Reviewer #4 (Public Review):

I am a new reviewer for this manuscript, which has been reviewed before. The authors provide a variational autoencoder that has three objectives in the loss: linear reconstruction of behavior from embeddings, reconstruction of neural data, and KL divergence term related to the variational model elements. They take the output of the VAE as the "behaviorally relevant" part of neural data and call the residual "behaviorally irrelevant". Results aim to inspect the linear versus nonlinear behavior decoding using the original raw neural data versus the inferred behaviorally relevant and irrelevant parts of the signal.

Overall, studying neural computations that are behaviorally relevant or not is an important problem, which several previous studies have explored (for example PSID in (Sani et al. 2021), TNDM in (Hurwitz et al. 2021), TAME-GP in (Balzani et al. 2023), pi-VAE in (Zhou and Wei 2020), and dPCA in (Kobak et al. 2016), etc). However, this manuscript does not properly put their work in the context of such prior works. For example, the abstract states "One solution is to accurately separate behaviorally-relevant and irrelevant signals, but this approach remains elusive", which is not the case given that these prior works have done that. The same is true for various claims in the main text, for example "Furthermore, we found that the dimensionality of primary subspace of raw signals (26, 64, and 45 for datasets A, B, and C) is significantly higher than that of behaviorally-relevant signals (7, 13, and 9), indicating that using raw signals to estimate the neural dimensionality of behaviors leads to an overestimation" (line 321). This finding was presented in (Sani et al. 2021) and (Hurwitz et al. 2021), which is not clarified here. This issue of putting the work in context has been brought up by other reviewers previously but seems to remain largely unaddressed. The introduction is inaccurate also in that it mixes up methods that were designed for separation of behaviorally relevant information with those that are unsupervised and do not aim to do so (e.g., LFADS). The introduction should be significantly revised to explicitly discuss prior models/works that specifically formulated this behavior separation and what these prior studies found, and how this study differs.

Beyond the above, some of the main claims/conclusions made by the manuscript are not properly supported by the analyses and results, which has also been brought up by other reviewers but not fully addressed. First, the analyses here do not support the linear readout from the motor cortex because i) by construction, the VAE here is trained to have a linear readout from its embedding in its loss, which can bias its outputs toward doing well with a linear decoder/readout, and ii) the overall mapping from neural data to behavior includes both the VAE and the linear readout and thus is always nonlinear (even when a linear Kalman filter is used for decoding). This claim is also vague as there is no definition of readout from "motor cortex" or what it means. Why is the readout from the bottleneck of this particular VAE the readout of motor cortex? Second, other claims about properties of individual neurons are also confounded because the VAE is a population-level model that extracts the bottleneck from all neurons. Thus, information can leak from any set of neurons to other sets of neurons during the inference of behaviorally relevant parts of signals. Overall, the results do not convincingly support the claims, and thus the claims should be carefully revised and significantly tempered to avoid misinterpretation by readers.

Below I briefly expand on these as well as other issues, and provide suggestions:

1) Claims about linearity of "motor cortex" readout are not supported by results yet stated even in the abstract. Instead, what the results support is that for decoding behavior from the output of the dVAE model -- that is trained specifically to have a linear behavior readout from its embedding -- a nonlinear readout does not help. This result can be biased by the very construction of the dVAE's loss that encourages a linear readout/decoding from embeddings, and thus does not imply a finding about motor cortex.

2) Related to the above, it is unclear what the manuscript means by readout from motor cortex. A clearer definition of "readout" (a mapping from what to what?) in general is needed. The mapping that the linearity/nonlinearity claims refer to is from the *inferred* behaviorally relevant neural signals, which themselves are inferred nonlinearly using the VAE. This should be explicitly clarified in all claims, i.e., that only the mapping from distilled signals to behavior is linear, not the whole mapping from neural data to behavior. Again, to say the readout from motor cortex is linear is not supported, including in the abstract.

3) Claims about individual neurons are also confounded. The d-VAE distilling processing is a population level embedding so the individual distilled neurons are not obtainable on their own without using the population data. This population level approach also raises the possibility that information can leak from one neuron to another during distillation, which is indeed what the authors hope would recover true information about individual neurons that wasn't there in the recording (the pixel denoising example). The authors acknowledge the possibility that information could leak to a neuron that didn't truly have that information and try to rule it out to some extent with some simulations and by comparing the distilled behaviorally relevant signals to the original neural signals. But ultimately, the distilled signals are different enough from the original signals to substantially improve decoding of low information neurons, and one cannot be sure if all of the information in distilled signals from any individual neuron truly belongs to that neuron. It is still quite likely that some of the improved behavior prediction of the distilled version of low-information neurons is due to leakage of behaviorally relevant information from other neurons, not the former's inherent behavioral information. This should be explicitly acknowledged in the manuscript.

4) Given the nuances involved in appropriate comparisons across methods and since two of the datasets are public, the authors should provide their complete code (not just the dVAE method code), including the code for data loading, data preprocessing, model fitting and model evaluation for all methods and public datasets. This will alleviate concerns and allow readers to confirm conclusions (e.g., figure 2) for themselves down the line.

5) Related to 1) above, the authors should explore the results if the affine network h(.) (from embedding to behavior) was replaced with a nonlinear ANN. Perhaps linear decoders would no longer be as close to nonlinear decoders. Regardless, the claim of linearity should be revised as described in 1) and 2) above, and all caveats should be discussed.

6) The beginning of the section on the "smaller R2 neurons" should clearly define what R2 is being discussed. Based on the response to previous reviewers, this R2 "signifies the proportion of neuronal activity variance explained by the linear encoding model, calculated using raw signals". This should be mentioned and made clear in the main text whenever this R2 is referred to.

7) Various terms require clear definitions. The authors sometimes use vague terminology (e.g., "useless") without a clear definition. Similarly, discussions regarding dimensionality could benefit from more precise definitions. How is neural dimensionality defined? For example, how is "neural dimensionality of specific behaviors" (line 590) defined? Related to this, I agree with Reviewer 2 that a clear definition of irrelevant should be mentioned that clarifies that relevance is roughly taken as "correlated or predictive with a fixed time lag". The analyses do not explore relevance with arbitrary time lags between neural and behavior data.

8) CEBRA itself doesn't provide a neural reconstruction from its embeddings, but one could obtain one via a regression from extracted CEBRA embeddings to neural data. In addition to decoding results of CEBRA (figure S3), the neural reconstruction of CEBRA should be computed and CEBRA should be added to Figure 2 to see how the behaviorally relevant and irrelevant signals from CEBRA compare to other methods.

References:

Kobak, Dmitry, Wieland Brendel, Christos Constantinidis, Claudia E Feierstein, Adam Kepecs, Zachary F Mainen, Xue-Lian Qi, Ranulfo Romo, Naoshige Uchida, and Christian K Machens. 2016. "Demixed Principal Component Analysis of Neural Population Data." Edited by Mark CW van Rossum. eLife 5 (April): e10989. https://doi.org/10.7554/eLife.10989.

Sani, Omid G., Hamidreza Abbaspourazad, Yan T. Wong, Bijan Pesaran, and Maryam M. Shanechi. 2021. "Modeling Behaviorally Relevant Neural Dynamics Enabled by Preferential Subspace Identification." Nature Neuroscience 24 (1): 140-49. https://doi.org/10.1038/s41593-020-00733-0.

Zhou, Ding, and Xue-Xin Wei. 2020. "Learning Identifiable and Interpretable Latent Models of High-Dimensional Neural Activity Using Pi-VAE." In Advances in Neural Information Processing Systems, 33:7234-47. Curran Associates, Inc. https://proceedings.neurips.cc/paper/2020/hash/510f2318f324cf07fce24c3a4b89c771-Abstract.html.

Hurwitz, Cole, Akash Srivastava, Kai Xu, Justin Jude, Matthew Perich, Lee Miller, and Matthias Hennig. 2021. "Targeted Neural Dynamical Modeling." In Advances in Neural Information Processing Systems. Vol. 34. https://proceedings.neurips.cc/paper/2021/hash/f5cfbc876972bd0d031c8abc37344c28-Abstract.html.

Balzani, Edoardo, Jean-Paul G. Noel, Pedro Herrero-Vidal, Dora E. Angelaki, and Cristina Savin. 2023. "A Probabilistic Framework for Task-Aligned Intra- and Inter-Area Neural Manifold Estimation." In . https://openreview.net/forum?id=kt-dcBQcSA.

"clients" generally refer to computer applications or devices that access services or resources from a server in a network.

can range from web browsers accessing internet pages, to applications fetching data from a database server, or devices like smartphones or computers running software that interacts with remote servers.

central to much of modern computing and internet-based services.

Reviewer #3 (Public Review):

Summary:<br /> The authors propose to invert a mechanistic model of phototransduction in mouse and rod photoreceptors to derive stimuli that compensate for nonlinearities in these cells. They fit the model to a large set of photoreceptor recordings and show in additional data that the compensation works. This can allow the exclusion of photoreceptors as a source of nonlinear computation in the retina, as desired to pinpoint nonlinearities in retinal computation. Overall, the recordings made by the authors are impressive and I appreciate the simplicity and elegance of the idea. The data support the authors' conclusions but the presentation can be improved.

Strengths:<br /> - The authors collected an impressive set of recordings from mouse and primate photoreceptors, which is very challenging to obtain.<br /> - The authors propose to exploit mechanistic mathematical models of well-understood phototransduction to design light stimuli that compensate for nonlinearities.<br /> - The authors demonstrate through additional experiments that their proposed approach works.

Weaknesses:<br /> - The authors use numerical optimization for fitting the parameters of the photoreceptor model to the data. Recently, the field of simulation-based inference has developed methods to do so, including quantification of the uncertainty of the resulting estimates. Since the authors state that two different procedures were used due to the different amounts of data collected from different cells, it may be worthwhile to rather test these methods, as implemented e.g. in the SBI toolbox (https://joss.theoj.org/papers/10.21105/joss.02505). This would also allow them to directly identify dependencies between parameters, and obtain associated uncertainty estimates. This would also make the discussion of how well constrained the parameters are by the data or how much they vary more principled because the SBI uncertainty estimates could be used.

- In several places, the authors refer the reader to look up specific values e.g. of parameters in the associated MATLAB code. I don't think this is appropriate, important values/findings/facts should be in the paper (lines 142, 114, 168). I would even find the precise values that the authors measure interesting, so I think the authors should show them in a figure/table. In general, I would like to see also the average variance explained by different models summarized in a table and precise mean/median values for all important quantities (like the response amplitude ratios in Figures 6/9).

- If the proposed model is supposed to model photoreceptor adaptation on a longer time scale, I fail to see why this can be an invertible model. Could the authors explain this better? I suspect that the model is mainly about nonlinearities as the authors also discuss in lines 360ff.

- The important Figures 6-8 are very hard to read, as it is not easy to see what the stimulus is, the modified stimulus, the response with and without modification, what the desired output looks like, and what is measured for part B. Reworking these figures would be highly recommended.

- If I understand Figure 6 correctly, part B is about quantifying the relative size of the response to the little first flash to the little second flash. While clearly, the response amplitude of the second flash is only 50% for the second flash compared to the first flash in primate rod and cones in the original condition, the modified stimulus seems to overcompensate and result in 130% response for the second flash. How do the authors explain this? A similar effect occurs in Figure 9, which the authors should also discuss.

Not like a static site generator, that's for sure. Normal people don't a step in between input source code and the output. They don't want a difference between input and output at all. Programmers want a compilation step, because they're programmers.

I do think this is something to consider and question, because often dress-codes are more directed toward female students than male. What I was wondering as well is can a parent take legal action if they disagree with the school's dress code?

I would like to be mindful of this as I become a teacher because I think it is important to adhere to what is expected of me.

python, c++ are programs written by programs. Of course those programs are much more harder for their syntax is broader allowing to be more precise when communicating with hardware/the computer.

of my 4 years of knowing how to code, I would say code is a sort of communication framework between a user and a computer. We code because even though we can put 2+2 together we can give a computer a function where a user inputs two different numbers and now the computer can add things way faster than we can.

If you can't explain something you're supposed to be good at are you doing a good job?

Résumé vidéo [00:48:26][^1^][1] - [01:11:02][^2^][2]:

La troisième partie de la vidéo traite de la notion de valeurs de la République, de son origine, de sa définition et de sa transmission. L'intervenant, Ismaël Ferhat, présente une analyse sémantique, juridique et pédagogique de cette notion, en prenant l'exemple de la laïcité.

Points forts: + [00:48:26][^3^][3] L'émergence récente des valeurs de la République * Une notion peu utilisée avant les années 80 * Une augmentation dans les publications, les médias et les codes juridiques * Une rupture liée aux débats sur l'immigration, le multiculturalisme et le séparatisme + [00:54:01][^4^][4] L'absence de définition constitutionnelle des valeurs de la République * Aucun texte ni décision du Conseil constitutionnel sur le sujet * Un avis du Conseil d'État qui distingue les valeurs des principes * Une définition circonscrite dans le code de l'éducation + [01:00:00][^5^][5] L'enjeu de l'éducation aux valeurs de la République * Une montée de la laïcité dans les programmes scolaires depuis les années 90 * Une nécessité d'expliquer les règles et de déconstruire les oppositions * Une évaluation positive de la maîtrise du principe de laïcité par les élèves

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Reply to the reviewers

1. General Statements [optional]

All four reviewers have positive comments on the paper. We totally agree with their comments, and proposed controls and experiments. Most of them are already introduced in the present text and several new figures added, as we had the controls/experiments proposed. Few others are now being done and we hope to have the complete set of experiments ready in 2-3 months.

2. Description of the planned revisions

Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

Reviewer #1

Most comments of this reviewer have already been done and included in the transferred manuscript, except for part of the first comment:

1.1 b. Is it possible that the loss of function of Wengen on its own has a phenotype? If so, that would suggest that Wgn in addition to its role in regeneration might be implicated in pro-survival processes in homeostatic conditions?

This issue is very important to understand the differential role of Wgn and Grnd. First of all, Wengen knock out (wgnKO; Andersen et al., 2015) is viable in homozygosis. However, in this paper we have focused on inducible mutants. Therefore, we have now crossed the flies to get the genotype hh-Gal UAS RNAi wgn and we will check for apoptotic phenotype, as suggested. This will take us few weeks of work.

Reviewer #2 Most comments have been already carried out and included in the transferred manuscript, except these ones:

*2.3. Aside from wgn, other RNAi experiments are not validated through independent RNAi lines. I suggest expanding the Supplemental Figures to reproduce a few key findings with independent RNAi lines. *

We have recently received a set of independent RNAi line to repeat the experiments for Traf1, Traf2, Ask1 from Bloomington Stock Center. And We did not do it before mainly because we wanted to focus on wgn and grnd. However, we agree with the Reviewer 2 and we will do the experiments. Another RNAi from VDRC for grnd and Tak1 have been ordered. These experiments will take about 2 months from the crosses to the analysis of results (some flies still to arrive, and many crosses will be done at 17ºC).

