lysosome function restore the early hatching phenotype or the sensitivity to DNA damage? Fam83fa rescue experiments would be very difficult to interpret - please see comments above and the corresponding changes to our manuscript.

In terms of over-expressing some of the downregulated genes identified in the RNA-seq and qRT-PCR to see if the phenotype can be rescued, we feel these are excellent suggestions and we hope other researchers in future will attempt such experiments.

Minor technical issues:

-Methods line 203, clarify how many embryos were used per sample for RNA-seq (this was only described as 15 embryos in the main body results text).

Text has been amended to clarify this. We thank the reviewer for noticing this oversight.

-Comment about the expansion of fam83f orthologs in mammals (8) as opposed to only 2 in zebrafish

We apologize for any confusion: mammals do not have 8 fam83f orthologs. Mammals and zebrafish have 8 FAM83 genes (FAM83A-FAM83H). Zebrafish, unlike mammals, have genome duplication and although mammals have only one FAM83F gene, zebrafish have two: Fam83fa and Fam83fb. We trust this clarifies this issue and believe this to be clear in our main text. However, we are happy to make any suggested amendments should the reviewer consider our wording confusing.

-Supplementary figure 1C: please include representative images of secondary axis formation in fam83fa overexpressed Xenopus embryos.

We have not included any images as these are already published in our related paper on FAM83F (Dunbar et al., 2020) which we refer to in the figure legend text. No additional images were captured specifically for this publication.

-Provide more information about the mis-regulated genes in the RNA-seq analysis, how many are up or down regulated? Perhaps a better plot than a Venn diagram can be an MA-plot with the Venn diagram moved to a supplementary figure.

The Venn diagrams in Figure 5A-C are to illustrate the number of differentially expressed genes that are shared between KO1 and KO2 (whether up or down regulated), and only those that are common to both lines are taken forward. Following the reviewer's comments, we have now displayed the behavior of the common genes across all replicates in one heatmap, with the data normalized to the WT untreated samples, and the normalized variance stabilized count indicates whether a gene is up or down regulated across each of the replicates and conditions. We believe this addresses the reviewer's comment as these data are now displayed in a more direct way and the genes that are consistently up or downregulated across all replicates (and indeed those that are not) can be clearly seen. We thank the reviewer for raising this and improving our data representation.

-A better comparison of mis-regulated genes in the fam83f knockouts would be a comparison of KO2 and perhaps KO3, as the compensatory effects in KO1 can lead to additional indirect effect on the transcriptome. We understand the time and cost involved in this experiment and suggest that the differential gene expression analysis be performed individually on up or down regulated genes from KO2, or a comparison of such analysis will be provided with the differential gene expression analysis that was performed on shared mis-regulated genes between KO1 and KO2.

The reviewer raises an excellent point. At the time of experimental design, we were concerned that omitting KO1 in favor of another line (e.g. KO3) would bias our results by excluding potentially important data. Similarly, as transcriptional adaptation occurs in a sequence specific manner, and the phenotype was present in KO1 regardless, we didn't want to exclude these data. However, with hindsight, we agree that it may have been prudent to exclude KO1 on this basis, and we may have seen an increased concordance of differentially expressed genes (DEGs) between KO2 and KO3. However, this is not possible to repeat now due to the Smith lab closing, and our documented findings are valid and important regardless. We acknowledge however that, with hindsight, what the reviewer suggests may have been better experimental design.

-Can you confirm with the RNA-seq analysis that fam83g is upregulated in KO1 as opposed to KO2? (i.e. can the compensatory analysis you have observed with qRT-PCR be confirmed with the RNA-seq data?)

This is an excellent question, and we thank the reviewer for raising this. fam83fb passed our threshold for significance to be deemed as differentially expressed (upregulated) in KO1 only, in accordance with our qRT-PCR data. fam83g did not pass the significance threshold, but perhaps this is not surprising as both fam83fb and fam83g are expressed at particularly low levels to start with and would probably require much greater sequencing depth to be detected.

Reviewer #3 (Significance (Required)):

There is fundamental value in clarifying the in vivo function of poorly characterized protein-coding genes. This study fills a gap in the literature, but the broader conceptual impact is limited. The authors do a thorough job at generating and characterizing CRISPR/Cas9 mediated knock-out zebrafish animals. It is further commended that the authors do a meticulous job in a quantitative description of the resulting phenotype. This is a thorough study, with the only major concern being the lack of rescue experiments that would be needed to substantiate the the role of fam83f in sensitivity to DNA damage and lysosome function.

We thank the reviewer for their comments and trust we have addressed the issues concerned with the changes described above.

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

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

In their manuscript "Zebrafish reveal new roles for Fam83f in hatching and the DNA damage-mediated autophagic response", Jones et al. provide an interesting exploration for the function of a poorly studied protein, Fam83f in embryonic development. Using the zebrafish as a model organism, the study combines loss-of-function genetics, phenotypic analysis and RNA-sequencing to characterize and explore the result of Fam83f loss. Upon critical review of the manuscript and the results we offer suggestions to improve the manuscript (see 'minor technical issues'). Additionally, we would like to highlight a weakness of the study in making the connection between Fam83f to the observed phenotype (increased sensitivity to DNA damage), see 'major issues'.

Major issues:

Most of our concern stems from relatively incomplete connection of the loss of fam83f to increased sensitivity to DNA-damage and lysosome function.

Is the increased sensitivity in fam83f KO embryos a direct effect to fam83f loss? A rescue experiment (by introduction of Fam83fa mRNA into their KO2 fish line) in the presence of ionizing radiation would help us understand the functional role of this protein in this process. Furthermore, can overexpression of any of the down-regulated genes involved in lysosome function restore the early hatching phenotype or the sensitivity to DNA damage?

Minor technical issues:

• Methods line 203, clarify how many embryos were used per sample for RNA-seq (this was only described as 15 embryos in the main body results text).
• Comment about the expansion of fam83f orthologs in mammals (8) as opposed to only 2 in zebrafish
• Supplementary figure 1C: please include representative images of secondary axis formation in fam83fa overexpressed Xenopus embryos.
• Provide more information about the mis-regulated genes in the RNA-seq analysis, how many are up or down regulated? Perhaps a better plot than a Venn diagram can be an MA-plot with the Venn diagram moved to a supplementary figure.
• A better comparison of mis-regulated genes in the fam83f knockouts would be a comparison of KO2 and perhaps KO3, as the compensatory effects in KO1 can lead to additional indirect effect on the transcriptome. We understand the time and cost involved in this experiment and suggest that the differential gene expression analysis be performed individually on up or down regulated genes from KO2, or a comparison of such analysis will be provided with the differential gene expression analysis that was performed on shared mis-regulated genes between KO1 and KO2.
• Can you confirm with the RNA-seq analysis that fam83g is upregulated in KO1 as opposed to KO2? (i.e. can the compensatory analysis you have observed with qRT-PCR be confirmed with the RNA-seq data?)

Significance

There is fundamental value in clarifying the in vivo function of poorly characterized protein-coding genes. This study fills a gap in the literature, but the broader conceptual impact is limited. The authors do a thorough job at generating and characterizing CRISPR/Cas9 mediated knock-out zebrafish animals. It is further commended that the authors do a meticulous job in a quantitative description of the resulting phenotype. This is a thorough study, with the only major concern being the lack of rescue experiments that would be needed to substantiate the the role of fam83f in sensitivity to DNA damage and lysosome function.

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

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

Fam83f is one of the proteins about which little is known. The authors Jones et al., tried to shed light on Fam83f function by knocking out the gene in zebrafish. Here they found that fam83 is expressed in the hatching gland and that larvae without Fam83f hatch significantly earlier than wild-type animals. The authors furthermore investigated the response of fam83f knock-out animals to DNA damage and found increased sensitivity to ionizing radiation and MMS. In order to find out more about Fam83f function in the DNA damage response, the authors performed RNA-seq after employing DNA damage and here they saw upregulation of several autophagy/lysosome-associated proteins and downregulation of some phosphatidylinositol-3-phosphate binding proteins, among others. Finally, the authors found that Fam83f is targeted to the lysosome. The manuscript is overall well written and clear in its general statement. In the manuscript, the authors describe the investigation of several aspects of Fam83f function and particularly the role in hatching seems to be important for Fam83f as the gene is strongly expressed in the hatching gland and its absence leads to a clear and considerable earlier hatching. Unfortunately, all aspects of Fam83f function that are described in the manuscript are investigated very superficially, the conclusions are not supported by data and important controls are lacking. As such, the RNA-seq results are not confirmed by qRT-PCR, the role of the Fam83f LIR domain is not confirmed by co-IPs and it has not been investigated whether the presence of Fam83f in lysosomes is due to its degradation or whether it has a function in this cellular compartment. Also, there is no leading concept in the manuscript. Starting from a role in hatching, the authors go to the DNA damage response and finally to the presence of Fam83f in lysosomes. How are these different aspects linked? Is the presence of Fam83f in lysosomes important for the suppression of hatching and how does Fam83f delays this process? (One would have wished that the authors would not have been that broad and were more focused on a particular aspect which then could have been investigated in depth.)

Specific comments:

• All materials should be described in material and methods including the antibodies that have been used
• Abbreviations should be explained
• Figure 4A: Levels of p53 should also be shown for untreated fam83f -/-KO1 and KO2 animals
• Some references are missing (e.g. page 17, lane 320/321: As this group of cells arises....)
• Lane 369: The authors write about 4 KO lines but only two are shown in the figure.
• Lane 374/375: The NMD is not proven
• Lane 380: how can RNA levels of fam83fa be upregulated when the gene has been knocked out? Why are these genes only upregulated in KO1? How relevant is this?
• Figure 3C is not mentioned in the text and lacks any labelling
• Lane 434/435: all relevant data should be shown (can be done as supplementary figure)
• Lane 434: The reference to the figure seems to be incorrect (5A<->4A)
• Figure 4C and 4D: what is the difference?
• S5C and S5D: why are there 3 clusters?
• Lane 495 to 505: What does this mean that the GO analysis shows upregulation and downregulation of endopeptidases and why "in contrast"?

Significance

The strength of the manuscript is certainly that it provides inside into Fam83f function as there is not much known about Fam83f.

These study is probably most interesting for people in the zebrafish and related fields as the authors convincingly show the expression of Fam83f in the hatching gland and also the earlier hatching in the absence of the protein is very clear.

The weakness of the study is clearly that it does not provide an in-depth analysis. As such, it shows that Fam83f is involved in hatching and can delay the process but it remains elusive how this is achieved. (Likwise, also the investigation into the DNA damage response remains very superficial and does not prove a specific role for Fam83f in the DNA damage response or whether the increased sensitivity is more unspecifically caused by the absence of a gene or eventually even connected to the earlier hatching.

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

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

In this manuscript, Jones et al. report on a potential role for fam83fa in zebrafish hatching, radiation response and autophagy. The authors are commended for generating multiple KO lines and maternal-zygotic embryos for analysis. However, important controls are lacking and the data is circumstantial throughout with very little mechanistic insight into the precise roles, if any, of fam83f in these processes.

1. Validation of the KO phenotypes (hatching, IR sensitivity) requires rescue with WT fam83fa WT mRNA, but not 1-500 or fam83fb mRNA.
2. While the hatching phenotype (Fig 3) is convincing, there is no data on HG development in the null embryos. Does the HG develop normally in the absence of fam83fb? If so, this would support the authors conclusions that the role of fam83fb is functional rather than developmental (indirect effect). In situs as in Fig.1 might be helpful here.
3. While the IR sensitivity phenotype (Fig S4) is convincing, IR-induced cell death/apoptosis was not analyzed. There is a large literature describing straightforward assays for cell death/apoptosis detection in zebrafish with assays such as acridine orange or TUNEL labeling, or active casp3 whole-mount IF. Is IR-induced cell death enhanced in fam83fa KOs?
4. Similarly, there are multiple tools to assay autophagy in zebrafish (e.g., Moss et al., Histochem Cell Biol 2020, PMC7609422; Mathai et al., Cells 2017, PMC5617967). Is autophagy affected in the KOs, with or without IR? These experiments might directly implicate fam83fa in autophagy.
5. Figure 4: Isn't there a slight reduction in p53 induction at 10 hours?
6. Given the widely documented, dominant role of p53 in zebrafish IR-sensitivity, the authors should test if the IR sensitivity of fam83fa KO animals is p53-dependent, ideally via a cross into p53 null, but at least via injection of p53 morpholinos.
7. Do autophagy inhibitors phenocopy the hatching and IR-sensitivity defects of fam83fa embryos? Do the inhibitors exacerbate the mutant phenotypes or synergize with M or Z mutant phenotypes? (I may have missed this but do M and Z fam83fa null embryos have any phenotype? Or do the phenotypes only manifest in MZ embryos?)

Significance

The role of Fam83f is not known. This study in zebrafish might be the first to clarify the function of this protein in vivo.

Based on the 1962 publication date of Pale Fire, it's a leading contender for the project Nabokov might have been working on during his photo session with Carl Mydans for LIFE Magazine in 1958.

joe is gone

This is what I was looking for - "you have obtained appropriate licenses...to use the Processed Documents..."

e

to believe i sprokened to call dis an AESOP miracle just for a second; as if the Valdeez and Yuan had found Arabian Coffers spilled all over the Carribean Galactica ... or something

maybe didn't... and Shakespeare ties that same story of some kind of ... betrayal ... to his famous words and those words somehow in my mind directly link to the kiss of Judas. Another table, another era; and one less "mesa" ... but again here we are staring at what is supposed to be an obvious and clear "technical revolution" of the progress of democracy--one that's clearly been stalled and halted by things like "not amending the Constitution properly" (that's according to me, and that's before really seeing you) ... and then here even in a place where we can acknowledge that those laws are archaic and backwards and not "up to speed" with the current needs of a technocratic civilization, we still fail to do anything about "voting for ideas over people" something he once said was a victory--or about the world's software degrading to something less than even remotely "unclear" ... communication itself is clearly being lost, and it's clearly something to do with "censorship" and something to do with "a secret" and something to do with ...

If I could tell you, I really can't discerne what exactly it is that Dave says in his songs stands between "what we see" and "what we do."

[a chanukah miracle!]

---

To be standing here and having an actual well schooled and intelligent rabbi trying to rewrite the meaning of the word "anarchy" ... in order to promote it; to say it's something other than ... "anarchy" that's the crux of what I see--a world that just wants to skip ahead, to fast forward through all the work and the struggle of actually rebuilding or building something that works; to "assuming" the system that allowed for this place to be ... so poorly managed, that it must be everything it says or shows or makes believe it truly isn't capable of ... "emancipation" of something that doesn't like the word water, and doesn't like the idea of being masses, or massless; and on top of all that doesn't even want to put it's two cents in--other than to say, I'm with Ivan or I'm with Taylor or "long live Bianca" and through it all, words like "now that you're gone, I can finally step up and move along" ...