*2 4. In Figure 1E, the authors show that wgn RNAi enhances cell death caused by hh>egr. What is missing here is a wgn RNAi control without hh>egr. Is there any cell death caused by the loss of wgn alone (without hh>egr)? *

This control is now in progress. Expected to have it complete in 2 weeks.

*2.5. If wgn RNAi causes some degree of cell death, is the observed effect with hh>egr a significant genetic interaction, or merely additive? *

The result from the previous comment will help us to respond this point.

• 1. Is the wgn-p38 pathway sufficient to block egr induced cell death? The authors could test this by having hh>egr in the licT1.1 background. The authors have a more complex experiment in Figure 3, where licT1.1 is introduced into the hh>egr, wgn RNAi background. However, testing the effect of licT1.1 without wgn would establish a more direct relationship between egr and wgn-p38. *

We have set the crosses for the experiment hh>egr and licT1.1 as suggested. The results will be included in the new version of the manuscript. 1 month.

Reviewer #3

All comments already carried out and included in the transferred manuscript. See next sections.

__Reviewer #4 __

Major comments:

*4.3 In Figure 5, the cells expressing Rpr appeared to be pulled/extruded basally as expected. It would be beneficial to quantify Wgn and Grnd signals along cross-sections and provide higher magnification images of domain boundaries to illustrate differences in TNFR localization and levels. ** The micrographs for Grnd Figure 5B,D, F capture substantial signal from the peripodial epithelium where the salE/Pv> driver is likely not active? *

We will do a thorough quantification of high-resolution stacks of images and include higher magnification of the analyzed stacks. To this aim, we need some more weeks to collect the images of each genotype, processed and quantify them. We propose to do have this work done in two months.

*4.4 The non-autonomous induction of Wgn seems stronger when facing dying Rpr overexpressing cells simultaneously depleted of Eiger compared to Rpr OE alone. Should this be a reproducible, could the authors discuss potential reason for this observation? *

It is difficult to respond this question, without quantification. The quantification suggested in the previous point, will allow us to state if Wgn is more accumulated in rpr +egr than rpr alone. Therefore, the previous point will tell us if there are significant differences and if, so it will help us to discuss it.

Timing: The entire plan can be executed in 2-3 month.

3. Description of the revisions that have already been incorporated in the transferred manuscript

Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

__Reviewer #1 __

1.1 a- *The result in Fig1.H is somehow surprising. How does the overexpression of Egr induce caspase activation in the absence of its receptor Grnd? *

The results of Fig. 1H, in which egr+grndRNAi+wgnRNAi results in high apoptosis indicates that wgn down regulation compromises survival even in the absence of grnd. The reviewer correctly points that “How does the overexpression of Egr induce caspase activation in the absence of its receptor Grnd?”.

There is evidence that Eiger is involved in the regulation of the pro-apoptotic gene head involution defective (hid) in primordial germ cells (Maezawa 2009 Dev. Growth Differ., 51 (4) (2009), pp. 453-461) and in the elimination of damaged neurons during development (Shklover et al., 2015). Moreover, Eiger is necessary for HID stabilization and regulates HID-induced apoptosis independently of JNK signaling (Shklover et al., 2015). Therefore, in our discs egr activation in the absence of grnd and wgn can still result in apoptosis because of the absence of wgn’s survival signal and, presumably, activation of hid.

We have introduced this issue in the text as:

“To check for epistasis between grnd and wgn, we activated hh> egrweak and knocked down both TNFRs. We found high levels of cell death compared to wgn RNAi alone (Fig. 1H and 1I), which demonstrates that wgn down-regulation is dominant over grnd. This is surprising as it is generally assumed that Egr interacts with Grnd to induce apoptosis via JNK, which in turn activates the proapoptotic gene hid (Andersen et al., 2015; Diwanji & Bergmann, 2020; Fogarty et al., 2016; Igaki et al., 2002; Moreno, Yan, et al., 2002; Sanchez et al., 2019; Shlevkov & Morata, 2012). Interestingly, Egr is necessary for HID stabilization and can regulate HID-induced apoptosis independently of JNK (Shklover et al., 2015). Therefore, cells egrweak that downregulate grnd and wgn can still be eliminated because the lack of both Wgn-survival signal and the pro-apoptotic Grnd/JNK signal could result in an alternative pathway of apoptosis.”

*1.2- In Fig.6, it would be relevant to include wengen inactivation within the domain where rpr is expressed to show that wengen is not required autonomously for regeneration (sal>rpr + wgn RNAi). What is the phenotype of the adult wing of sal-lexA>rpr + nub-gal4 >wgn RNAi animals.? *

We have already added a new figure (Fig. S4C) containing this data. As shown, both wgnRNAi alone and wgn RNAi + rpr do not show relevant anomalies and regenerate normally. Therefore, we conclude that wgn is not autonomously required for regeneration.

The adult wings sal-lexA>rpr + nub-gal4 >wgn RNA result in a strong aberration, as regeneration is inhibited. This experiment has been also added in another figure (Fig. S4B) it is done.

*1.4 Minor- In fig.1I, it is surprising that knockdown of neither Grnd nor dTRAF2 significantly affects Egr-induced apoptosis *

After applying a One-Way ANOVA test to compare all the groups against all the groups in fig. 1B no significative differences were detected between Control and RNAi grnd or RNAi dTRAF2 (p>0,05). But if we apply a Student’s T test, which is less restrictive, we obtain, indeed, significative differences:

Control vs. RNAigrnd p=9,48x10-7

Control vs. RNAi dTRAF2 p=2,47x10-7

We have now added in the text:

“Note that when egrweak cells downregulated dTRAF2 or grnd the cell death area ratio is slightly lower than egrweak alone (Fig. 1I), comfirming that dTRAF2 and Grnd contribute to apoptosis in egrweak cells.”

*1.5 Minor The ability of the wing disc to regenerate has been associated with the induction of a developmental delay mediated by Dilp8. Are the authors observing this developmental delay is the case of sal-lexA>rpr + Ap-gal4 >wgn RNAi or sal-lexA>rpr + Ci-Gal4>wgn RNAi *

The developmental delay due to Dilp8 has been observed by many laboratories, indeed. The question of the reviewer is relevant because if there is no delay in pupariation, regeneration could be compromised not because regeneration has been affected but because after pupariation regeneration is impeded.

However, delay in pupariation has been found in our experiments. Usually for 11hrs of heat shock (to induce apoptosis) we found 1-2 days of delay.

We have added the following text:

“The ability of the wing disc to regenerate after genetic ablation has been associated with the induction of a developmental delay (Colombani et al., 2012; Garelli et al., 2012; Jaszczak et al., 2015; Katsuyama et al., 2015; Smith-Bolton et al., 2009). All genotypes analyzed in figure 6 showed a similar developmental delay of 1-2 days (at 17ºC) after genetic ablation in comparison to the animals of the same genotype in which no genetic ablation was induced, i.e. developed continuously at 17ºC (Fig. S4A). After the adults emerged, the wings were dissected, and regeneration was analyzed.”

*1.7 Minor - The investigation of the evolutionary origin of TNFR in drosophila included in Fig.2 is cutting a bit the flow of the results. *

The evolutionary origin starts now with a sentence that can smoothen the flow and few changes in that paragraph have been made:

“Opposing roles between proteins of the TNFR superfamily suggests that they have an ancient origin and have followed divergent evolutionary paths. To track the differences observed between grnd and wgn, we decided to investigate the evolutionary origin of these two Drosophila genes.”

*1.8 Minor The authors could explain in more details the double transactivation system for non-fly specialists. *

The entire section has been re-written in Material and Methods.

*1.9 Minor - It can be interesting to include and/or discuss these few references: *

*PLoS Genet. 2019 Aug; 15(8): e1008133. ** PLoS Genet. 2022 Dec 5;18(12):e1010533. FEBS Lett. 2023 Oct;597(19):2416-2432. *

*Curr Biol. 2016 Mar 7;26(5):575-84. *

*Nat Commun. 2020 Jul 20;11(1):3631. **

*

All these references, and few others, have been introduced in the text.

__Reviewer #2 __ *2. 1. The authors find that wgn RNAi enhances hh>egr-induced apoptosis. They validate the results with two independent RNAi lines in Figure S1. However, Figure S1 is missing a control without wgn RNAi, and therefore, the results are difficult to assess. *

Fig S1A now contains this control.

• 1. Are the two independent wgn RNAi lines targeting different regions of the coding sequence? *

As the regions targeted by the 2 RNAi’s are different, see below, we have included in the text:

“This observation was corroborated with an independent RNAi-wgn strain targeting a different region in the coding sequence (Fig. S1A and S1B). “

Bloomington BL55275 (dsRNA-HMCO3962)

VDRC GD9152 (dsRNA-GD3427)

*2.7. In Figure 4, the authors show that egr expression induces ROS and performs anti-oxidant experiments. This part could be strengthened if they show that the ROS sensor signal disappears after Sod::Cat expression. *

We had done this experiment and there is a definitively drop in Mitosox in discs in which the weak allele of egr is active. We have included this new image in Figure 4G and in the text.

*2.8. How effective is egr RNAi? In Figure 5E, F, the authors knock down egr and obtain negative results. Based on this, the authors argue that Wgn localization occurs through an egr-independent mechanism. Drawing strong conclusions based on a negative result with egr RNAi is not a good practice since one cannot rule out residual egr activity that mediates the effect (of course , because there is cell death as well, death cells express egr). I suggest either finding ways to completely abolish egr function, or tone down the conclusion. *

We have used ‘after knocking down eiger’ instead of in the ‘absence’ or ‘abolish’ eiger.

• 1. Figure 6 shows that wgn RNAi aggravates the reaper phenotype. What's missing is a control that expresses wgn RNAi but not reaper. *

Control experiments using the UAS-wgnRNAi in the absence of rpr are now shown in figure S4C.

Reviewer #3 ____ 3.1.Minor Fig 6C-E would need a control disc without induced apoptosis (ie wildtype disc) stained for phospho-p38 as a baseline comparison. This is important to judge the significance of the remaining phospho-p38 in panel E where wgn is knocked down. The authors write ** " However, after knocking down wgn, phosphorylated p38 in the wing pouch ** surrounding the apoptotic cells was abolished (Fig. 6E)." *Depending on the amount of phospho-p38 in control discs, this may need to be rephrased to "strongly reduced" instead of "abolished". *

A control disc stained with P-p38 has been added in Figure 6.

We have changes ‘abolished’ by ‘strongly reduced’.

* 3.2. This sentence in the Intro needs fixing because TNFa doesn't transduce the signal from TNFR to Ask1 since it's upstream of TNFR: "TNFα can transduce the TRAF-mediated signal from TNFR to Ask1 to modulate its activity (Hoeflich et al., 1999; Nishitoh et al., 1998, p. 0; Obsil & Obsilova, 2017; Shiizaki et al., 2013)." *

We have rephrased this sentence by:

“TNFα binds to TNFRs which in turn interact with TRAFs to transduce the signal to Ask1 to modulate its activity”.

*3.3a In the results section, the authors start by ectopically overexpressing Eiger. Are there conditions where Eiger expression is induced in the wing? If yes, it would be helpful for the reader to mention that this system with the genetically GAL4-induced expression of Eiger aims to phenocopy one of these conditions. *

Eiger ectopic expression has been induced in the wing to generate apoptosis. This is a common technique in Drosophila, and the Reviewer3 is right that a sentence should be useful for the reader.

A sentence has been introduced at the beginning of the results section:

“Ectopic expression of egr in Drosophila imaginal discs results in JNK-dependent apoptosis (Brodsky et al., 2004; Igaki et al., 2002; Moreno, Yan, et al., 2002).”

*3.3b Fig 2C is not very self-explanatory: it is worth writing out what Hsa (H. sapiens), Bla and Sco stand for (there is plenty of space). *

We have re-designed figure 2 to make it more self-explanatory.

*3.4. This sentence is confusing: ** " ...Wgn localization were due to ROS or to the expression of egr, we used RNAi to knock down egr in the apoptotic cells and found that reduced Egr/TNFα had no effect on Wgn localization (Fig. 5E, 5F)." The authors may want to specify that Wgn is still accumulated even without Egr. ("No effect" is unclear). *

This sentence has been modifies as:

“Wgn localization were due to ROS or to the expression of egr, we used RNAi to knock down egr in the apoptotic cells and found that Wgn accumulation was not altered by the knocking down Egr/TNFα (Fig. 5E, 5F). “

*3.5 Comment. It discovers that Wengen is activated by ROS. In fact, since Wengen binds TNF with an affinity that is several orders of magnitude lower than Grindelwald, and since Wengen is not even located at the cell membrane, these data call into question whether Wengen is a TNF receptor, or a ROS receptor? Could the authors comment on this ? Could it be that the results obtained in the past showing that Wengen is activated by TNF were actually due to TNF inducing apoptosis, leading to production of ROS, leading to activation of Wengen?

*

We totally agree with Reviewer#3. We have added a final paragraph in the discussion section.

“We speculate that the subcellular location of Wgn and Grnd may contribute to the different functions of both receptors. Grnd is more exposed at the apical side of the plasma membrane, which makes this receptor more accessible for ligand interactions (Palmerini et al., 2021). Wgn, embedded in cytoplasmic vesicles, is less accessible to the ligand and could be more restricted to being activated by local sources of signaling molecules, such as ROS. In contrast to initial reports (Kanda et al., 2002; Kauppila et al., 2003), los-of-function of wgn does not rescue Egr-induced apoptosis in the Drosophila eye (Andersen et al., 2015), which supports our observation in the wing that Wgn is not required for Egr-induced apoptosis. Instead, Egr-induced apoptosis generates ROS which target intracellular Wgn to foster a cell survival program of cells close to the apoptotic zone.”

__Reviewer #4 __

*4.1 b Are phospho-p38 levels increased in cells expressing Egr[weak]? *

We have the results of these experiments. To respond to this point, a new figure has been added (Fig. S4) in which we show the P-p38 levels are increased (non-autonomously) in egrw, as previously found for reaper. In addition, we show that egrw + activation of p35 and egrw + activation of Sod1::Cat results in strong reduction of P-p38. This indicates that P-p38 is stimulated by the ROS produced by apoptotic cells.

The text now:

“It is worth noting that cells egrw induce phosphorylation of p38 in neighboring cells (Fig. S4A) and that, as previously found for rpr (REF), depends on ROS generated by egrw apoptotic cells (Fig. S4B, C).”

*4.2 In Figure 4C it appears that the Dcp-1 positive cells move apically rather than basally. Including nuclear staining would be very informative allowing assessment of tissue morphology. ** The authors focus on the pouch region of the wing imaginal disc, where phenotypes are strong and obvious. However, the hh-Gal4 driver also affects posterior cells in the hinge and notum, where the effects of Eiger[weak] overexpression seem weaker (e.g., minimal to no MitoSox signal in hinge and notum posterior cells). Could the authors explain this observation? *

Point 1: Actually, cells move more basally, though some move more apical as well. Depending on the section cells the image could be confusing. To solve that, we show now a plane on these discs at apical and a plane basal. Both high magnifications. There one can see that there is more concentration of pyknotic nuclei basally. We have added this observation in a new supplementary figure (Fig. S3) and the corresponding text in page 5: “Apoptotic cells in egrweak are characterized by pyknotic nuclei and are positive for Dcp1. These cells tend to concentrate in the basal side of the epithelium, although some are scattered apically (Fig. S3). Accumulation of Wgn was observed in healthy anterior cells adjacent to both apical and basal egrweak cells (Fig. 4, Fig. S3A, B).”

Point 2 Comment on MitoSOX in notum: At the stages of the imaginal discs used in this study, almost all notum cells are anterior compartment. The hh positive cells in notum much less abundant, therefore most of the staining was found in the posterior compartment of the wing pouch.

*4.5 Figure 6 C-E. Does WgnRNAi potentiates and GrndRNAi suppress Rpr-induced apoptosis similarly to their effects when knocked down in Eiger[weak]OE cells? *

The areas controlled by salE/Pv >rpr (dotted lines) are full of pycnotic nuclei, which indicates concentration of apoptotic cells in all genotypes shown.

Thus, in the conditions generated here, apoptosis is not inhibited and grnd RNAi does not interfere with the activation of P-p38. In wgn knock down, phospho-p38 is strongly inhibited, indicating the importance of wgn in phosphorylation of p38.

To clarify this point, we have added in the text: “Note that rpr-induced apoptosis is not suppressed after knocking down grnd or wgn.” Also in the figure legend we added: “White lines in the confocal images outline the salE/Pv-LHG,LexO-rpr dark area full of pyknotic nuclei of apoptotic cells.”

4.6 The activation of p38 following salE/Pv>rpr-mediated ablation as shown by immunostaining is noteworthy. While loss Grnd knockdown leads to phospho-p38 signal enrichment around the rpr-expressing cells, WgnRNAi results in reduced phospho-p38 signal in the wing pouch but also beyond the nub-expression domain. Do salE/Pv>rpr nub>WgnRNAi cells still generate ROS?

So far there is no evidence of Wengen as a ROS scavenger. We have evidence that ROS (using MitoSox probe) are produced in egrweak + Wgn RNAi cells. Thus, the inhibition of wgn expression does not block ROS production. A new figure shows this observation (Figure S4A).

4.7 Are ROS responsible for the long-range signaling and p38 activation, referring to authors' previous work Santabarbara-Ruiz et al., 2019, PLoS Genet 15(1): e1007926. https://doi.org/10.1371/journal. pgen.1007926, Figure 5G?

ROS are responsible for p38 activation as shown in a new figure (Fig. S4). In this new figure egrweak is activated in hh, and p38 is most of cells in the posterior compartment, and also anterior. However, after blocking apoptosis or ROS production, this P-p38 is reduced.

4.8 Minor I propose rephrasing the description of "UAS-Egr[weak] transgene, a strain that produces a reduced Egr/TNFα activity". It could imply a loss of function strain rather than a transgene that causes mild/moderate Egr overexpression.

The sentence has been rephrases as suggested (End of the first paragragraph in results section).

*4.9 Minor. I recommend the authors to revise the charts for improved clarity in genotype representation. For example, in Figure 1I, the label "control-GFP" might be misleading. It would be beneficial to specify that "control" refers to Eiger[weak] alone with other manipulations being done simultaneously with Eiger[weak] overexpression. *

All charts have been revised.

4.10 Minor. Additionally, considering that individuals with color blindness may struggle to differentiate between red and green colors, I strongly suggest using a color-blind-friendly palette, especially in Figure 4A, C, G, and Figure 4A, C, E." ** All images have been revised for color blind code.

• 11 Minor. Providing detailed information regarding the reagents used in the study, such as Catalogue Numbers or RRIDs, is beneficial for enhancing reproducibility. *

We have added the RRID and Cat #. If no ID was available, we added the reference or contact.

4.12 This reviewer points two limitations that we are now trying to solve:

*Limitations: *

*Quality of the imaging – higher magnification images and quantification would enhance the study. ** The summarizing model may contain excessive speculations that lack support from the data or references to the existing literature. *

Quality of imaging. We have now an extra supplemental figure with higher magnifications. Extra higher magnifications will be included in the next version as well as quantification, as exposed for the Revision Plan points 4.3 and 4.4.

Model: We have re-written the paragraph on the model, introduced references and drop some speculations. We hope the current version will be more convincing for the reader.

4. Description of analyses that authors prefer not to carry out

Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

Reviewer 1

*1.3. Is the overexpression of Wengen sufficient to increase tissue regeneration? *

The suggestion of the reviewer is a key point in regeneration biology: how to accelerate regeneration?

We have demonstrated that Wengen is upstream the Ask1-p38 axis that drives regeneration. The reviewer wonders if Wengen overexpression can result in increase in regeneration. In a previous work we have demonstrated that p38 activation is key for regeneration but its overexpression can be deleterious (Esteban-Collado et al., 2021). Only in discs that sensitized for low p38 (starvation, low Akt, Ask1S83A mutant), the overexpression rescues regeneration. Therefore, the levels of the Wgn-Ask1-p38 have to be very tightly controlled. An excess will be deleterious. We are aware of the importance of the question, but at this point we do not have the technology to finely control Wgn-Ask1-p38 levels to do this experiment.

1.6 Minor - It possible to test the induction of apoptosis in a wgn null mutant background to see if the phenotype is even stronger than the one observed with RNAi (the wgn RNAi is induced at the same time than egr or rpr overexpression).

Flies wgnKO survive, but they gave us problems when carrying transgenes for our design of genetic ablation. Indeed, we tried to generate wgnKO carrying Gal4+tubGal80+eigerweak without success.

In addition, the reason we have used an inducible mutant is because it allows us to work in time and space without altering expression in other cell types beyond wing discs. Wgn is required in other organs during development like gut, trachea and axon growth, etc.., and thus, we ensure the affected cells belong to the tissue analyzed.

Résumé vidéo [00:00:00][^1^][1] - [00:22:32][^2^][2]:

Cette vidéo explique ce qu'est la dotation horaire globale (DHG) et comment elle est répartie entre les établissements scolaires du second degré. Elle aborde les aspects juridiques, les étapes du processus, les éléments de calcul, les conséquences pour les enseignants et les élèves, et les problèmes rencontrés.

Points forts: + [00:00:00][^3^][3] La DHG, c'est quoi ? * Une enveloppe d'heures d'enseignement attribuée à chaque établissement * Un élément d'autonomie pédagogique et éducative * Un sujet sensible qui impacte les conditions de travail et d'apprentissage + [00:00:44][^4^][4] Le cadre juridique de la DHG * Le code de l'éducation et les arrêtés ministériels qui définissent les horaires réglementaires, les dotations spécifiques, les heures d'autonomie, etc. * Le rôle du chef d'établissement, du conseil pédagogique et du conseil d'administration dans la répartition des heures + [00:05:00][^5^][5] Les étapes du processus de la DHG * La loi de finances qui fixe le budget de l'éducation nationale * La répartition des dotations nationales entre les académies, les départements et les établissements * La communication de la DHG aux chefs d'établissement entre janvier et février * La construction du tableau de répartition des moyens donnés (TRMD) en concertation avec les équipes pédagogiques * Le vote du TRMD en conseil d'administration * Les ajustements possibles en fonction des effectifs réels et des besoins spécifiques + [00:15:01][^6^][6] Les éléments de calcul de la DHG * Les heures réglementaires minimales par discipline et par niveau * Les heures statutaires liées aux obligations de service des enseignants * Les heures spécifiques liées aux projets et aux dispositifs de l'établissement * Les heures d'autonomie qui permettent de moduler la répartition des heures et de proposer des enseignements complémentaires ou facultatifs * Les heures supplémentaires annuelles (HSA) ou effectives (HSE) qui compensent le manque d'heures postes + [00:19:20][^7^][7] Les conséquences de la DHG pour les enseignants et les élèves * Les compléments de service donnés ou reçus qui obligent les enseignants à se partager entre plusieurs établissements * Les suppressions de postes ou de divisions qui entraînent la disparition ou la réduction de certaines disciplines * Les heures dans une autre discipline qui sont proposées aux enseignants sous-service * Les effectifs surchargés qui nuisent à la qualité de l'enseignement et à la sécurité des élèves * Les élèves fictifs qui sont créés pour gonfler les effectifs et sauver les moyens

Impossible à comprendre où va cette ligne de code. Il faut être plus précis, ça devient fatigant. Nous sommes en apprentissage on ne devrait pas avoir à deviner les choses ou a perdre du temps à chercher une information qui devrait nous être fournie.

Ah yes, more restricting rules for women. Code #13 does seem reasonable and it explains why women can't act irrational. However, the rest of the codes that follow BELOW seem like women are deemed as children and irresponsible.

"The Laws of Manu" certainly NOT similar to the "Code of Hammurabi" since the Laws of Manu focuses more so on women.

This a strong law to start with, especially with women being unvalued compared to other code of laws like the Code of Hammurabi. Analyzing from this

This code of law seems familiar because this is how Hispanic and Asian families depict a "ruined" woman. My mother would very much agree. Ha!

From this law code, it seems they value their women. Despite of the woman's low status on the caste they still seem to be valued with their gods.

This page provides an introduction to the concept and utility of functions in Python. The progression from basic functions to more complex ones with parameters and return values is well-structured, making it accessible for beginners like me. Particularly helpful are the practical examples and clear explanation of benefits such as code reusability, ease of maintenance, and improved code organization.

Using a for loop to run a defined function makes it easy for me. I don't need to repeatedly type a large paragraph for it to be paraphrased. At the same time, the def function allows me to be more efficient. By doing this, I can reuse it when I need to, making changes to the code easier as well.

Cadre juridique posé par l'auteur- argument épistémique

This demonstrates the potential of coordinated online campaigns to disrupt corporate behavior and amplify worker demand. By weaponizing data through the mass submission of fake job applications, protesters have leveraged technology to make a tangible impact. This tactic, while controversial, demonstrates a novel form of digital strike that blurs the line between activism and online disruption and raises questions about the ethics and effectiveness of such methods in labor disputes.

Author Response

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

Public Reviews:

Reviewer #1 (Public Review):

The present study provides a phylogenetic analysis of the size prefrontal areas in primates, aiming to investigate whether relative size of the rostral prefrontal cortex (frontal pole) and dorsolateral prefrontal cortex volume vary according to known ecological or social variables.

I am very much in favor of the general approach taken in this study. Neuroimaging now allows us to obtain more detailed anatomical data in a much larger range of species than ever before and this study shows the questions that can be asked using these types of data. In general, the study is conducted with care, focusing on anatomical precision in definition of the cortical areas and using appropriate statistical techniques, such as PGLS. That said, there are some points where I feel the authors could have taken their care a bit further and, as a result, inform the community even more about what is in their data.

We thank the reviewer for this globally positive evaluation of our work, and we appreciate the advices to improve our manuscript.

The introduction sets up the contrast of 'ecological' (mostly foraging) and social variables of a primate's life that can be reflected in the relative size of brain regions. This debate is for a large part a relic of the literature and the authors themselves state in a number of places that perhaps the contrast is a bit artificial. I feel that they could go further in this. Social behavior could easily be a solution to foraging problems, making them variables that are not in competition, but simply different levels of explanation. This point has been made in some of the recent work by Robin Dunbar and Susanne Shultz.

Thank you for this constructive comment, and we acknowledge that the contrast between social vs ecological brain is relatively marginal here. Based also on the first remark by reviewer 3, we have reformulated the introduction to emphasize what we think is actually more critical: the link between cognitive functions as defined in laboratory conditions and socio-ecological variables measured in natural conditions. And the fact that here, we use brain measures as a potential tool to relate these laboratory vs natural variables through a common scenario. Also, we were already mentioning the potential interaction between social and foraging processes in the discussion, but we are happy to add a reference to recent studies by S. Shultz and R. Dunbar (2022), which is indeed directly relevant. We thank the reviewer for pointing out this literature.

In a similar vein, the hypotheses of relating frontal pole to 'meta-cognition' and dorsolateral PFC to 'working memory' is a dramatic oversimplification of the complexity of cognitive function and does a disservice to the careful approach of the rest of the manuscript.

We agree that the formulation of which functions we were attributing to the distinct brain regions might not have been clear enough, but the functional relation between frontal pole and metacognition in the one hand, and DLPFC and working memory on the other hand, have been firmly established in the literature, both through laboratory studies and through clinical data. Clearly, no single brain region is necessary and sufficient for any cognitive operation, but decades of neuropsychology have demonstrated the differential implication of distinct brain regions in distinct functions, which is all we mean here. We have made a specific point on that topic in the discussion (cf p. 16). We have also reformulated the introduction to clarify that, even if the relation between these regions and their functions (FP/ metacognition; DLPFC/ working memory) was clear in laboratory conditions, it was not clear whether this mapping could be used for real life conditions. And therefore whether that simplification was somehow justified beyond the lab (and the clinics), and whether these neuro-cognitive concepts could be applied to natural conditions, are indeed critical questions that we wanted to address. The central goal of the present study was precisely to evaluate the extent to which this brain/cognition relation could be used to understand more natural behaviors and functions, and we hope that it appears more clearly now.

One can also question the predicted relationship between frontal pole meta-cognition and social abilities versus foraging, as Passingham and Wise show in their 2012 book that it is frontal pole size that correlates with learning ability-an argument that they used to relate this part of the brain to foraging abilities. I would strongly suggest the authors refrain from using such descriptive terms. Why not simply use the names of the variables actually showing significant correlations with relative size of the areas?

We basically agree with the reviewer, and we acknowledge the lack of clarity in the introduction of the previous manuscript. There were indeed lots of ambiguity in what we were referring to as ‘function’, associated with a given brain region. « Function » referred to way to many things! We have reformulated the introduction not only to clarify the different types of functions that were attributed to distinct brain regions in the literature but also to clarify how this study was addressing the question: by trying to articulate concepts from neuroscience laboratory studies with concepts from behavioral ecology and evolution using intuitive scenarios. We hope that the present version of the introduction makes that point clearer.