Back to the sky; I suppose ... is what it is that the "river" means to Taylor, and I just wonder in our song what's left here--if I was wrong to assume our system wouldn't have allowed the mass slavery of entire civilization and to believe that your "brains" must be simulated ... as in "not actually here" but rather there--or just to assume you wouldn't be so damned hubratically sure that whatever you are ... it's so far above and so far advanced of the "things you pretend to be here" that you can just disregard the mass enslavement of them or yourselves or whatever it truly is; to look around and show you in this place, it's clear we care more for animals than for each other, and it's surely obvious that in the grand scheme---were we not so wrong here--we might have been right about "being so much more advanced." It's that leap to something so much smarter and so much ... more capable; like growing fingers and exiting the womb, that's the kind of experience and the magical leap forward I envision the ascension and the singularity to be--except here we are,

NEGA-OIL: in a symphonic creschendo straight out of Carnegie's "Old Bailey" ...

` "Voilà! In view, a humble vaudevillian veteran cast vicariously as both victim and villain by the vicissitudes of Fate. This visage, no mere veneer of vanity, is a vestige of the vox populi, now vacant, vanished. However, this valorous visitation of a bygone vexation stands vivified, and has vowed to vanquish these venal and virulent vermin vanguarding vice and vouchsafing the violently vicious and voracious violation of volition!

The only verdict is vengeance; a vendetta, held as a votive not in vain, for the value and veracity of such shall one day vindicate the vigilant and the virtuous.

Verily, this vichyssoise of verbiage veers most verbose, so let me simply add that it's my very good honor to meet you and you may call me V." ` https://bookroo.com/quotes/v-for-vendetta

Test typing and adjustments

• Ring and Cylinder adjustment (distance of platen from typeface; the type shouldn't touch the platen or you'll find you're imprinting on your paper, making holes in the paper and/or ribbon, which isn't good) [Interestingly Dul doesn't mention this particular adjustment]
• evenness of the letters (on feet)
• alignment between capital and lower case (motion)
• margin consistency
• line scale height
• adjust escapement "so that it's moving the proper distance both up and down to prevent skipping or missing spaces"
• adjust the height of the ribbon, this is done to make sure the entire letter is printed (and presumably so the bichrome is properly usable when available)

How A Rusty 1930s Royal Typewriter Is Professionally Restored | Refurbished | Insider

Done by Lucas Dul. Some particularly interesting portions on adjustments after restoration. He generally touches on the order of adjustments he makes, but in brief rather than completely.

example hypothesis annotation

By default this runs the lawlibrary site.

To run another site (e.g., open_by_laws, change the os.environ.setdefault("DJANGO_SETTINGS_MODULE", "lawlibrary.settings") line in manage.py.

nice

huh

what the context .. context define by goal ?

Context switch can lead to Leads to decision fatigue

---

‍No. Multitasking actually does more bad to productivity than good.

This is really good but do not have time for it

Richard M.Ryan: Is Human Autonomy a Western Ideology or a Basic Need?

The Internet is generally considered as a global technological system of networked computer networks, as the network of networks working with TCP/IP. Such definitions see the Internet as a purely technological system, they forget that knowledgeable human activities make the Internet work, the technological structure can't be separated from its human use and the permanent creation and communication of meaningful information through the Internet. The technical process of data transmission in the Internet known as routing is a mechanistic one. Self-organizing systems involve certain degrees of freedom, chance, irreducibility, unpredictability, and indeterminacy, hence when considering the Internet a purely technological system, it can't be characterized as self-organizing. Social self-organization is a self-referential, mutual process where structural media and human actions produce each other. The Internet is a global socio-technological system that is based on a technological structure consisting of networked computer networks that works with the help of the TCP/IP protocol and stores objectified human knowledge, human actors permanently re-create this global knowledge storage mechanism by producing new informational content, communicating in the system, and consuming existing informational content in the system; the technological infrastructure enables and constrains human communication. The Internet consists of both a technological infrastructure and communicating human actors. Together these two parts form a socio-technological system, the technological structure functions as a structural mass medium that produces and reproduces networked communicative actions and is itself produced and reproduced by communicative actions. The technical structure is medium and outcome of human agency, it enables and constrains human activity and thinking and is the result of productive social communication processes. Important qualities that are connected with the Internet as a sociotechnological system are Open Source, Virtual Reality, globalization, and many-to-many dialogue. Traditional mass media have been based on one-to-many-communication, whereas the Internet is based on many-to-many-communication. Hence the Internet has a large intrinsic democratic potential. In the terminology of Vilem Flusser it can be said that it could support a shift from discursive media society to dialogic media society.

By whom?

Author checks and approves proofs.

And revised manuscript.

What preparation do we anticipate?

maybe mention accessibility here?

(of an action) carried out without real interest, feeling, or effort: he gave a perfunctory nod

demand or specify (a requirement), typically as part of an agreement: he stipulated certain conditions before their marriage

The phrase "have got to do something" is an informal way of saying "must do something" or "need to do something."

a lawyer who takes claims or disputes to a law court

quick and light in movement or action

warn or reprimand someone firmly

quarrel noisily over a trivial matter

anxiety or fear that something bad or unpleasant will happen

| ɪnˈdʌɪtm(ə)nt |

noun

mainly North American English a formal charge or accusation of a serious crime: an indictment for conspiracy.

a word or phrase that is not formal or literary and is used in ordinary or familiar conversation

interpret (a person's words or actions) wrongly: my advice was deliberately misconstrued

destroy or ruin (a plan or project)

| kəˈləʊkwɪəl |

adjective

(of language) used in ordinary or familiar conversation; not formal or literary: colloquial and everyday language | colloquial phrases.

a formal statement by or on behalf of a defendant or prisoner, stating guilt or innocence in response to a charge, offering an allegation of fact, or claiming that a point of law should apply

accustomed to particular conditions; experienced

subject (someone) to trial

proceeding in a convoluted or undirected fashion

convolute | ˈkɒnvəluːt |

verb [with object]

make (an argument, story, etc.) complex and difficult to follow.

a member of a jury

remove from employment or office, typically on the grounds of unsatisfactory performance: the prime minister dismissed five members of his cabinet.

| əˈtəːni |

noun (plural attorneys)

mainly US English a lawyer

have a private discussion; confer

carry out or commit (a harmful, illegal, or immoral act): a crime has been perpetrated against a sovereign state.

For instructional faculty who are receiving an initial secondary appointment that includes compensation, the FAS Dean's Office will typically send a letter that the candidate will countersign and return. The FAS Dean's Office will circulate the final version of the letter to the secondary department for processing.

For instructional faculty being reappointed, the FAS Dean's Office will return the Secondary Appointment Form (and, if applicable, the faculty compensation approval form) to the secondary department for processing.

For all ladder faculty, the FAS Dean’s Office will return the Secondary Appointment Form (and, if applicable, the faculty compensation approval form) to the secondary department for processing.

Secondary Appointment Form with approvals from the primary department chair or school dean

If an initial appointment: Candidate CV

Not clear what this means.

maybe co-something? collaborating, cooperating, cowriting. Co-writing the classroom? I don't hate shifting the narrative but I worry that it is too value neutral.

subverts?

https://www.nytimes.com/2024/06/24/world/asia/china-flooding-landslides-weather.html

https://nautil.us/its-time-to-take-the-gaia-hypothesis-seriously-236490/

This is okay, and I would be in favor of making it even stronger, Also, I think it is more likely that people would want to use it for synthesis, rather than research. For example, giving it a chapter and asking it to write the learning objectives.

I love this list and agree with everything in it, but I think it should be a separate document.

This article provides an overview of the issues related to missing data in research in higher education. The authors point out that much of the literature on handling missing data is in the fields of psychology and sociology and the majority of research in higher education does not mention methods for dealing with missing data.

The authors suggest that traditional methods of listwise deletion, pairwise deletion, imputation, and dummy-variable adjustment are all inadequate as they lead to biased results that underestimate the variance in parameters and standard errors.

They suggest that maximum likelihood (ML) and multiple imputation (MI) be used whenever possible to provide more accurate estimates of the population. If possible, Full-Information Maximum-Likelihood (FIML) procedures should be used if interaction variables are of interest. The authors recommend that all studies use MI as the default method of dealing with missing data if possible unless the context calls for a different approach.

They then complete all of these missing data procedures on an actual dataset from higher education, arriving at the conclusion that EM was the most appropriate approach due to their context, but that MI is usually the most appropriate approach, particularly if the data will be subject to regression analysis.

https://muse-jhu-edu.wv-o-ursus-proxy02.ursus.maine.edu/article/539119

Cox, B. E., McIntosh, K., Reason, R. D., & Terenzini, P. T. (2014). Working with missing data in higher education research: A primer and real-world example. The Review of Higher Education, 37(3), 377–402.

Integrative Dynamics

Supra-urban

Faszinierender Versuch von Ford Doolittle, die Gaia-Hypothese evolutiontheoretisch zu unterlegen

https://taz.de/Britischer-Supreme-Court-entscheidet/!6016107/

https://taz.de/Britischer-Supreme-Court-entscheidet/!6016107/

Example of feedback

I can demo these steps if needed.

Allogenic

https://taz.de/Maja-Goepel-in-taz-FUTURZWEI/!vn6018893/

https://www.derstandard.de/story/3000000225600/daempfer-fuer-e-autos-volkswagen-investiert-60-milliarden-euro-in-verbrenner

シンタックスに問題があります。

サンプルプログラムで日本語が出るのはここが最初です。

MatPlotlibの日本語化Appendixのことをここでも訳注で追記してはどうでしょうか。

ちなみに第2版のときとはIPAフォントのダウンロードサイトのURLが変わって、以下のURLのようです。

https://moji.or.jp/ipafont/ipafontdownload/

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

Learn more at Review Commons

---

Reply to the reviewers

We thank the reviewer for their careful evaluation and constructive criticisms of our manuscript. We also appreciate the positive review by all three reviewers. The reviewers noted:

• "The computational model in this manuscript can be a tool to discover unknown molecular pathways interactions in cardiomyocyte proliferation."
• "This is an interesting study reporting the generation of a computational model of cardiomyocyte proliferation, which predicts molecular drivers of cell cycle progression."
• "The model provides a convenient systems framework to prioritize potential signaling drivers of therapeutic modulators of cardiomyocyte proliferation." We have responded to all reviewer comments and have outlined the corresponding additions and changes to the manuscript.

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary:

In the manuscript by Harris et al. titled "Dynamic map illuminates Hippo to cMyc module crosstalk driving cardiomyocyte proliferation," the authors developed a computational model of cardiac proliferation signaling that incorporates various regulatory networks (cytokinesis, mitosis, DNA replication, etc.) to predict molecular drivers (genes) that support cardiomyocyte proliferation. Published research articles on cardiomyocyte proliferation in multiple contexts (different species, ages, in vitro and in vivo, etc) were used to build and validate the computational model. The authors found using their model that different processes during cardiomyocyte proliferation may or may not be context-dependent. For example, DNA replication is regulated differently in conditions with high Neuregulin compared to high YAP, whereas mitosis and cytokinesis regulation is similar in these conditions. To experimentally validate their model, the authors used an in vitro system to test the effects of YAP on 3 connected pathways; in the context of YAP activation, inhibition of PI3K, cMyc, or FoxM1 was combined to assay cell-cycle markers in cultured neonatal rat ventricular cardiomyocytes. Cell-cycle marker expression in cardiomyocytes was attenuated by inhibition of cMyc or PI3K, suggesting that these pathways are involved in YAP-mediated cardiomyocyte proliferation. While this model can be a good tool to gain new insights on interactions between molecular pathways, there are a few questions to be addressed prior to publication.

We appreciate the Reviewer's positive remarks about important findings in our manuscript and the ability of our model to be a tool to gain insights on interactions between molecular pathways to regulate cardiomyocyte proliferation. We have strived to address their points, as shown below.

Major Comments:

1. One of the potential uses for this computational model is to discover new interactions between known pathways that are involved in cardiomyocyte proliferation. However, this would be more powerful if factors such as species, age (neonate vs. adult), and experimental design (in vivo vs. in vitro) were accounted for, as new node inputs or a combination of existing node input activity values. This is very important because cardiomyocyte proliferation can drastically vary depending on these experimental factors. We agree that future extensions of this model accounting for species, age, and experimental design may enable an understanding of how these factors regulate proliferation. While this model's predictions are most relevant to immature cardiomyocytes, we note that it is the first systems model of the molecular network regulating cardiomyocyte proliferation. We extensively validated it against neonatal cardiomyocyte literature and then made new predictions regarding Hippo-cMyc pathways, which we validated in new cardiomyocyte experiments and against data in adult mice. This provides a strong foundation for future extensions. We now address this potential in the Discussion:

"While our model's predictions are most relevant to immature cardiomyocytes, it is the first systems model of the molecular network regulating cardiomyocytes. In the future, we hope that we and others may extend this model to identify how factors like species, age, and experimental design regulate proliferation. However, these endeavors would span multiple manuscripts, and the field currently lacks sufficient stage-specific data. For example, a previous foundational computational model of cardiomyocyte electrophysiology (Luo and Rudy, Circ Res 1994) focused on adult guinea pigs. This model became the foundation for a range of developmental and species-specific models in electrophysiology (Tusscher et al, AJP 2004,; Courtemanche eta al, AJP 1998; Paci et al, ABME 2013). We believe the open availability of our code will enable similar dissemination and extension for additional factors." Line 651-661

For reference:

Luo and Rudy, Circ Res 1994, >2.1k citations; Tusscher et al, AJP 2004, >1.7k citations; Courtemanche et al, AJP 1998, 1.5K citations; Paci et al, ABME 2013, 147 citations

The finding that cardiomyocyte proliferation is context-dependent is very exciting and warrants further investigation/validation. The authors state that different sets of nodes/modules are affected by neuregulin activation compared to YAP activation. This should be experimentally validated - qPCR/Western blots on sets of genes that are predicted to be differentially regulated in the high neuregulin context vs the high YAP context.

We agree that the model's prediction of context-dependent cardiomyocyte proliferation is very exciting. To further validate these predictions, we have performed additional experiments to validate context-dependent changes of phospho-ERK treated with Nrg and TT10. Using a high throughput capillary electrophoresis western blot system, we observed that with a short treatment of 30 min, Nrg induces greater phosphor-ERK compared to TT10, which validates our model predictions at short time intervals. Additionally, the model predicted greater p-AKT with 30 min treatment with Nrg compared to TT10. To validate this prediction, we now compare to Western blots from Hara et al. examining p-AKT in Nrg and TT10-treated cells. Validating our model predictions, their data show that Nrg induces greater p-AKT than with TT10. We have added new panels C, D, and E to Figure 4.

Figure 4: Influence of node knockdowns shifts with context, revealing crosstalk from Hippo to Growth Factor modules.