The major methodological judgements in this paper are of course in the delineation of the frontal pole and dorsolateral prefrontal cortex. As I said above, I appreciate how carefully the authors describe their anatomical procedure, allowing researchers to replicate and extend their work. They are also careful not to relate their regions of interest to precise cytoarchitectonic areas, as such a claim would be impossible to make without more evidence. That said, there is a judgement call made in using the principal sulcus as a boundary defining landmark for FP in monkeys and the superior frontal sulcus in apes. I do not believe that these sulci are homologous. Indeed, the authors themselves go on to argue that dorsolateral prefrontal cortex, where studied using cytoarchitecture, stretches to the fundus of principal sulcus in monkeys, but all the way to the inferior frontal sulcus in apes. That means that using the fundus of PS is not a good landmark.

We thank the reviewer for his kind remarks on our careful descriptions. But then, it is not clear whether our choice of using the principal sulcus as a boundary for FP in monkeys vs the superior frontal sulcus in apes is actually a judgement call. First, and foremost, there is no clear and unambiguous definition of what should be the boundaries of the FP. By contrast with cytoarchitectonic maps, but clearly this is out of reach here. In humans and great apes we used Bludau et al 2014 (i.e. sup frontal sulcus), and in monkeys, we chose a conservative landmark that eliminated area 9, which is traditionally associated with the DLPFC (Petrides, 2005; Petrides et al, 2012; Semendeferi et al, 2001).

Of course, any definition will attract criticism, so the best solution might be to run the analysis multiple times, using different definitions for the areas, and see how this affects results.

Indeed, functional maps indicate that dorsal part of anterior PFC in monkeys is functionally part of FP. But again, cytoarchitectonic maps also indicate that this part of the brain includes BA 9, which is traditionally associated with DLPFC (Petrides, 2005; Petrides et al, 2012). As already pointed out in the discussion, there is a functional continuum between FP and DLPFC and our goal when using PS as dorsal border was to be very conservative and to exclude the ambiguous area. But we agree with the reviewer that given that this decision is arbitrary, it was worth exploring other definitions of the FP volume. So, we did complete a new analysis with a less conservative definition of the FP, to include this ambiguous dorsal area, and it is now included in the supplementary material. Maybe as expected, including the ambiguous area in the FP volume shifted the relation with socio-ecological variables towards the pattern displayed by the DLPFC (ie the influence of population density decreased). The most parsimonious interpretation of this results is that when extending the border of the FP region to cover a part of the brain which might belong to the DLPFC, or which might be somehow functionally intermediate between the 2, the specific relation of the FP with socio-ecological variables decreases. Thus, even if we agree that it was important to conduct this analysis, we believe that it only confirms the difficulty to identify a clear boundary between FP and DLPFC. Again, we have clearly explained throughout the manuscript that we admit the lack of precision in our definitions of the functional brain regions. In that frame, the conservative option seems more appropriate and for the sake of clarity, the results of the additional analysis of a FP volume that includes the ambiguous area is only included in the supplementary material.

If I understand correctly, the PGLS was run separately for the three brain measure (whole brain, FP, DLPFC). However, given that the measures are so highly correlated, is there an argument for an analysis that allows testing on residuals. In other words, to test effects of relative size of FP and DLPFC over and above brain size?

Generally, using residuals as “data” (or pseudo-data) is not recommended in statistical analyses. Two widely cited references from the ecological literature are:

Garcia-Berthou E. (2001) On the Misuse of Residuals in Ecology: Testing Regression Residuals vs. the Analysis of Covariance. Journal of Animal Ecology, 70 (4): 708-711.

Freckleton RP. (2002). On the misuse of residuals in ecology: regression of residuals vs. multiple regression. Journal of Animal Ecology 71: 542–545. https://doi.org/10.1046/ j.1365-2656.2002.00618.x.

The main reason for this recommendation is that residuals are dependent on the fitted model, and thus on the particular sample under consideration and the eventual significant effects that can be inferred.

In the discussion and introduction, the authors discuss how size of the area is a proxy for number of neurons. However, as shown by Herculano-Houzel, this assumption does not hold across species. Across monkeys and apes, for instance, there is a different in how many neurons can be packed per volume of brain. There is even earlier work from Semendeferi showing how frontal pole especially shows distinct neuron-to-volume ratios.

We appreciate the reviewer’s comment, but the references to Herculano-Houzel that we have in mind do indicate that the assumption is legitimate within primates.

Herculano-Houzel et al (2007) show that the neuronal density of the cortex is well conserved across primate species (but only monkeys were studied). The conclusion of that study is that using volumes as a proxy for number of neurons, as a measure of computational capacity, should be avoided between rodents and primates (and as they showed later, even more so with birds, for which neuronal density is higher). BUT within primates, since neuronal densities are conserved, volume is a good predictor of number of neurons. Gabi et al (2016) provide evidence that the neuronal density of the PFC is well conserved between humans and non-human primates, which implies that including humans and great apes in the comparison is legitimate. In addition, the brain regions included in the analysis presumably include very similar architectonic regions (e.g. BA 10 for FP, BA 9/46 for DLPFC), which also suggests that the neuronal density should be relatively well conserved across species. Altogether, we believe that there is sufficient evidence to support the idea that the volume of a PFC region in primates is a good proxy for the number of neurons in that region, and therefore of its computational capacity.

Semendeferi and colleagues (2001) pointed out some differences in cytoarchitectonic properties across parts of the FP and discussed how these properties could 1) be used to identify area 10 across species 2) be associated with distinct computational properties, with the idea that thicker ‘cell body free’ layers would leave more space for establishing connections (across dendrites and axons). This pioneering work, together with more recent imaging studies on functional connectivity (e.g. Sallet et al, 2013) emphasize the critical contribution of connectivity pattern as a tool for comparative anatomy. But unfortunately, as pointed out in the discussion already, this is currently out of reach for us.

We acknowledge the limitations, and to be fair, the notion of computational capacity itself is hard to define operationally. Based on the work of Herculano-Houzel et al, average density is conserved enough across primates (including humans) to justify our approximation. We have tried to define our regions of interest using both anatomical and functional maps and, thanks to the reviewer’s suggestions, we even tried several ways to segment these regions. Functional maps in macaques and humans do not exactly match cytoarchitectonic maps, presumably because functions rely not only upon the cytoarchitectonics but also on connectivity patterns (e.g. Sallet et al, 2013).

In sum, we appreciate the reviewer’s point but feel that, given the current understanding of brain functions and the relative conservation of neuronal density across primate PFC regions, the volume of a PFC region seems to be reasonable proxy for its number of neurons, and therefore its computational capacity. We have added these points to the discussions, and we hope that the reader will be able to get a fair sense of how legitimate is that position, given the literature.

Overall, I think this is a very valuable approach and the study demonstrates what can now be achieved in evolutionary neuroscience. I do believe that they authors can be even more thorough and precise in their measurements and claims.

Reviewer #2 (Public Review):

In the manuscript entitled "Linking the evolution of two prefrontal brain regions to social and foraging challenges in primates" the authors measure the volume of the frontal pole (FP, related to metacognition) and the dorsolateral prefrontal cortex (DLPFC, related to working memory) in 16 primate species to evaluate the influence of socio-ecological factors on the size of these cortical regions. The authors select 11 socio-ecological variables and use a phylogenetic generalized least squares (PGLS) approach to evaluate the joint influence of these socio-ecological variables on the neuro-anatomical variability of FP and DLPFC across the 16 selected primate species; in this way, the authors take into account the phylogenetic relations across primate species in their attempt to discover the influence of socio-ecological variables on FP and DLPF evolution.

The authors run their studies on brains collected from 1920 to 1970 and preserved in formalin solution. Also, they obtained data from the Mussée National d´Histoire Naturelle in Paris and from the Allen Brain Institute in California. The main findings consist in showing that the volume of the FP, the DLPFC, and the Rest of the Brain (ROB) across the 16 selected primate species is related to three socio-ecological variables: body mass, daily traveled distance, and population density. The authors conclude that metacognition and working memory are critical for foraging in primates and that FP volume is more sensitive to social constraints than DLPFC volume.

The topic addressed in the present manuscript is relevant for understanding human brain evolution from the point of view of primate research, which, unfortunately, is a shrinking field in neuroscience.

We must not have been clear enough in our manuscript, because our goal is precisely not to separate humans from other primates. This is why, in contrast to other studies, we have included human and non-human primates in the same models. If our goal had been to study human evolution, we would have included fossil data (endocasts) from the human lineage.

But the experimental design has two major weak points: the absence of lissencephalic primates among the selected species and the delimitation of FP and DLPFC. Also, a general theoretical and experimental frame linking evolution (phylogeny) and development (ontogeny) is lacking.

We admit that lissencephalic species could not be included in this study because we use sulci as key landmarks. We believe that including lissencephalic primates would have introduced a bias and noise in our comparisons, as the delimitations and landmarks would have been different for gyrencephalic and lissencephalic primates. Concerning development, it is simply beyond the scope of our study.

Major comments.

1) Is the brain modular? Is there modularity in brain evolution?: The entire manuscript is organized around the idea that the brain is a mosaic of units that have separate evolutionary trajectories:

"In terms of evolution, the functional heterogeneity of distinct brain regions is captured by the notion of 'mosaic brain', where distinct brain regions could show a specific relation with various socio-ecological challenges, and therefore have relatively separate evolutionary trajectories".

This hypothesis is problematic for several reasons. One of them is that each evolutionary module of the brain mosaic should originate in embryological development from a defined progenitor (or progenitors) domain [see García-Calero and Puelles (2020)]. Also, each evolutionary module should comprise connections with other modules; in the present case, FP and DLPFC have not evolved alone but in concert with, at least, their corresponding thalamic nuclei and striatal sector. Did those nuclei and sectors also expand across the selected primate species? Can the authors relate FP and DLPFC expansion to a shared progenitor domain across the analyzed species? This would be key to proposing homology hypotheses for FP and DLPFC across the selected species. The authors use all the time the comparative approach but never explicitly their criteria for defining homology of the cerebral cortex sectors analyzed.

We do not understand what the referee is referring to with the word ‘module’, and why it relates to development. Same thing for the anatomical relation with subcortical structures. Yes, the identity of distinct functional cortical regions relies upon subcortical inputs during development, but clearly this is neither technically feasible, nor relevant here anyways.

We acknowledge, however, that our definition of functional regions was not precise enough, and we have updated the introduction to clarify that point. In short, we clearly do not want to make a strong case for the functional borders that we chose for the regions of interest here (FP and DLPFC), but rather use those regions as proxies for their corresponding functions as defined in laboratory conditions for a couple of species (rhesus macaques and humans, essentially).

Contemporary developmental biology has showed that the selection of morphological brain features happens within severe developmental constrains. Thus, the authors need a hypothesis linking the evolutionary expansion of FP and DLPFC during development. Otherwise, the claims form the mosaic brain and modularity lack fundamental support.

Once again, we do not think that our definition of modules matches what the reviewer has in mind, i.e. modules defined by populations of neurons that developed together (e.g. visual thalamic neurons innervating visual cortices, themselves innervating visual thalamic neurons). Rather, the notion of mosaic brain refers to the fact that different parts of the brain are susceptible to distinct (but not necessarily exclusive) sources of selective pressures. The extent to which these ‘developmental’ modules are related to ‘evolutionary’ modules is clearly beyond the scope of this paper.

Our goal here was to evaluate the extent to which modules that were defined based on cognitive operations identified in laboratory conditions could be related (across species) to socio-ecological factors as measured in wild animals. Again, we agree that the way these modules/ functional maps were defined in the paper were confusing, and we hope that the new version of the manuscript makes this point clearer.

Also, the authors refer most of the time to brain regions, which is confusing because they are analyzing cerebral cortex regions.

We do not understand why the term ‘brain’ is more confusing than ‘cerebral cortex’, especially for a wide audience.

2) Definition and delimitation of FP and DLPFC: The precedent questions are also related to the definition and parcellation of FP and DLPFC. How homologous cortical sectors are defined across primate species? And then, how are those sectors parcellated?

The authors delimited the FP:

"...according to different criteria: it should match the functional anatomy for known species (macaques and humans, essentially) and be reliable enough to be applied to other species using macroscopic neuroanatomical landmarks".

There is an implicit homology criterion here: two cortical regions in two primate species are homologs if these regions have similar functional anatomy based on cortico-cortical connections. Also, macroscopic neuroanatomical landmarks serve to limit the homologs across species.

This is highly problematic. First, because similar function means analogy and not necessarily homology [for further explanation see Puelles et al. (2019); García-Cabezas et al. (2022)].

We are not sure to follow the Reviewer’s point here. First, it is not clear what would be the evolutionary scenario implied by this comment (evolutionary divergence followed by reversion leading to convergence?). Second, based on the literature, both the DLPFC and the FP display strong similarities between macaques and humans, in terms of connectivity patterns (Sallet et al, 2013), in terms of lesion-induced deficit and in terms of task-related activity (Mansouri et al, 2017). These criteria are usually sufficient to call 2 regions functionally equivalent. We do not see how this explanation is "highly problematic" as it is clearly the most parsimonious based on our current knowledge.

Second, because there are several lissencephalic primate species; in these primates, like marmosets and squirrel monkeys, the whole approach of the authors could not have been implemented. Should we suppose that lissencephalic primates lack FP or DLPFC?

We understand neither the reviewer’s logic, nor the tone. We understand that the reviewer is concerned by the debate on whether some laboratory species are more relevant than others for studying the human prefrontal cortex, but this is clearly not the objective of our work. As explained in the manuscript, we identified FP and DLPFC based on functional maps in humans and laboratory monkeys (macaques), and we used specific gyri as landmarks that could be reliably used in other species. And, as rightfully pointed out by reviewer 1, this is in and off itself not so trivial. Of course, lissencephalic animals could not be studied because we could not find these landmarks, but why would it mean that they do not have a prefrontal cortex? The reviewer implies that species that we did not study do not have a prefrontal cortex, which makes little sense. Standards in the field of comparative anatomy of the PFC, especially when it implies rodents (lissencephalic also) include cytoarchitectonic and connectivity criteria, but obviously we are not in a position to address it here. We have, however, included references to the seminal work of Angela Roberts and collaborator in the discussion on marmosets prefrontal functions, to reinforce the idea that the functional organization is relatively well conserved across all primates (with or without gyri on their brain) (Dias et al, 1996; Roberts et al, 2007).

Do these primates have significantly more simplistic ways of life than gyrencephalic primates? Marmosets and squirrel monkeys have quite small brains; does it imply that they have not experience the influence of socio-ecological factors on the size of FP, DLPFC, and the rest of the brain?

Again, none of this is relevant here, because we could not draw conclusions on species that we cannot study for methodological reasons. The reviewer seems to believe that an absence of evidence is equivalent to an evidence of absence, but we do not.