(A) Total influence of node knockdowns on the DNA replication, mitosis, and cytokinesis modules, compared across multiple signaling contexts: baseline, high Nrg, and high YAP. Total influence sums the overall effect of a node knockdown on a network module. (B) The total influence of each network module varies depending on whether a basal state, high Nrg, or high YAP signaling context is applied. (C) Capillary electrophoresis western blot for phosphorylated ERK, beta-actin, and GAPDH from neonatal cardiomyocytes treated with Nrg or TT10 for 30 min. (D) Model predictions of AKT and ERK activity of acute response to Nrg or TT10 (time constants for gene expression set to 100). (E) Quantification of effects of Nrg or TT10 treatment on p-ERK (from Western blot in panel C, n = 3) or p-AKT (from Western blot from (Hara et al., 2018), n = 1).

The overall description of the model can be improved. For example, how are the input and parameters set to validate or predict different experimental observations? What is the steady state activity of each of the nodes and does this make sense biologically? Including a few more sentences to explain the model would help with overall understanding for an uninformed reader.

We have addressed the following questions provided by the reviewer in the methods and results section of the manuscript:

How are the input and parameters set to validate or predict different experimental observations?

__ __"At baseline, input reaction weight parameters (w) were set based on information from the literature describing the baseline state of these inputs in the heart (each input reaction weight can be found in Supplemental File 1). To simulate experiments with biochemical stimuli, input reaction weights were increased to 0.8 or 1. To simulate experiments with inhibition or knockdown, the corresponding maximum species value (ymax) was set to 0.1 or 0. Complete annotations for all validation simulations are provided in Supplemental File 2." Line 154-160

What is the steady-state activity of each of the nodes and does this make sense biologically?

"Steady-state activity of model nodes was obtained by running the model until there was a __ __

Minor Comments:

Line 124 - The use of "species" and "reactions" is confusing to uninformed readers. Do you mean nodes and interactions/bridges?

We now further clarify these terms in the manuscript:

"As in past network models (Zeigler et al., PMID 27017945; Tan et al., PMID 29131824; Kraeutler et al PMID 21087478), species (or nodes) refer to a small molecule, gene, protein, or process. Reactions (or edges) are activating or inhibiting relationships between network species." Line 143-146

Line 130 - I could not find Supplementary File 2, which includes the references

We apologize for the error. Supplementary File 2 references articles and resources used to build the model. These files are now attached.

Line 257 - What is the meaning of the directional arrows in Fig 1A?

We clarified the Fig 1A legend:

"Arrows between modules represent one or more reactions that link species from one module to species in another module. " Line 594-595

Line 301 - Unclear what default values mean here. Please elaborate and provide an example of how this is reasonable.

We have added further descriptions of default values in reference to the parameters to the manuscript.

"A previous study identified default values of the parameters (ymax, EC50, W, etc.) that most accurately predict the results of knockdown screens compared to a model where all biochemical parameters were measured experimentally (Kraeutler et al 2010). Subsequent studies started from these default values and further demonstrated that model accuracy was robust to random variation in the parameters (Tan et all 2017, Zeigler et al 2017). Consistent with these prior models, we performed robustness analysis that demonstrates that the CM proliferation model accuracy (compared against 78 experiments) is maintained at >80% with up to 35% variation in ymax, 30% variation with w, and a variation of >50% with EC50 (Figure S4)." Line 305-312

---

Supplemental FigS2 - Why would knockdown of PKA, Lats1 or SMAD3 have the exact same effects on node activation? This is seen with multiple other genes was well (IGF and FGF for example).

PKA, Lats1, and SMAD3 all inhibit cell cycle progression in part through cMyc. Therefore, their knockdown have similar effects on downstream signaling and proliferation. Similarly, IGF and FGF both stimulate Ras and PI3K via similar mechanisms, which is consistent with experimental studies of IGF- and FGF-dependent proliferation.

---

Reviewer #1 (Significance (Required)):

---

The computational model in this manuscript can be a tool to discover unknown molecular pathway interactions in cardiomyocyte proliferation. The novelty lies in the ability to adjust any parameter or the entire setting/context. While this sounds very exciting, improvement of the model to account for age, experimental conditions (in vivo vs in vitro), and species (human, pig, mouse) could lead to increase prediction accuracy. Additionally, more robust validation of context-dependent interactions between signaling pathways would also increase overall enthusiasm for the manuscript. Readers interested in a systems biology approach to cardiomyocyte proliferation, or researchers probing molecular interactions during cardiomyocyte proliferation would be interested in using such a model to discover novel contexts/combinations in which cardiomyocyte proliferation is more likely.

---

The reviewer comes from a varied training background and is qualified to evaluate this manuscript in full - BS in biomedical engineering and mathematics. PhD in biomedical engineering (molecular biology, cardiac electrophysiology). Postdoctoral training in cardiac regeneration and immunity.

---

We appreciate the positive comments about our model of the cardiomyocyte proliferation network. As described above, we believe that we have addressed the concerns with additional experimental validation.

---

The manuscript submitted by Harris and colleagues collates a molecular map of cardiomyocyte cell cycle activation through mathematical modeling of previously published experimental results. They attempt to validate the constructed model several ways: 1) through testing results compiled from additional literature, 2) through in vitro analysis, and 3) through in vivo supporting data. When validating through additional literature the model proves quite reliable particularly for prediction of effects on synthesis, mitosis, and cytokinetic entry, but was less reliable (or insufficiently tested) at predicting completion of these stages as determined by polyploidization and multinucleation. A potentially novel observation which arose from the model - that hippo nodule connects to the growth factor nodule through PI3K, Myc, and FoxM1 - was partially confirmed with in vitro experiments, though a few experiments are warranted.

We appreciate the reviewer's recognition of the important contributions of this model of the cardiomyocyte proliferation network. We have addressed the concerns below.

Major comments:

• The model is admittedly weakest in its handling of completion of cytokinesis resulting in new daughter cells (i.e. proliferation) versus failure to complete either M phase or cytokinesis resulting in the much more common cellular phenotypes - polyploidy and multinucleation. Notably, very few molecules were "tested" for this output (figure 2) and this proved the least reliable aspect of the model/map. I wonder if the authors consulted the literature on somatic polyploidization at all when building the model (files not provided as indicated, see minor comment 1 below )? And if not, would doing so help strengthen this arm of their map? There are some great reviews on the topic (see PMIDs 25921783, 23849927, 30021843) - while admittedly much of the work is done on other cell types (i.e. trophoblast giant cells and hepatocytes) maybe understanding the molecular intricacies in these cells could be incorporated to strengthen the predictive model in cardiomyocytes. Notably, PMID 23849927 even provides a table of citations about key nodes in the model influencing polyploidy. To validate this model, we used entirely cardiomyocyte specific studies. We appreciate the reviewer's reference to PMID 23849927, which enabled us to add two additional experiments to the validation table in Figure 2. That paper found that overexpression of either cMyc or cyclin D increases polyploidy, which both matched our new simulations in the updated Figure 2.

Motivated by the reviewer's citation of PMID 23849927, we further validated the model against polyploidization data from multiple cell types, finding an 85.7% accuracy (6 of 7 experiments) as now shown in Supplementary Figure S7.

We included an additional discussion of polyploidization in the manuscript.

"Our model validation is notably weakest in predicting experiments on polyploidization, indicating a need to better characterize polyploidy and cytokinesis pathways. Because such data are limited in cardiomyocytes, we performed an additional validation against polyploidization experiments from other cell types as summarized in Pandit et al. Our CM proliferation model predicted 85% (6 of 7) experiments. Future experiments are needed to identify conserved or differential mechanisms of polyploidization and cytokinesis in cardiomyocytes." Line 587-594

• Paragraph on the cytokinesis module (lines 364-377) is confusing - not sure what the takeaway message is. Also, while progression through G1/S and G2/M are "required" for cytokinesis they on their own are not sufficient (lines 366-368), this perhaps goes back to major comment 1. We agree this sentence was confusing, it was meant to be introductory rather than stating a particular result. We removed that sentence and further revised our description of the output module to clarify the model structure:

"The output module interlinks the phenotypic outputs of the other modules, representing how experimentally measured aspects of cell cycle activity (DNA replication by EdU or Ki67), mitosis by phospho-Histone 3 (pHH3), abscission by cytokinetic midbody converge on polyploidy, binucleation, or cytokinesis (e.g. completed proliferation) (Figure 1G)." Line 283-286

Minor comments:

• Use of the word "Proliferation" should be reserved for situations where the authors can clearly say a new daughter cell was born. In many instances, "cell cycle activation" or "cell cycle progression" might be better terms. As suggested by the reviewer, we now use "cell cycle progression" in 7 instances, reserving "proliferation" for cell cycle progression through cytokinesis. In the remaining 90 instances, we refer to proliferation based on the model's predictions of completed cell division based on the combined DNA replication, mitosis, and cytokinesis pathways in the "output module". We retain "proliferation" in the title because the model encompasses the entire proliferation process from cell cycle entry through cytokinesis.

• Supplementary Files 1 & 2 or Supplementary Document 2 were not provided or not found during review, thus we were unable to confirm which literature were used to build and validate the model. Thank you, we have included Supplementary Files 1 and 2 along with supplementary document 2 in the submission.

• Figures are too small, particular Figure 1 We have enlarged Figure 1.

• "E2F" should be specified as E2F1-3 yield quite distinct results from E2F7/8. We have changed "E2F" to "E2F123"

• Text corresponding to Figure 5 does not reference most of the panels in the Figure. i.e. figures are not "cited" in the text We have made sure that each panel in Figure 5 is referenced in the text addressing the figure. We have also bolded all references to Figure 5.

---

• Figure 5C - why is there no bars for PI3K. Text claims it was predicted by the model, but the data are missing? We apologize for the confusion regarding Figure 5C, in which the bar for PI3K was near zero. We now clarify this in the legend.

"Predicted DNA replication and mitosis activity is close to zero when PI3K is inhibited alone and when PI3K is inhibited in combination with TT10 treatment."

• Data provided in figure 5D & E are insufficient on their own to claim "proliferation". Perhaps adding total cardiomyocyte numbers, where one would expect expansion compared to control. We agree that Ki67 and pH3 are not sufficient to claim "proliferation", so we modified the Figure 5 legend to:

"Prediction and experimental validation of cardiomyocyte cell cycle progression mediated by the Hippo pathway via PI3K, cMyc, and FoxM1."

We previously found that cardiomyocyte numbers without live tracking are not sufficient to robustly measure proliferation (Woo et al, J Mol Cardiol, 2019).

---

• Consider adding a details about the p-values to the figure legend in figure 5. Thank you for this suggestion p-value information has been added to the legend of Figure 5. We use *** Our literature-based validation in Figure 2 focused on 78 experiments that examined well-established and corroborated aspects of cardiomyocyte proliferation. Later in the paper, we focused on a newly predicted mechanism of cardiomyocyte proliferation involving small number of comparisons that would naturally have a lower a priori probability of validation in vitro neonatal experiments (Figure 5) and adult mouse experiments (Figure 6). Therefore, in the revised text we focus on the specific comparisons rather than statistics.

"Based on predictions from this validated model, we hypothesized that YAP drove proliferation via PI3K, cMyc and FoxM1. To test this model-driven hypothesis, we accurately predicted TT10-induced DNA replication that is suppressed by inhibition of PI3K, cMyc and to a lesser extent FoxM1 (Figure 5D). These model predictions were further validated using RNA-seq and ATAC-seq data from adult mouse hearts showing that constitutively active YAPS5A induces expression of Myc and FoxM1 as well as increased chromatin accessibility at PI3Kca and Myc." Line 454-468

In the discussion, we add:

"Further model revision is needed based on these molecular mechanisms of YAP-TEAD-Myc interactions to distinguish between chromatin accessibility, transcription factor binding, and gene expression." Line 649-651

As it stands now, the generated map largely constitutes already known details offering few if any new insights; however, if updated as new results arise AND made available as a public tool, the model could prove to be a highly valuable resource to the field.

We thank the reviewer for recognizing our model as a valuable resource and public tool. We have made our model publicly available on GitHub at https://github.com/saucermanlab/Cardiomyocyte-Proliferation-Network.

The virtual knockdown screens in Figure 3, 4 and 5 provide a wide range of new insights, which we clarify in new text.

"Because this is a literature-based network model, each component or direct interaction has been studied individually. However, our model makes much broader predictions of how these components interact to regulate proliferation, beyond the ~30 papers available for validation on the response of this system to perturbations shown in Figure 3. For example, Supplemental Figure S3 provides ~5000 predictions of how each protein responds to knockdown of every other protein. These predictions led to new insights into how YAP regulates proliferation via cMyc (experimentally validated in Figure 5 in vitro and Figure 6 in vivo), as well as many other insights that can be validated in future studies. These future studies will be aided by the open-source availability of our model on GitHub." Line 563-571

__ I have expertise in cardiomyocyte cell cycle and polyploidization.__

---

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

The authors generated a computational model of cardiomyocyte proliferation, which predicts molecular drivers of cell cycle progression. Interestingly, the model correctly predicts the outcome of 95% independent experiments from the literature. The model also elucidated crosstalk between the growth factor and Hippo modules and the authors identified key hubs for which the Hippo signaling pathway regulates cardiomyocyte proliferation. The model provides a convenient systems framework to prioritize potential signaling drivers of therapeutic modulators of cardiomyocyte proliferation.

Reviewer #3 (Significance (Required)):

This is an interesting study reporting the generation of a computational model of cardiomyocyte proliferation, which predicts molecular drivers of cell cycle progression. The program may provide a convenient framework prioritizing potential signaling drivers of therapeutic modulators of cardiomyocyte proliferation. However, the overall impact of the study appears modest since it is unclear whether the study allows elucidation of the unique properties of cardiomyocyte proliferation in adult hearts (i.e. they hardly proliferate) and the validation study was conducted only in neonatal myocytes. The field has seen many studies with neonatal myocytes but the findings are not always translatable to adult cardiomyocytes.

We thank the reviewer for recognizing the importance of our work that provides a framework for prioritizing potential signaling drivers of therapeutic modulators of CM proliferation.

Neonatal studies are the most prevalent with cardiomyocyte proliferation literature, making it the most robust starting point that allows for rigorous validation. Based on the high performance of the model against neonatal data, in the future we expect this model to be a stepping stone towards adaptions to understand differences in the adult cardiomyocyte proliferation network. We have updated our model discussion on future directions on this point.

"While our model's predictions are most relevant to immature cardiomyocytes, it is the first molecular network model of cardiomyocyte proliferation. In the future, this model will enable extensions to identify how factors like species, age and experimental design regulate proliferation. However, such endeavors would span multiple manuscripts and the field currently lacks sufficient stage-specific data. For example, the highly influential computational model of Luo and Rudy focused on adult guinea-pig cardiomyocyte electrophysiology (Luo and Rudy, Circ Res 1994). That model became the foundation for a wide range of development- and species-specific models in electrophysiology (Tusscher et al, AJP 2004,; Courtemanche eta al, AJP 1998; Paci et al, ABME 2013). We believe the open availability of our code will enable similar dissemination and extension for additional factors regulating cardiomyocyte proliferation." Line 655-665

The authors described that "Literature articles used for model development came from multiple cell types due to limited CM data." It is unclear whether this would allow the identification of unique mechanisms present in cardiomyocytes. As the authors admitted, the fact that the model predictions and experimental observations for polyploidization did not match clearly suggests the complexity surrounding the possibility of cell phenotypes in cardiomyocyte populations. The authors could have addressed whether this model allows the identification of unique mechanisms mediating cardiomyocyte proliferation in the adult heart.