The authors state that:

"the strong development of executive functions in species with larger prefrontal cortices is related to an absolute increase in number of neurons, rather than in an increase in the ration between the number of neurons in the PFC vs the rest of the brain".

How does it apply to marmosets and squirrel monkeys?

Again, we do not understand the reviewer’s point, since it is widely admitted that lissencephalic monkeys display both a prefrontal cortex and executive functions (again, see the work of Angela Roberts cited above). Our goal here was certainly not to get into the debate of what is the prefrontal cortex in a handful of laboratory species, but to evaluate the relevance of laboratory based neuro-cognitive concepts for understanding primates in general, and in their natural environment.

References:

García-Cabezas MA, Hacker JL, Zikopoulos B (2022) Homology of neocortical areas in rats and primates based on cortical type analysis: an update of the Hypothesis on the Dual Origin of the Neocortex. Brain structure & function Online ahead of print. doi:doi.org/ 10.1007/s00429-022-02548-0

García-Calero E, Puelles L (2020) Histogenetic radial models as aids to understanding complex brain structures: The amygdalar radial model as a recent example. Front Neuroanat 14:590011. doi:10.3389/fnana.2020.590011

Nieuwenhuys R, Puelles L (2016) Towards a New Neuromorphology. doi:10.1007/978-3-319-25693-1

Puelles L, Alonso A, Garcia-Calero E, Martinez-de-la-Torre M (2019) Concentric ring topology of mammalian cortical sectors and relevance for patterning studies. J Comp Neurol 527 (10):1731-1752. doi:10.1002/cne.24650

Reviewer #3 (Public Review):

This is an interesting manuscript that addresses a longstanding debate in evolutionary biology - whether social or ecological factors are primarily responsible for the evolution of the large human brain. To address this, the authors examine the relationship between the size of two prefrontal regions involved in metacognition and working memory (DLPFC and FP) and socioecological variables across 16 primate species. I recommend major revisions to this manuscript due to: 1) a lack of clarity surrounding model construction; and 2) an inappropriate treatment of the relative importance of different predictors (due to a lack of scaling/normalization of predictor variables prior to analysis). My comments are organized by section below:

We thank the reviewer for the globally positive evaluation and for the constructive remarks. Introduction:

• Well written and thorough, but the questions presented could use restructuring.

Again, we thank the reviewer, and we believe that this is coherent with some of the remarks of reviewer 1. We have extensively revised the introduction, toning down the social vs ecological brain issue to focus more on what is the objective of the work (evaluating the relevance of lab based neuro-cognitive concepts for understanding natural behavior in primates).

Methods:

• It is unclear which combinations of models were compared or why only population density and distance travelled tested appear to have been included.

The details of the model comparison analysis were presented as a table in the supplementary material (#3, details of the model comparison data), but we understand that this was not clear enough. We have provided more explanation both in the main manuscript and in the supplements. All variables were considered a priori; however, we proceeded beforehand to an exploratory analyses which led us to exclude some variables because of their lack of resolution (not enough categories for qualitative variables) or strong cross-correlations with other quantitative variables. There were much more than three variables included in the models but the combination of these 3 (body mass, daily traveled distance and population density) best predicted (had the smallest AIC) the size of the brain regions. We provide additional information about these exploratory analyses in the supplementary material, sections 2 and 3.

• Brain size (vs. body size) should be used as a predictor in the models.

We do not understand the theoretical reason for replacing body size by brain size in the models. Brain size is not a socio-ecological variable. And of course, that would be impossible for modeling brain size itself. Or is it that the reviewer suggests to use brain size as a covariate to evaluate the effects of other variables in the model over and above the effect on brain size? But what is the theoretical basis for this?

• It is not appropriate to compare the impact of different predictors using their coefficients if the variables were not scaled prior to analysis.

We thank the Reviewer for this comment; however, standardized coefficients are not unproblematic because their calculations are based on the estimated standard-deviations of the variables which are likely to be affected by sampling (in effect more than the means). We note that the methods of standardized coefficients have attracted several criticisms in the literature (see the References section in https://en.wikipedia.org/wiki/Standardized_coefficient). Nevertheless, we now provide a table with these coefficients which makes an easy comparison for the present study. We also updated tables 1, 2 and 3 to include standardized beta values.

Reviewer #1 (Recommendations For The Authors):

N/A

Reviewer #2 (Recommendations For The Authors):

Contemporary developmental biology has showed that the brain of all mammals, including primates, develops out of a bauplan (or blueprint) made of several fundamental morphological units that have invariant topological relations across species (Nieuwenhuys and Puelles 2016).

At some point in the discussion the authors acknowledge that:

"Our aim here was clearly not to provide a clear identification of anatomical boundaries across brain regions in individual species, as others have done using much finer neuroanatomical methods. Such a fine neuroanatomical characterization appears impossible to carry on for a sample size of species compatible with PGLS".

I do not think it would be impossible to carry such neuroanatomical characterization. It would take time and effort, but it is feasible. Such characterization, if performed within the framework of contemporary developmental biology, would allow for well-founded definition and delineation of cortical sectors across primate species, including lissencephalic ones, and would allow for meaningful homologies and interspecies comparisons.

We do not see how our work would benefit from developmental biology at that point, because it is concerned with evolution, and these are very distinct biological phenomena. We do not understand the reviewer’s focus on lissencephalic species, because they are not so prevalent across primates, and it is unlikely that adding a couple of lissencephalic species will change much to the conclusions.

Minor points:

• Please, format references according to the instructions of the journal.

Ok - done

• The authors could use the same color code across Figures 1, 2, and 3.

Ok – done

• The authors say that group hunting "only occurs in a few primate species", but it also occurs in wolves, whales, and other mammalian species.

We focus on primates here, these other species are irrelevant. Again, this is beside the point.

Reviewer #3 (Recommendations For The Authors):

My comments are organized by section below:

Introduction:

• Well written and thorough

• The two questions presented towards the end of the intro are not clear and do not guide the structure of the methods/results sections. I believe one it would be more appropriate to ask if: 1) the relative proportions of the FP and DLPFC (relative to ROB) are consistent across primates; and 2) if the relative size of these region is best predicted by social and/ or ecological variables. Then, the results sections could be organized according to these questions (current results section 1 = 1; current results sections 2, 3, 4 = 2.1, 2.2, 2.3)

As explained above, we agree with the reviewer that the introduction was somehow misleading and we have edited it extensively. We do not, however, agree with the reviewer regarding the relative (vs absolute) measure. We have discussed this in our response to reviewer 1 regarding the comparison of regional volumes as proxies for number of neurons. The best predictor of the computing capacity of a brain region is its number of neurons, but there is no reason to believe that this capacity should decrease if the rest of the brain increases, as implied by the relative measure that the reviewer proposes. That debate is probably critical in the field of comparative neuroanatomy, and confronting different perspectives would surely be both interesting and insightful, but we feel that it is beyond the scope of the present article.

Methods:

• While the methods are straightforward and generally well described, it is unclear which combinations of models were compared or why only population density and distance travelled tested appear to have been included (in e.g., Fig SI 3.1) even though many more variables were collected.

We agree that this was not clear enough, and we have tried to improve the description of our model comparison approach, both in the main text and in the supplementary material.

• Why was body mass rather than ROB used as a predictor in the models? The authors should instead/also include analyses using ROB (so the analysis is of FP and DLPFC size relative to brain size). Using body mass confounds the analyses since they will be impacted by differences in brain size relative body size.


Again, we have addressed this issue above. First, body size is a socio-ecological variable (if anything, it especially predicts energetic needs and energy expenditure), but ROB is clearly not. We do not see the theoretical relevance of ROB in a socio-ecological model. Second, from a neurobiological point of view, since within primates the volume of a given brain region is directly related to its number of neurons (again, see work of Herculano-Houzel), which is a good proxy for its computing capacity, we do not see the theoretical reason for considering ROB.

• It is not appropriate to compare the impact of different predictors using their coefficients if the variables were not scaled prior to analysis. The authors need to implement this in their approach to make such claims.

We thank the reviewer again for pointing that out. We have addressed this question above.

• Differences across primates in terms of frontal lobe networks throughout the brain should be acknowledged (e.g., Barrett et al. 2020, J Neurosci).

We have added that reference to the discussion, together with other references showing that the difference between human and non-human primates is significant, but essentially quantitative, rather than qualitative (the building blocks are relatively well conserved, but their relative weight differs a lot). Thank you for pointing it out.

I hope the authors find my comments helpful in revising their manuscript.

And we thank again the reviewer for the helpful and constructive comments.

Author Response

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

eLife assessment

This manuscript provides a fundamental contribution to the understanding of the role of intrinsically disordered proteins in circadian clocks and the potential involvement of phase separation mechanisms. The authors convincingly report on the structural and biochemical aspects and the molecular interactions of the intrinsically disordered protein FRQ. This paper will be of interest to scientists focusing on circadian clock regulation, liquid-liquid phase separation, and phosphorylation.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

"Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock" is a convincing manuscript that delves into the structural and biochemical aspects of FRQ and the FFC under both LLPS and non-LLPS conditions. Circadian clocks serve as adaptations to the daily rhythms of sunlight, providing a reliable internal representation of local time.

All circadian clocks are composed of positive and negative components. The FFC contributes negative feedback to the Neurospora circadian oscillator. It consists of FRQ, CK1, and FRH. The FFC facilitates close interaction between CK1 and the WCC, with CK1-mediated phosphorylation disrupting WCC:c-box interactions necessary for restarting the circadian cycle.

Despite the significance of FRQ and the FFC, challenges associated with purifying and stabilizing FRQ have hindered in vitro studies. Here, researchers successfully developed a protocol for purifying recombinant FRQ expressed in E. coli.

Armed with full-length FRQ, they utilized spin-labeled FRQ, CK1, and FRH to gain structural insights into FRQ and the FFC using ESR. These studies revealed a somewhat ordered core and a disordered periphery in FRQ, consistent with prior investigations using limited proteolysis assays. Additionally, p-FRQ exhibited greater conformational flexibility than np-FRQ, and CK1 and FRH were found in close proximity within the FFC. The study further demonstrated that under LLPS conditions in vitro, FRQ undergoes phase separation, encapsulating FRH and CK1 within LLPS droplets, ultimately diminishing CK1 activity within the FFC. Intriguingly, higher temperatures enhanced LLPS formation, suggesting a potential role of LLPS in the fungal clock's temperature compensation mechanism.

Biological significance was supported by live imaging of Neurospora, revealing FRQ foci at the periphery of nuclei consistent with LLPS. The amino acid sequence of FRQ conferred LLPS properties, and a comparison of clock repressor protein sequences in other eukaryotes indicated that LLPS formation might be a conserved process within the negative arms of these circadian clocks.

In summary, this manuscript represents a valuable advancement with solid evidence in the understanding of a circadian clock system that has proven challenging to characterize structurally due to obstacles linked to FRQ purification and stability. The implications of LLPS formation in the negative arm of other eukaryotic clocks and its role in temperature compensation are highly intriguing.

Strengths:

The strengths of the manuscript include the scientific rigor of the experiments, the importance of the topic to the field of chronobiology, and new mechanistic insights obtained.

Weaknesses:

This reviewer had questions regarding some of the conclusions reached.

Recommendations For The Authors:

The reviewer has a few questions for the authors:

1) Concerning the reduced activity of sequestered CK1 within LLPS droplets with FRQ, to what extent is this decrease attributed to distinct buffer conditions for LLPS formation compared to non-LLPS conditions?

We don’t believe that these buffer conditions significantly influence the change in FRQ phosphorylation by CK1 observed at elevated temperatures. The pH and ionic strength of the buffer are in keeping with physiological conditions (300 mM NaCl, 50 mM sodium phosphate, 10 mM MgCl2, pH 7.5); CK1 autophosphorylation is robust and generally increases with temperature under these conditions (Figure 7B). However, as LLPS increases CK1 autophosphorylation remains high, whereas phosphorylation of FRQ dramatically decreases. In fact, we chose to alter temperature specifically to induce changes in phase behavior under constant buffer conditions. In this way LLPS could be increased, and FRQ phosphorylation evaluated, without altering the solution composition. Thus, we believe that the reduced CK1 kinase activity toward FRQ as a substrate is directly due to the impact of the generated LLPS milieu, i.e. the changes in structural/dynamic properties of FRQ and/or CK1 induced by the effects of being a phase separate microenvironment, which could be substantially different from non-phase separated buffer environment. For example, previous work done on the disordered region of DDX4 [Brady et al. 2017, and Nott et al. 2015] show that even the amount of water content and stability of biomolecules such as double strand nucleic acids encapsulated within the droplets differ between non- and phase separated DDX4 samples.

Nott T.J. et al. Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles. Mol. Cell. 2015 57 936-947.

Brady J.P. et al. Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation. PNAS 2017 114 8194-8203.

In the results section we have clarified the use of temperature to control LLPS, “We compared the phosphorylation of FRQ by CK1 in a buffer that supports phase separation under different temperatures, using the latter as a means to control the degree of LLPS without altering the solution composition.”

On p.16 of the discussion we have elaborated on the above point, “We believe that the reduced CK1 kinase activity toward FRQ as a substrate is directly due to the impact of the generated LLPS milieu, i.e. the changes in structural/dynamic properties of FRQ and/or CK1 induced by the effects of being a phase separate microenvironment, which could be substantially different from non-phase separated buffer environment. For example, previous work done on the disordered region of DDX4 {Brady, 2017 #130;Nott, 2015 #131} show that even the amount of water content and stability of biomolecules such as double strand nucleic acids encapsulated within the droplets differ between non- and phase separated DDX4 samples. Indeed, the spin-labeling experiments indicate that the dynamics of FRQ have been altered by LLPS (Fig. 7D).”

2) The DEER technique demonstrated spatial proximity between FRH and CK1 when bound to FRQ in the FFC. Is there evidence suggesting their lack of proximity in the absence of FRQ? Also, how important is this spatial proximity to FFC function?

We have additional data substantiating that FRH and CK1 do not interact in the absence of FRQ. In the revised paper we have included the results of a SEC-MALS experiment showing that FRH and CK1 elute separately when mixed in equimolar amounts and applied to an analytical S200 column coupled to a MALS detector (Figure 1 below and Fig. S8). The importance of the FRH and CK1 proximity is currently unknown, but there are reasons to believe that it could have functional consequences. For example, CK1, as recruited by FRQ, phosphorylates the White-Collar Complex (WCC) in the repressive arm of the circadian oscillator [e.g. He et al. Genes Dev. 20, 2552 (2006); Wang et al, Mol. Cell 74, 771 (2019)]. Interactions between the WCC and the FFC are mediated at least in part by FRH binding to White Collar-2 [Conrad et al. EMBO J. 35, 1707 (2016)]. Thus, FRH:FRQ may effectively bridge CK1 to the WCC to facilitate the phosphorylation of the latter by the former.