Although we necessarily included literature on other cell types to support network reactions, all of the experimental validation in Figure 2 was with cardiomyocyte data (~33 publications). 80% of experiments were from neonatal CMs, 10% from adult CMs, 5% from in vivo studies, and the other 5% from hiPSC-derived cardiomyocytes as annotated in Supplemental File 3.

At this time, there is insufficient data from which to make a model focused only on adult CMs. The mode's open-source availability enables future extensions that examine age and species-dependent mechanisms of cardiomyocyte proliferation. We updated the manuscript, addressing the ability of our model to adapt to new information.

"This model provides an initial network framework for integrating additional discoveries in cardiomyocyte proliferation. As more information becomes available in cardiomyocyte proliferation literature the model can be adapted. Additionally, the field can use our open-sourced model to adapt this model to other developmental stages or species." Line 671-674

Acknowledging the limited data on cardiomyocyte polyploidy, we performed a new separate validation of 7 experiments in non-myocytes from PMID 23849927, finding an 85.7% accuracy (new Supplementary Figure S7).

Please provide more information regarding the rationale for having six modules in the authors' model, including the growth factor and the Hippo pathway.

We revised the text to clarify the motivation for the six modules:

"Our initial review of the literature indicated multiple complex molecular pathways that regulate cardiomyocyte proliferation, including growth factors, Hippo signaling, G1/S transition, G2/M transition, or cytokinesis pathways (Hashmi and Ahmad, PMID: 31205684; Payan et al, PMID: 30930108; Moral et al., PMID: 35008660; Wang et al., PMID: 30111784; Johnson et al., PMID: 34360531). Several review articles (Zheng et al, PMID: 32664346; Mia and Singh, PMID: 31632964; Diaz Del Moral et al, PMID: 35008660; Besson et al, PMID: 18267085; Wang et al, PMID: 19216791)) also organized the literature based on these distinct pathways or processes, which we used to define the boundaries of the six modules. However, how these molecular pathways work together is not well characterized. Therefore, we designed the model to incorporate each of these established modules and how they work together to drive cardiomyocyte proliferation." Line 550-557

---

The extent of cardiomyocyte proliferation at baseline is very low in the adult heart. The model identified 25 nodes that may influence baseline proliferation. Is there any evidence to support the involvement of these mechanisms in baseline cardiomyocyte proliferation in vivo?

We agree with the reviewer that proliferation at baseline is very low in the adult heart, and also rather low in neonatal cardiomyocytes. As shown in Figure S4A, we performed a virtual knockdown screen under baseline conditions that showed that no genetic knockdowns caused a substantial decrease in DNA replication or cytokinesis, consistent with a low baseline proliferation rate.

We describe this point about baseline proliferation in revised text:

"A complete virtual knockdown screen of the model was done under baseline conditions in Figure S4A, which showed that no knockdowns caused substantial decreases in DNA replication or cytokinesis. This is consistent with a low baseline proliferation rate described in cardiomyocyte literature." Line 354-357

The validation study was conducted with neonatal rat ventricular cardiomyocytes. This study could have been repeated with adult cardiomyocytes since they are more resistant to proliferation and, thus, the Myc may not work as expected. In addition, the authors could have commented on the mechanism through which chromatin opening and YAP allow transcription of Myc in the heart.

We agree that Myc is likely less proliferative in adult hearts. While our model was extensively validated against neonatal cardiomyocytes (Figure 2 for literature, Figure 5 for new neonatal experiments), only 10% of literature-based validations in Figure 2 are from adult cardiomyocytes due to limited data. However, in Figure 6 we validate YAP-dependent signaling to Myc, PI3K, and FOXM1 using RNA-seq and ATAC-seq data from Monroe et al. from adult mouse cardiomyocytes in vivo. While molecular mechanisms of YAP regulation of Myc are not characterized in the heart, based on the reviewer's suggestion, we add new discussion on YAP-Myc interaction in other cells:

"Overexpression of Myc induces cardiomyocyte proliferation in vitro and in vivo in several contexts, with open chromatin and Myc binding near mitotic genes (PMID: 32286286). But to our knowledge, crosstalk of YAP with Myc has not been reported in the heart. Our model prediction and experiments in neonatal cardiomyocytes support a YAP-TEAD-Myc pathway for cardiomyocyte proliferation. Further, our analysis of ATAC-seq and RNA-seq data from Monroe et al. validate that YAP induces Myc chromatin availability and gene expression in adult mouse hearts.

In MDA-MB-231 breast cancer cells, YAP/TAZ/TEAD bind directly to Myc enhancers through chromatin looping, with decreased acetylation of H3K27 and cell proliferation upon YAP/TAZ knockdown (26258633). YAP-TEAD-Myc signaling regulates the proliferation of cancer cells (26258633), tumorigenesis (29416644), and the growth of Drosophila imaginal discs (20951343). In the future, computational models and experiments are needed to better resolve how YAP promotes proliferation via Myc in the adult heart, including regulation by Mycn (30315164), cyclin T1 (32286286)."Line 632-644

---

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

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

The authors generated a computational model of cardiomyocyte proliferation, which predicts molecular drivers of cell cycle progression. Interestingly, the model correctly predicts the outcome of 95% independent experiments from the literature. The model also elucidated crosstalk between the growth factor and Hippo modules and the authors identified key hubs for which the Hippo signaling pathway regulates cardiomyocyte proliferation. The model provides a convenient systems framework to prioritize potential signaling drivers of therapeutic modulators of cardiomyocyte proliferation.

Significance

This is an interesting study reporting the generation of a computational model of cardiomyocyte proliferation, which predicts molecular drivers of cell cycle progression. The program may provide a convenient framework prioritizing potential signaling drivers of therapeutic modulators of cardiomyocyte proliferation. However, the overall impact of the study appears modest since it is unclear whether the study allows elucidation of the unique properties of cardiomyocyte proliferation in adult hearts (i.e. they hardly proliferate) and the validation study was conducted only in neonatal myocytes. The field has seen many studies with neonatal myocytes but the findings are not always translatable to adult cardiomyocytes.

The authors described that "Literature articles used for model development came from multiple cell types due to limited CM data." It is unclear whether this would allow the identification of unique mechanisms present in cardiomyocytes. As the authors admitted, the fact that the model predictions and experimental observations for polyploidization did not match clearly suggests the complexity surrounding the possibility of cell phenotypes in cardiomyocyte populations. The authors could have addressed whether this model allows the identification of unique mechanisms mediating cardiomyocyte proliferation in the adult heart.

Please provide more information regarding the rationale for having six modules in the authors' model, including the growth factor and the Hippo pathway.

The extent of cardiomyocyte proliferation at baseline is very low in the adult heart. The model identified 25 nodes that may influence baseline proliferation. Is there any evidence to support the involvement of these mechanisms in baseline cardiomyocyte proliferation in vivo?

The validation study was conducted with neonatal rat ventricular cardiomyocytes. This study could have been repeated with adult cardiomyocytes since they are more resistant to proliferation and, thus, the Myc may not work as expected. In addition, the authors could have commented on the mechanism through which chromatin opening and YAP allow transcription of Myc in the heart.