He et al. CKI and CKII mediate the FREQUENCY-dependent phosphorylation of the WHITE COLLAR complex to close the Neurospora circadian negative feedback loop. Genes Dev. 2006 20, 2552-2565.

Wang B. et al. The Phospho-Code Determining Circadian Feedback Loop Closure and Output in Neurospora Mol. Cell 2019 74, 771-784.

Conrad et al. Structure of the frequency-interacting RNA helicase: a protein interaction hub for the circadian clock. EMBO J. 2016 35, 1707-1719.

Author response image 1.

Size-exclusion chromatography- multiangle light scattering (SEC-MALS) of a mixture of purified FRH and CK1. The proteins elute separately as monomers with no evidence of co-migration.

3) Is there any indication that impairing FRQ's ability to undergo LLPS disrupts clock function?

We do not currently have direct evidence that LLPS of FRQ is essential for clock function. These experiments are ongoing, but complicated by the fact that changes to FRQ predicted to alter LLPS behavior also have the potential to perturb its many other clock-related functions that include dynamic interactions with partners, dynamic post-translational modification and rates of synthesis and degradation. That said, the intrinsic disorder of FRQ is important for it to act as a protein interaction hub, and large intrinsically disordered regions (IDRs) very often mediate LLPS, as is certainly the case here. In this work, we argue that the ability of FRQ to sequester clock proteins during the TTFL may involve LLPS. Additionally, we show that the phosphorylation state of FRQ, which is a critical factor in clock period determination, depends on LLPS. Given that the conditions under which FRQ phase separates are physiological in nature and that live-cell imaging is consistent with FRQ phase separation in the nucleus, it seems likely that FRQ does phase separate in Neurospora. Furthermore, given that the sequence features of FRQ that mediate phase-separation are conserved not only across FRQ homologs but also in other functionally related clock proteins, it is probable, albeit worthy of further investigation, that LLPS has functional consequences for the clock. See the response to reviewer 3 for more discussion on this topic.

Minor Points:

• It might be worth noting that the cyanobacterial circadian clock also regulates LLPS, albeit involving metabolic enzymes (https://doi.org/10.1016/j.celrep.2020.108032).

Indeed, we have included a reference to this paper on p. 3: “Emerging studies in plants (Jung, et al., 2020), flies (Xiao, et al., 2021) and cyanobacteria (Cohen, et al., 2014; Pattanayak, et al., 2020) implicate LLPS in circadian clocks, and in Neurospora it has recently been shown that the Period-2 (PRD-2) RNA-binding protein influences frq mRNA localization through a mechanism potentially mediated by LLPS (Bartholomai, et al., 2022).”

• On page 9, six lines from the top, please insert "of" between "distributions" and "p-FRQ".

We have corrected this typo.

Reviewer #2 (Public Review):

Summary:

This study presents data from a broad range of methods (biochemical, EPR, SAXS, microscopy, etc.) on the large, disordered protein FRQ relevant to circadian clocks and its interaction partners FRH and CK1, providing novel and fundamental insight into oligomerization state, local dynamics, and overall structure as a function of phosphorylation and association. Liquid-liquid phase separation is observed. These findings have bearings on the mechanistic understanding of circadian clocks, and on functional aspects of disordered proteins in general.

Strengths:

This is a thorough work that is well presented. The data are of overall high quality given the difficulty of working with an intrinsically disordered protein, and the conclusions are sufficiently circumspect and qualitative to not overinterpret the mostly low-resolution data.

Weaknesses:

None

Recommendations For The Authors:

1)Fig.2B: Beyond the SEC part (absorbance vs elution volume), I don't understand this plot, in particular the horizontal lines. They appear to be correlating molecular weight with normalized absorption at 280 nm, but the chromatogram amplitudes are different. Clarify, or modify the plot. There are also some disconnected line segments between 10-11 mL - these seem to be spurious.

We apologize for the confusion. The horizontal lines are meant to only denote the average molecular weights of the elution peaks and not correlate with the A280 values. The disconnected lines are the light-scattering molecular weight readouts from which the horizontal lines are derived. The problematic nature of the figure is that the full elution traces and MALS traces across the peaks call for different scales to best depict the relevant features of the data. We have reworked the figure and legend to make the key points more clear.

2) It could be useful to add AF2 secondary structure predictions, pLDDT, and the helical propensity analysis to the sequence ribbon in Fig.1C.

Thank you for the suggestion, we have updated the figure to incorporate the pLDDT scores into the linear sequence map, as well as the secondary structure predictions.

3) Fig.3D: It would be better to show the raw data rather than the fits. At the same time, I appreciate the fact that the authors resisted the temptation to show distance distributions.

Yes, we agree that it is important to show the raw data; it is included in the supplementary section. Depicting the raw data here unfortunately obscures the differences in the traces and we believe that showing the data as a superposition is quite useful to convey the main differences among the sites. However, we have now explicitly stated in the figure legend that the corresponding raw data traces are given in Figures S5-6.

4) Fig.5: For all distance distributions, error intervals should be added (typically done in terms of shaded bands around the best-fit distribution). As shown, precision is visually overstated. The error analysis shown in the SI is dubious, as it shows some distances have no error whatsoever (e.g. 6nm in 370C-490C), which is not possible.

We did previously show the error intervals in the SI, but we agree that it is better to include them here as well, and have done so in the new Figure 5. With respect to the error analysis, we are following the methodology described in the following paper:

Srivastava, M. and Freed J., Singular Value Decomposition Method To Determine Distance Distributions in Pulsed Dipolar Electron Spin Resonance: II. Estimating Uncertainty. J. Phys Chem A (2019) 123:359-370. doi: 10.1021/acs.jpca.8b07673.

Briefly, the uncertainty we are plotting is showing the "range" of singular values over which the singular value decomposition (SVD) solution remains converged. For most of the data displayed in this paper we only used the first few singular values (SVs) and the solution remained converged for ± 1 or 2 SVs near the optimum solution. For example, if the optimum solution was 4 SVs then the range in which the solution remained converged is ~3-6 SVs. We plot three lines - lowest range of SVs, highest range of SVs and optimum number of SVs – in the SI figures the optimum SV solution is shown in black and the region between the converged solutions with the highest and lowest number of SVs is shaded in red. Owing to the point-wise reconstruction of the distance distribution, the SVD method enables localized uncertainty at each distance value. Therefore, some points will have high uncertainty, whereas others low. The distance that may appear to have no uncertainty has actually very low uncertainty; which can be seen at close inspection. In these cases, we observe this "isosbestic" type behavior where the P(r) appears to change little across the acceptable solutions and hence there is only a small range of P(r) values at that particular r. This behavior results from multimodal distributions wherein the change in SVs shifts neighboring peaks to lower and higher distances respectively, producing an apparent cancelation effect. What we believe is most important for the biochemical interpretation, and accurately reflected by this analysis, is the general width of the uncertainty across the distribution and how this impacts the error in both the mean and the overall skewing of the distribution at short or long distances.

Details of the error treatment as described above have been added to the supplementary methods section.

5) The Discussion (p.13) states that the SAXS and DEER data show that disorder is greater than in a molten globule and smaller than in a denatured protein. Evidence to support this statement (molten globule DEER/SAXS reference data etc.) should be made explicit.

We will make the statement more explicit by changing it to the following: “Notably, the shape of the Kratky plots generated from the SAXS data suggest a degree of disorder that is substantially greater than that expected of a molten globule (Kataoka, et al., 1997), but far from that of a completely denatured protein (Kikhney, et al., 2015; Martin, Erik W., et al., 2021). Similarly, the DEER distributions, though non-uniform across the various sites examined, indicate more disorder than that of a molten globule (Selmke et al., 2018) but more order than a completely unfolded protein (van Son et al. 2015).”

van Son, M., et al. Double Electron−Electron Spin Resonance Tracks Flavodoxin Folding, J. Phys. Chem. B 2015, 119, 13507−13514. doi: 10.1021/acs.jpcb.5b00856.

Selmke, B. et al. Open and Closed Form of Maltose Binding Protein in Its Native and Molten Globule State As Studied by Electron Paramagnetic Resonance Spectroscopy. Biochemistry 2018, 57, 5507−5512 doi: 10.1021/acs.biochem.8b00322.

6) Fig. S11B could be promoted to the main paper.

This comment makes a good point. Figure 8 is now an updated scheme, similar to the previous Fig. S11B. Thank you for the suggestion.

Minor corrections:

p.1: "composed from" -> "composed of"

p.2: TFFLs -> TTFLs

p.2: "and CK1 via" => "and to CK1 via"

p.5: "Nickel" -> "nickel"

p.5: "Size Exclusion Chromatography" -> "Size exclusion chromatography"

p.5: "Multi Angle Light Scattering" -> "multi-angle light scattering"

Fig.2 caption: "non-phosphorylated (np-FRQ)" -> "non-phosphorylated FRQ (np-FRQ)"

Fig. S3: What are the units on the horizontal axis?

Fig. 5H is too small

Fig. S8, S9: all distance distribution plots show a spurious "1"

Fig. 6A has font sizes that are too small to read

p.11: "cytoplasm facing" -> "cytoplasm-facing"

p.11: "temperature dependent" -> "temperature-dependent"

p.12: "substrate-sequestration and product-release" -> "substrate sequestration and product release"

p.12: "depend highly buffer composition" -> "depend highly on buffer composition"

We thank the reviewer for finding these errors and their attention to detail. All of these minor points have been addressed in the revised manuscript.

Reviewer #3 (Public Review):

Summary:

The manuscript from Tariq and Maurici et al. presents important biochemical and biophysical data linking protein phosphorylation to phase separation behavior in the repressive arm of the Neurospora circadian clock. This is an important topic that contributes to what is likely a conceptual shift in the field. While I find the connection to the in vivo physiology of the clock to be still unclear, this can be a topic handled in future studies.

Strengths:

The ability to prepare purified versions of unphosphorylated FRQ and P-FRQ phosphorylated by CK-1 is a major advance that allowed the authors to characterize the role of phosphorylation in structural changes in FRQ and its impact on phase separation in vitro.

Weaknesses:

The major question that remains unanswered from my perspective is whether phase separation plays a key role in the feedback loop that sustains oscillation (for example by creating a nonlinear dependence on overall FRQ phosphorylation) or whether it has a distinct physiological role that is not required for sustained oscillation.

The reviewer raises the key question regarding data suggesting LLPS and phase separated regions in circadian systems. To date condensates have been seen in cyanobacteria (Cohen et al, 2014, Pattanayak et al, 2020) where there are foci containing KaiA/C during the night, in Drosophila (Xiao et al, 2021) where PER and dCLK colocalize in nuclear foci near the periphery during the repressive phase, and in Neurospora (Bartholomai et al, 2022) where the RNA binding protein PRD-2 sequesters frq and ck1a transcripts in perinuclear phase separated regions. Because the proteins responsible for the phase separation in cyanobacteria and Drosophila are not known, it is not possible to seamlessly disrupt the separation to test its biological significance (Yuan et al, 2022), so only in Neurospora has it been possible to associate loss of phase separation with clock effects. There, loss of PRD-2, or mutation of its RNA-binding domains, results in a ~3 hr period lengthening as well as loss of perinuclear localization of frq transcripts. A very recent manuscript (Xie et al., 2024) calls into question both the importance and very existence of LLPS of clock proteins at least as regards to mammalian cells, noting that it may be an artefact of overexpression in some places where it is seen, and that at normal levels of expression there is no evidence for elevated levels at the nuclear periphery. Artefacts resulting from overexpression plainly cannot be a problem for our study nor for Xiao et al. 2021 as in both cases the relevant clock protein, FRQ or PER, was labeled at the endogenous locus and expressed under its native promoter. Also, it may be worth noting that although we called attention to enrichment of FRQ[NeonGreen] at the nuclear periphery, there remained abundant FRQ within the core of the nucleus in our live-cell imaging.

Cohen SE, et al.: Dynamic localization of the cyanobacterial circadian clock proteins. Curr Biol 2014, 24:1836–1844, https://doi.org/10.1016/j.cub.2014.07.036.

Pattanayak GK, et al.: Daily cycles of reversible protein condensation in cyanobacteria. Cell Rep 2020, 32:108032, https://doi.org/10.1016/j.celrep.2020.108032.

Xiao Y, Yuan Y, Jimenez M, Soni N, Yadlapalli S: Clock proteins regulate spatiotemporal organization of clock genes to control circadian rhythms. Proc Natl Acad Sci U S A 2021, 118, https://doi.org/10.1073/pnas.2019756118.

Bartholomai BM, Gladfelter AS, Loros JJ, Dunlap JC. 2022 PRD-2 mediates clock-regulated perinuclear localization of clock gene RNAs within the circadian cycle of Neurospora. Proc Natl Acad Sci U S A. 119(31):e2203078119. doi: 10.1073/pnas.2203078119.

Yuan et al., Curr Biol 78: 102129, 2022. https://doi.org/10.1016/j.ceb.2022.102129

Pancheng Xie, Xiaowen Xie, Congrong Ye, Kevin M. Dean, Isara Laothamatas , S K Tahajjul T Taufique, Joseph Takahashi, Shin Yamazaki, Ying Xu, and Yi Liu (2024). Mammalian circadian clock proteins form dynamic interacting microbodies distinct from phase separation. Proc. Nat. Acad. Sci. USA. In press.

We have updated the discussion on p. 15 accordingly:

“Live cell imaging of fluorescently-tagged FRQ proteins is consistent with FRQ phase separation in N. crassa nuclei. FRQ is plainly not homogenously dispersed within nuclei, and the concentrated foci observed at specific positions in the nuclei indicate condensate behavior similar to that observed for other phase separating proteins (Bartholomai, et al., 2022; Caragliano, et al., 2022; Gonzalez, A., et al., 2021; Tatavosian, et al., 2019; Xiao, et al., 2021). While ongoing experiments are exploring more deeply the spatiotemporal dynamics of FRQ condensates in nuclei, the small size of fungal nuclei as well as their rapid movement with cytoplasmic bulk flow through the hyphal syncytium makes these experiments difficult. Of particular interest is drawing comparisons between FRQ and the Drosophila Period protein, which has been observed in similar foci that change in size and subnuclear localization throughout the circadian cycle (Meyer, et al., 2006; Xiao, et al., 2021), although it must be noted that the foci we observed are considerably more dynamic in size and shape than those reported for PER in Drosophila (Xiao, et al., 2021). A very recent manuscript (Xie, et al., 2024) calls into question the importance and very existence of LLPS of clock proteins at least in regards to mammalian cells, noting that it may be an artifact of overexpression in some instances where it is seen, and that at normal levels of expression there is no evidence for elevated levels at the nuclear periphery. Artifacts resulting from overexpression are unlikely to be a problem for our study and that of Xiao et al as in both cases clock proteins were tagged at their endogenous locus and expressed from their native promoters. Although we noted enrichment of FRQmNeonGreen near the nuclear envelope in our live-cell imaging, there remained abundant FRQ within the core of the nucleus.”

Recommendations For The Authors:

The data in Fig 6 showing microscopy of Neurospora is suggestive but needs more information/controls. Does the strain that expresses FRQ-mNeonGreen have normal circadian rhythms? How were the cultures handled (in terms of circadian entrainment etc.) for imaging? Do samples taken at different clock times appear different in terms of punctate structures in microscopy? The authors cite the Xiao 2021 paper in Drosophila, but would be good to see if the in vivo picture is fundamentally similar in Neurospora.

All of the live-cell images we report were from cells grown in constant light; in the dark, strains bearing FRQ[NeonGreen] have normally robust rhythms with a slightly elongated period length as measured by a frq Cbox-luc reporter. Although we are interested, of course, in whether and if so how the punctate structures changed as function of circadian time, this is work in progress and beyond the scope of the present study. This said, it is plain to see from the movie included as a Supplemental file here that the puncta we see are moving and fusing/splitting on a scale of seconds whereas those reported in Drosophila by Xiao et al. (Xiao et al, 2021, above) were stable for many minutes; thus the FRQ foci seen in Neurospora are quite a bit more dynamic than those in Drosophila.

We have updated the results section on p. 11 to provide this information more clearly: “FRQ thus tagged and driven by its own promoter is expressed at physiologically normal levels, and strains bearing FRQmNeonGreen as the only source of FRQ are robustly rhythmic with a slightly longer than normal period length. Live-cell imaging in Neurospora crassa offers atypical challenges because the mycelia grow as syncytia, with continuous rapid nuclei motion during the time of imaging. This constant movement of nuclei is compounded by the very low intranuclear abundance of FRQ and the small size of fungal nuclei, making not readily feasible visualization of intranuclear droplet fission/fusion cycles or intranuclear fluorescent photobleaching recovery experiments (FRAP) that could report on liquid-like properties. Nonetheless, bright and dynamic foci-like spots were observed well inside the nucleus and near the nuclear periphery, which is delineated by the cytoplasm-facing nucleoporin Son-1 tagged with mApple at its C-terminus (Fig. 6D,E, Movie S1). Such foci are characteristic of phase separated IDPs (Bartholomai, et al., 2022; Caragliano, et al., 2022; Gonzalez, A., et al., 2021; Tatavosian, et al., 2019) and share similar patterning to that seen for clock proteins in Drosophila (Meyer, et al., 2006; Xiao, et al., 2021), although the foci we observed are substantially more dynamic than those reported in Drosophila.”

Another issue where some commentary would be helpful: Fig 7 shows that phase separation behavior is strongly temperature dependent (not biophysically surprising). Is that at odds with the known temperature compensation of the circadian rhythm if LLPS indeed plays a key role in the oscillator?

We believe that the dependence of CK1-mediated FRQ phosphorylation on temperature, as manifested by FRQ phase separation, is consistent with temperature compensation within the Neurospora circadian oscillator. The phenomenon of temperature compensation by circadian clocks involves the intransigence of the oscillator period to temperature change. Stability of period with temperature change would not necessarily be expected of a generic chemical oscillator, which would run faster (shorter period) at higher temperature owing to Arrhenius behavior of the underlying chemical reactions. Circadian phosphorylation of FRQ is one such chemical process that contributes to the oscillation of FRQ abundance on which the clock is based. Reduced CK1 phosphorylation of FRQ causes both longer periods [Mehra et al., 2009] and loss of temperature compensation (manifested as a reduction of period length at higher temperature) [Liu et al, Nat Comm, 10, 4352 (2019); Hu et al, mBio, 12, e01425 (2021)]. Thus, the ability of increased LLPS formation at elevated temperature to reduce FRQ phosphorylation by CK1 (but not intrinsic CK1 autophosphorylation) would be a means to counter a decreasing period length that would otherwise manifest in an under compensated system. As further negative feedback on the system, LLPS is also promoted by FRQ phosphorylation itself, which in turn will reduce phosphorylation by CK1. Thus, both increased FRQ phosphorylation and temperature will couple to increased LLPS and mitigate period shortening through reduction of CK1 activity.

Mehra et al., A Role for Casein Kinase 2 in the Mechanism Underlying Circadian Temperature Compensation. May 15, 2009. Cell 137, 749–760,

Liu et al. FRQ-CK1 interaction determines the period of circadian rhythms in Neurospora. Nat Comm. 2019, 10 4352.

Hu et al FRQ-CK1 Interaction Underlies Temperature Compensation of the Neurospora Circadian Clock mBio 2021 12 WOS:000693451600006.

We have added Figure 8 to clarify the interpretation of the temperature compensation implicaitons of our work, the legend of which reads:

“Figure 8: LLPS may play a role in temperature compensation of the clock through modulation of FRQ phosphorylation. Reduced CK1 phosphorylation of FRQ causes both longer periods (Mehra, et al., 2009) and loss of temperature compensation (manifested as a shortening of period at higher temperature) (Hu, et al., 2021; Liu, X., et al., 2019). Thus, the ability of increased LLPS at elevated temperature (larger grey circle) to reduce FRQ phosphorylation by CK1 will counter a shortening period that would otherwise manifest in an under compensated system. As further negative feedback, LLPS is also promoted by increased FRQ phosphorylation, which in turn will reduce phosphorylation by CK1. Thus, both increased FRQ phosphorylation and temperature favor LLPS and reduction of CK1 activity.”

one minor comment: The chemical structures in Fig 3A have some issues where the "N" and "S" are flipped. Would be good to remake these figures to fix this problem.

We apologize, the figure has been replaced with an improved version.

This section provides a helpful introduction to accessing Reddit data using the PRAW library and the significance of APIs in this process. However, it might be beneficial for readers if the text included specific examples of the types of requests one can make with PRAW and the kinds of data accessible through these requests. For instance, illustrating how to retrieve posts from a specific subreddit or how to analyze user comments could make the API's capabilities more tangible. Additionally, offering tips or resources on navigating the PRAW documentation would greatly assist beginners in overcoming the initial learning curve mentioned.

This is a new page note on code llama

Be careful on this.

Author Response

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

We would firstly like to thank all reviewers for their comments and support of this manuscript.

Reviewer #1 (Recommendations For The Authors):

No further recommendations.

Reviewer #2 (Recommendations For The Authors):

All of my comments have been sufficiently addressed.

Reviewer #3 (Recommendations For The Authors):

Thanks for responding to my former recommendations constructively. I believe these points have been fully addressed in this new version.

However, I have not seen any comments on the points I raised in my former public review concerning the I-2 dependence of the FonSIX4 cell death. Do you know whether FonSIX4 would trigger cell death in tissues not expressing any I-2?

We are a little confused concerning this comment. I-2 is a different class of resistance protein (NLR) that recognises Avr2 and this is likely to be intracellular. From the previous public review, we believe reviewer 3 may have been asking us to clarify the dependence of I (MM or M82) on FonSIX4 cell death. We have performed these controls by expressing FonSIX4 and associated FonSIX4/Avr1 chimeras in N. benthamiana (with the PR-1 signal peptide for efficient secretion of effectors) and it does not cause cell death in the absence of the I receptor – see S11F Fig. This was not explicitly conveyed in text so we have included the following in text: “Using the N. benthamiana assay we show FonSIX4 is recognised by I receptors from both cultivars (IM82 and iMoneymaker) and cell death is dependent on the presence of IM82 or iMoneymaker (Fig 5B, S11 Fig).”

I still recommend discussing whether the Avr1 residues crucial for Avr activity are in the same structural regions of the C-terminal domain where previous work has identified residues under diversifying selection in symbiotic fungal FOLD proteins.

The region important for recognition does encompass some residues within the structural region identified to be under diversifying selection in FOLD effectors from Rhizophagus irregularis previously reported (two residues within one beta-strand). However, we also see residues that don’t overlap to this area. We also note that the mycFOLD proteins analysed in symbiotic fungi are heavily skewed towards strong structurally similarity with FolSIX6 (similar cysteine spacing within both N and C-domains and structural orientation of the N and C-domains) rather than Avr1. We are under the impression that Avr1 was not included in the analysis of diversifying selection in symbiotic fungal FOLD proteins, it also is unclear to us if close Avr1 homologues are present. With this in mind, and considering our already lengthy discussion (as previously highlighted during reviewer), we have decided not to include further discussion concerning this point.

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

We would like to thank the editor(s) and reviewers for their work concerning our manuscript. Most of the suggested changes were related to text changes which we have incorporated into the revised version. Please find our response to reviewers below.

Reviewer #1 (Recommendations For The Authors):

I only have very minor suggestions for the authors. The first one comes from reading the manuscript and finding it very dense with so many acronyms. This will limit the audience that will read the study and appreciate its impact. This is more noticeable in the Results, with many passages that I would suggest moving to Methodology.

We thank reviewer 1 for their very positive review. We understand that due to the nature of this study, which includes many protein alleles/mutations that were expressed with different boundaries etc., it is difficult to achieve this. Reviewer 2 asked for more details to be provided. We hope we have achieved a nice balance in the revised manuscript.

Something else that would facilitate the reading of the manuscript is the effectors name. The authors use the SIX name or the Avr name for some effectors and it makes it difficult to follow up.

We have tried to make this consistent for Avr1 (SIX4), Avr2 (SIX3) and Avr3 (SIX1). Other SIX effectors are not known Avrs so the SIX names were used.

Reading the manuscript and seeing how in most of the sections the authors used a computational approach followed by an experimental approach, I wonder why Alphafold2-multimer was not used to investigate the interaction between the effector and the receptor?

This is a great suggestion, we have certainly investigated this, however to date there is no experimental evidence to directly support the direct interaction between I and Avr1. Post review, we spent some time trying to capture an interaction using a co-immunoprecipitation approach however to date we have not been able to obtain robust data that support this. We are currently looking to study this utilising protein biophysics/biochemistry but this work will take some time.

Reviewer #2 (Recommendations For The Authors):

We thank reviewer 2 for the very thorough editing and recommendations. We have incorporated all minor text edits below into the manuscript.

Line 43: perhaps "Effector recognition" instead of "Effector detection", to be consistent with line 51?

Line 60: Change to "leads".

Line 79: Italicise Avr2.

Line 94: Add the acronym ETI in parentheses after "effector-triggered immunity".

Line 106: "(Leptosphaeria Avirulence-Supressing)" should be "(Leptosphaeria Avirulence and Supressing)".

Line 112: Change "defined" to "define".

Line 119: Spell out the species name on first use.

Line 205: Glomeromycota is a division rather than a genus. Consistent with Fig 2, it also does not need to italicized.

Line 207: Change "basidiomycete" to "Division Basidiomycota", consistent with Fig 2.

Line 214: Change "alignment of Avr1, Avr3, SIX6 and SIX13" to "alignment of the mature Avr1, Avr3, SIX6 and SIX13 sequences".

Line 324: Change "solved structures" to "solved protein structures".

Line 335: Spell out acronyms like "MS" on first use in figure legends. Also dpi in other figure legends.

Line 341: replace "effector-triggered immunity (ETI)" with "(ETI)" - see comment on Line 94.

Line 370: Change "domains" to "domain".

Line 374: In the title, change "C-terminus" to C-domain", consistent with the rest of the figure legend.

Line 404: Change "(basidiomycetes and ascomycetes)" to "(Basidiomycota and Ascomycota fungi)", consistent with Fig 2C.

Line 416: Change "in" to "by".

Line 427: un-italicize the parentheses.

Line 519: First mention of NLR. Spell out the acronym on first use in main text. S5 and S11 figure titles should be bolded.

Line 852: Replace "@" with "at".

S4 Table: Gene names should be italicised.

S5 Table: Needs to be indicated that the primer sequences are in the 5´-3´ orientation.

With regards to the Agrobacterium tumefaciens-mediated transient expression assays involving co-expression of the Avr1 effector and I immune receptor, the authors need to make clear how many biological replicates were performed as this information is only provided for the ion leakage assay.

We have added these data to the figure legend

Line 57: For me, the text "Fol secretes a limited number of structurally related effectors" reads as Fol secretes structurally related effectors, but very few of them are structurally related. Perhaps it would be better to say that the effector repertoire of Fol is made up of proteins that adopt a limited number of structural folds, or that the effector repertoire can be classified into a reduced set of structural families?

This edit has been incorporated.

Lines 66-67: Subtle re-wording required for "The best-characterized pathosystem is F. oxysporum f. sp. lycopersici (Fol)", as a pathosystem is made up of a pathogen and its host. Perhaps "The best-characterized pathosystem involves F. oxysporum f. sp. lycopersici (Fol) and tomato".

Sentence has been reworded.

Line 113 and throughout: Stick with one of "resistance protein", "receptor", "immune receptor" and "immunity receptor" throughout the manuscript.

We have decided to use both receptor and immunity receptor as not all receptors investigated in the manuscript provide immunity.

Lines 149-150: The title does not fully represent what is shown in the figure. The text "that is unique among fungal effectors" can be deleted as there is nothing in Fig 1 that shows that the fold is unique to fungal effectors.

Figure title has been changed.

Line 173: The RMSD of Avr3 is stated as being 3.7 Å, but in S3 Fig it is stated as being 3.6 Å.

This was a mistake in the main text and has been corrected.

Lines 202-204: This sentence needs to be reworded, as the way that it is written implies that the Diversispora and Rhizophagus genera are in the Ascomycota division. Also, "Ascomycetes" should be changed to "Ascomycota fungi", consistent with Fig 2.

Sentence has been reworded.

Line 233: "Scores above 8". What type of scores? Z-scores?

These are Z-scores. This has been added in text.

Lines 242-246: It is stated that SIX9 and SIX11 share structural similarity to various RNA-binding proteins, but no scores used to make these assessments is given. The scores should be provided in the text.

Z-scores have been added.

Fig 4A: SIX3 should be Avr2, consistent with line 292. The gene names should be italicised in Fig 4A.

SIX3 was changed to Avr2. Gene names have been italicised.

Line 356: Subtle rewording required, as "co-infiltrated with both IM82 and iMoneymaker" implies that you infiltrated with protein rather than Agrobacterium strains.

Sentence has been reworded.

Fig 5A, Fig 5C and Line 380: Light blue is used, but this looks grey. Perhaps change colour, as grey is already used to show the pro-domain in Fig 5A (or simply change the colour used to highlight the pro-domain)?

Colour depicting the C-domain was changed.