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

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

The manuscript submitted by Harris and colleagues collates a molecular map of cardiomyocyte cell cycle activation through mathematical modeling of previously published experimental results. They attempt to validate the constructed model several ways: 1) through testing results compiled from additional literature, 2) through in vitro analysis, and 3) through in vivo supporting data. When validating through additional literature the model proves quite reliable particularly for prediction of effects on synthesis, mitosis, and cytokinetic entry, but was less reliable (or insufficiently tested) at predicting completion of these stages as determined by polyploidization and multinucleation. A potentially novel observation which arose from the model - that hippo nodule connects to the growth factor nodule through PI3K, Myc, and FoxM1 - was partially confirmed with in vitro experiments, though a few experiments are warranted.

Major comments:

1. The model is admittedly weakest in its handling of completion of cytokinesis resulting in new daughter cells (i.e. proliferation) versus failure to complete either M phase or cytokinesis resulting in the much more common cellular phenotypes - polyploidy and multinucleation. Notably, very few molecules were "tested" for this output (figure 2) and this proved the least reliable aspect of the model/map. I wonder if the authors consulted the literature on somatic polyploidization at all when building the model (files not provided as indicated, see minor comment 1 below)? And if not, would doing so help strengthen this arm of their map? There are some great reviews on the topic (see PMIDs 25921783, 23849927, 30021843) - while admittedly much of the work is done on other cell types (i.e. trophoblast giant cells and hepatocytes) maybe understanding the molecular intricacies in these cells could be incorporated to strengthen the predictive model in cardiomyocytes. Notably, PMID 23849927 even provides a table of citations about key nodes in the model influencing polyploidy.
2. Paragraph on the cytokinesis module (lines 364-377) is confusing - not sure what the takeaway message is. Also, while progression through G1/S and G2/M are "required" for cytokinesis they on their own are not sufficient (lines 366-368), this perhaps goes back to major comment 1.

Minor comments:

1. Use of the word "Proliferation" should be reserved for situations where the authors can clearly say a new daughter cell was born. In many instances "cell cycle activation" or "cell cycle progression" might be better terms.
2. Supplementary Files 1 & 2 or Supplementary Document 2 were not provided or not found during review, thus we were unable to confirm which literature were used to build and validate the model.
3. Figures are too small, particular Figure 1
4. "E2F" should be specified as E2F1-3 yield quite distinct results from E2F7/8.
5. Text corresponding to Figure 5 does not reference most of the panels in the Figure. i.e. figures are not "cited" in the text
6. Figure 5C - why is there no bars for PI3K. Text claims it was predicted by the model, but the data are missing?
7. Data provided in figure 5D & E are insufficient on their own to claim "proliferation". Perhaps adding total cardiomyocyte numbers, where one would expect expansion compared to control.
8. Consider adding a details about the p-values to the figure legend in figure 5.
9. Data presented in figure 6 do not "validate" the model. Rescue experiments as were provided in vitro would be necessary or at minimum YAP/TEAD binding to the promoters (ATAC insufficient). Alternatively, walking back these statements, might be easiest.
10. Validation studies through the literature suggested ~94% fidelity. The invitro validation suggests 66% reliability of model? The in vivo 33%? Perhaps this should be added as a discussion point - can the authors comment on the loss of fidelity as the rigor/complexity of the experiment increased?

Significance

As it stands now, the generated map largely constitutes already known details offering few if any new insights; however, if updated as new results arise AND made available as a public tool, the model could prove to be a highly valuable resource to the field.

I have expertise in cardiomyocyte cell cycle and polyploidization

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

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

Summary:

In the manuscript by Harris et al. titled "Dynamic map illuminates Hippo to cMyc module crosstalk driving cardiomyocyte proliferation," the authors developed a computational model of cardiac proliferation signaling that incorporates various regulatory networks (cytokinesis, mitosis, DNA replication, etc.) to predict molecular drivers (genes) that support cardiomyocyte proliferation. Published research articles on cardiomyocyte proliferation in multiple contexts (different species, ages, in vitro and in vivo, etc) were used to build and validate the computational model. The authors found using their model that different processes during cardiomyocyte proliferation may or may not be context-dependent. For example, DNA replication is regulated differently in conditions with high Neuregulin compared to high YAP, whereas mitosis and cytokinesis regulation is similar in these conditions. To experimentally validate their model, the authors used an in vitro system to test the effects of YAP on 3 connected pathways; in the context of YAP activation, inhibition of PI3K, cMyc, or FoxM1 was combined to assay cell-cycle markers in cultured neonatal rat ventricular cardiomyocytes. Cell-cycle marker expression in cardiomyocytes was attenuated by inhibition of cMyc or PI3K, suggesting that these pathways are involved in YAP-mediated cardiomyocyte proliferation. While this model can be a good tool to gain new insights on interactions between molecular pathways, there are a few questions to be addressed prior to publication.

Major Comments:

1. One of the potential uses for this computational model is to discover new interactions between known pathways that are involved in cardiomyocyte proliferation. However, this would be more powerful if factors such as species, age (neonate vs. adult), experimental design (in vivo vs. in vitro) are accounted for, as new node inputs or a combination of existing node input activity values. This is very important because cardiomyocyte proliferation can drastically vary depending on these experimental factors.
2. The finding that cardiomyocyte proliferation is context-dependent is very exciting and warrants further investigation/validation. The authors state that different sets of nodes/modules are affected by neuregulin activation compared to YAP activation. This should be experimentally validated - qPCR/Western blots on sets of genes that are predicted to be differentially regulated in the high neuregulin context vs the high YAP context.
3. The overall description of the model can be improved. How are the modules and overarching model built from published results? For example, how are the input and parameters set to validate or predict different experimental observations? What is the steady-state activity of each of the nodes and does this make sense biologically? Includng a few more sentences to explain the model would help with overall understanding for an uninformed reader.

Minor Comments:

Line 124 - The use of "species" and "reactions" is confusing to uninformed readers. Do you mean nodes and interactions/bridges?

Line 130 - I could not find Supplementary File 2, which includes the references?

Line 251 - "theseanal"

Line 257 - What is the meaning of the directional arrows in Fig 1A?

Line 301 - Unclear what default values mean here. Please elaborate and provide an example of how this is reasonable?

Supplemental Fig S2 - Why would knockdown of PKA, Lats1, or SMAD3 have the exact same effects on node activation? This is seen with multiple other genes as well (IGF and FGF for example).

Significance

The computational model in this manuscript can be a tool to discover unknown molecular pathway interactions in cardiomyocyte proliferation. The novelty lies in the ability to adjust any parameter or the entire setting/context. While this sounds very exciting, improvement of the model to account for age, experimental conditions (in vivo vs in vitro), and species (human, pig, mouse) could lead to increase prediction accuracy. Additionally, more robust validation of context-dependent interactions between signaling pathways would also increase overall enthusiasm for the manuscript. Readers interested in a systems biology approach to cardiomyocyte proliferation, or researchers probing molecular interactions during cardiomyocyte proliferation would be interested in using such a model to discover novel contexts/combinations in which cardiomyocyte proliferation is more likely.

The reviewer comes from a varied training background and is qualified to evaluate this manuscript in full - BS in biomedical engineering and mathematics. PhD in biomedical engineering (molecular biology, cardiac electrophysiology). Postdoctoral training in cardiac regeneration and immunity.

原文を尊重するべきですが、ディレクトリとフォルダーで表記ゆれがあるのが気になりました。