Lines 530-531: This text is no longer correct. Rlm4 and Rlm3 are now known to be alleles of Rlm9. See: Haddadi, P., Larkan, N. J., Van deWouw, A., Zhang, Y., Neik, T. X., Beynon, E., ... & Borhan, M. H. (2022). Brassica napus genes Rlm4 and Rlm7, conferring resistance to Leptosphaeria maculans, are alleles of the Rlm9 wall‐associated kinase‐like resistance locus. Plant Biotechnology Journal, 20(7), 1229.

We thank the reviewer for picking this up. This text has been updated.

Line 553: Provide more information on what the PR1 signal peptide is.

More information about the PR1 signal peptide has been added.

Lines 767-781: Descriptions and naming conventions of proteins throughout the figure legend need to be consistent and better reflect their makeup. For example, I think it would be best to put the sequence range after each protein mentioned - e.g. Avr118-242 or Avr159-242 instead of Avr1, PSL1_C37S18-111 instead of PSL1_C37S, etc. Furthermore, it is often stated that a protein is full-length when it lacks a signal peptide - my thought is that if a proteins lack its signal peptide, it is not full-length. The acronym "PD" also needs to be spelled out as "pro-domain (PD)" in the figure legend.

We have incorporated sequence range for proteins that were produced upon first use. Sequence ranges that were modelled in AlphaFold2 were not added in text because they can be found in Supplementary Table 3.

Lines 853-845: It is stated the sizes of proteins are indicated above the chromatogram in S10 Fig, but this is not the case. It is also not clear from S10B Fig that the faint peaks correspond to the peaks in the Fig 4B chromatogram. In S10D Fig, the stick of C58S is difficult to see. Perhaps change the colour or use an arrow/asterisk?

Protein size estimates have been added above the chromatogram. Added text to indicate that the faint peaks correspond to peaks in Fig 4B. Added an asterisk in S10D Fig to identify the location of C58.

S14 Fig is not mentioned/referenced in the main text of the manuscript.

This was a mistake and has been added.

The reference list needs to be updated to accommodate those referenced bioRxiv preprints that have now been published in peer-reviewed journals.

The reference list has been updated.

Reviewer #3 (Recommendations For The Authors):

It would be good to discuss whether the pro-domains affecting virulence or avirulence activity.

Kex2, the protease that cleaves the pro-domain functions in the golgi. We therefore suspect that the pro-domain is removed prior to secretion. For recombinant protein production in E. coli we find that these pro-domains are necessary to obtain soluble protein (doi: 10.1111/nph.17516). As we require the pro-domain for protein production and can not completely removing them from our preps, we cannot perform experiments to test this and subsequently comment further. In a paper that identified SIX effectors in tomato utilising proteomics approach (https://bsppjournals.onlinelibrary.wiley.com/doi/10.1111/j.1364-3703.2007.00384.x), it appears that the pro-domains were not captured in this analysis. This supports the conclusion that they are not associated with the mature/secreted protein.

The authors stated that the C-terminal domain of SIX6 has a single disulfide bond unique to SIX6. Please clarify in which context is it unique: in Fusarium or across all FOLD proteins?

This is in direct comparison to Avr1 and Avr3. The disulfide in the C-domain of SIX6 is unique compared to Avr1 and Avr3. This has been made clear in text.

The structural similarity of FOLD proteins to other known structures have been discussed (lines 460ff), but it is not clear whether all structures and models identified in this work would yield cysteine inhibitor and tumor necrosis factors as best structural matches in the database or whether this is specific to a single FOLD protein. Please consider discussing recently published findings by others (Teulet et al. 2023, New Phytologist) on this aspect.

This analysis was performed for Avr1, we obtained relatively low similarity hits for Avr3/Six6. We have updated this text accordingly… “Unfortunately, the FOLD effectors share little overall structural similarity with known structures in the PDB outside of the similarity with each other. At a domain level, the N-domain of the FOLD effector Avr1 has some structural similarities with cystatin cysteine protease inhibitors (PDB code: 4N6V, PDB code: 5ZC1) [60, 61], and the C-domain with tumour necrosis factors (PDB code: 6X83) [62] and carbohydrate-binding lectins (PDB code: 2WQ4) [63]. Relatively weak hits were observed for Avr3/Six6.”

It might be useful to clearly point out that the ToxA fold and the C-terminus of the FOLD fold are different.

We have secondary structural topology maps of the FOLD and ToxA-like families in S8 Fig which highlight the differences in topology between these two families.

Please add information to Fig.S8 listing the approach to generate the secondary structure topology maps.

We have added this information in the figure caption.

Author Response

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

eLife assessment

This work presents H3-OPT, a deep learning method that effectively combines existing techniques for the prediction of antibody structure. This work is important because the method can aid the design of antibodies, which are key tools in many research and industrial applications. The experiments for validation are solid.

Comments to Author:

Several points remain partially unclear, such as:

1). Which examples constitute proper validation;

Thank you for your kind reminder. We have modified the text of the experiments for validation to identify which examples constitute proper validation. We have corrected the “Finally, H3-OPT also shows lower Cα-RMSDs compared to AF2 or tFold-Ab for the majority of targets in an expanded benchmark dataset, including all antibody structures from CAMEO 2022” into “Finally, H3-OPT also shows lower Cα-RMSDs compared to AF2 or tFold-Ab for the majority (six of seven) of targets in an expanded benchmark dataset, including all antibody structures from CAMEO 2022” and added the following sentence in the experimental validation section of our revised manuscript to clarify which examples constitute proper validation: “AlphaFold2 outperformed IgFold on these targets”.

2) What the relevance of the molecular dynamics calculations as performed is;

Thank you for your comment, and I apologize for any confusion. The goal of our molecular dynamics calculations is to compare the differences in binding affinities, an important issue of antibody engineering, between AlphaFold2-predicted complexes and H3-OPT-predicted complexes. Molecular dynamics simulations enable the investigation of the dynamic behaviors and interactions of these complexes over time. Unlike other tools for predicting binding free energy, MM/PBSA or MM/GBSA calculations provide dynamic properties of complexes by sampling conformational space, which helps in obtaining more accurate estimates of binding free energy. In summary, our molecular dynamics calculations demonstrated that the binding free energies of H3-OPT-predicted complexes are closer to those of native complexes. We have included the following sentence in our manuscript to provide an explanation of the molecular dynamics calculations: “Since affinity prediction plays a crucial role in antibody therapeutics engineering, we performed MD simulations to compare the differences in binding affinities between AF2-predicted complexes and H3-OPT-predicted complexes.”.

3) The statistics for some of the comparisons;

Thank you for the comment. We have incorporated statistics for some of the comparisons in the revised version of our manuscript and added the following sentence in the Methods section: “We conducted two-sided t-test analyses to assess the statistical significance of differences between the various groups. Statistical significance was considered when the p-values were less than 0.05. These statistical analyses were carried out using Python 3.10 with the Scipy library (version 1.10.1).”.

4) The lack of comparison with other existing methods.

We appreciate your valuable comments and suggestions. Conducting comparisons with a broader set of existing methods can further facilitate discussions on the strengths and weaknesses of each method, as well as the accuracy of our method. In our study, we conducted a comparison of H3-OPT with many existing methods, including AlphaFold2, HelixFold-Single, ESMFold, and IgFold. We demonstrated that several protein structure prediction methods, such as ESMFold and HelixFold-Single, do not match the accuracy of AlphaFold2 in CDR-H3 prediction. Additionally, we performed a detailed comparison between H3-OPT, AlphaFold2, and IgFold (the latest antibody structure prediction method) for each target.

We sincerely thank the comment and have introduced a comparison with OmegaFold. The results have been incorporated into the relevant sections (Fig 4a-b) of the revised manuscript.

Author response image 1.

Public Reviews

Comments to Author:

Reviewer #1 (Public Review):

Summary:

The authors developed a deep learning method called H3-OPT, which combines the strength of AF2 and PLM to reach better prediction accuracy of antibody CDR-H3 loops than AF2 and IgFold. These improvements will have an impact on antibody structure prediction and design.

Strengths:

The training data are carefully selected and clustered, the network design is simple and effective.

The improvements include smaller average Ca RMSD, backbone RMSD, side chain RMSD, more accurate surface residues and/or SASA, and more accurate H3 loop-antigen contacts.

The performance is validated from multiple angles.

Weaknesses:

1) There are very limited prediction-then-validation cases, basically just one case.

Thanks for pointing out this issue. The number of prediction-then-validation cases is helpful to show the generalization ability of our model. However, obtaining experimental structures is both costly and labor-intensive. Furthermore, experimental validation cases only capture a limited portion of the sequence space in comparison to the broader diversity of antibody sequences.

To address this challenge, we have collected different datasets to serve as benchmarks for evaluating the performance of H3-OPT, including our non-redundant test set and the CAMEO dataset. The introduction of these datasets allows for effective assessments of H3-OPT’s performance without biases and tackles the obstacle of limited prediction-then-validation cases.

Reviewer #2 (Public Review):

This work provides a new tool (H3-Opt) for the prediction of antibody and nanobody structures, based on the combination of AlphaFold2 and a pre-trained protein language model, with a focus on predicting the challenging CDR-H3 loops with enhanced accuracy than previously developed approaches. This task is of high value for the development of new therapeutic antibodies. The paper provides an external validation consisting of 131 sequences, with further analysis of the results by segregating the test sets into three subsets of varying difficulty and comparison with other available methods. Furthermore, the approach was validated by comparing three experimentally solved 3D structures of anti-VEGF nanobodies with the H3-Opt predictions

Strengths:

The experimental design to train and validate the new approach has been clearly described, including the dataset compilation and its representative sampling into training, validation and test sets, and structure preparation. The results of the in-silico validation are quite convincing and support the authors' conclusions.

The datasets used to train and validate the tool and the code are made available by the authors, which ensures transparency and reproducibility, and allows future benchmarking exercises with incoming new tools.

Compared to AlphaFold2, the authors' optimization seems to produce better results for the most challenging subsets of the test set.

Weaknesses:

1) The scope of the binding affinity prediction using molecular dynamics is not that clearly justified in the paper.

We sincerely appreciate your valuable comment. We have added the following sentence in our manuscript to justify the scope of the molecular dynamics calculations: “Since affinity prediction plays a crucial role in antibody therapeutics engineering, we performed MD simulations to compare the differences in binding affinities between AF2-predicted complexes and H3-OPT-predicted complexes.”.

2) Some parts of the manuscript should be clarified, particularly the ones that relate to the experimental validation of the predictions made by the reported method. It is not absolutely clear whether the experimental validation is truly a prospective validation. Since the methodological aspects of the experimental determination are not provided here, it seems that this may not be the case. This is a key aspect of the manuscript that should be described more clearly.

Thank you for the reminder about experimental validation of our predictions. The sequence identities of the wild-type nanobody VH domain and H3 loop, when compared with the best template, are 0.816 and 0.647, respectively. As a result, these mutants exhibited low sequence similarity to our dataset, indicating the absence of prediction bias for these targets. Thus, H3-OPT outperformed IgFold on these mutants, demonstrating our model's strong generalization ability. In summary, the experimental validation actually serves as a prospective validation.

Thanks for your comments, we have added the following sentence to provide the methodological aspects of the experimental determination: “The protein expression, purification and crystallization experiments were described previously. The proteins used in the crystallization experiments were unlabeled. Upon thawing the frozen protein on ice, we performed a centrifugation step to eliminate any potential crystal nucleus and precipitants. Subsequently, we mixed the protein at a 1:1 ratio with commercial crystal condition kits using the sitting-drop vapor diffusion method facilitated by the Protein Crystallization Screening System (TTP LabTech, mosquito). After several days of optimization, single crystals were successfully cultivated at 21°C and promptly flash-frozen in liquid nitrogen. The diffraction data from various crystals were collected at the Shanghai Synchrotron Research Facility and subsequently processed using the aquarium pipeline.”

3) Some Figures would benefit from a clearer presentation.

We sincerely thanks for your careful reading. According to your comments, we have made extensive modifications to make our presentation more convincing and clearer (Fig 2c-f).

Author response image 2.

Reviewer #3 (Public Review):

Summary:

The manuscript introduces a new computational framework for choosing 'the best method' according to the case for getting the best possible structural prediction for the CDR-H3 loop. The authors show their strategy improves on average the accuracy of the predictions on datasets of increasing difficulty in comparison to several state-of-the-art methods. They also show the benefits of improving the structural predictions of the CDR-H3 in the evaluation of different properties that may be relevant for drug discovery and therapeutic design.

Strengths:

The authors introduce a novel framework, which can be easily adapted and improved. The authors use a well-defined dataset to test their new method. A modest average accuracy gain is obtained in comparison to other state-of-the art methods for the same task while avoiding testing different prediction approaches.

Weaknesses:

1) The accuracy gain is mainly ascribed to easy cases, while the accuracy and precision for moderate to challenging cases are comparable to other PLM methods (see Fig. 4b and Extended Data Fig. 2). That raises the question: how likely is it to be in a moderate or challenging scenario? For example, it is not clear whether the comparison to the solved X-ray structures of anti-VEGF nanobodies represents an easy or challenging case for H3-OPT. The mutant nanobodies seem not to provide any further validation as the single mutations are very far away from the CDR-H3 loop and they do not disrupt the structure in any way. Indeed, RMSD values follow the same trend in H3-OPT and IgFold predictions (Fig. 4c). A more challenging test and interesting application could be solving the structure of a designed or mutated CDR-H3 loop.

Thank you for your rigorous consideration. When the experimental structure is unavailable, it is difficult to directly determinate whether the target is easy-to-predict or challenging. We have conducted our non-redundant test set in which the number of easy-to-predict targets is comparable to the other two groups. Due to the limited availability of experimental antibody structures, especially nanobody structures, accurately predicting CDR-H3 remains a challenge. In our manuscript, we discuss the strengths and weakness of AlphaFold2 and other PLM-based methods, and we introduce H3-OPT as a comprehensive solution for antibody CDR3 modeling.

We also appreciate your comment on experimental structures. We fully agree with your opinion and made attempts to solve the experimental structures of seven mutants, including two mutants (Y95F and Q118N) which are close to CDR-H3 loop. Unfortunately, we tried seven different reagent kits with a total of 672 crystallization conditions, but were unable to obtain crystals for these mutants. Despite the mutants we successfully solved may not have significantly disrupted the structures of CDR-H3 loops, they have still provided valuable insights into the differences between MSA-based methods and MSA-free methods (such as IgFold) for antibody structure modeling.

We have further conducted a benchmarking study using two examples, PDBID 5U15 and 5U0R, both consisting of 18 residues in CDR-H3, to evaluate H3-OPT's performance in predicting mutated H3 loops. In the first case (target 5U15), AlphaFold2 failed to provide an accurate prediction of the extended orientation of the H3 loop, resulting in a less accurate prediction (Cα-RMSD = 10.25 Å) compared to H3-OPT (Cα-RMSD = 5.56 Å). In the second case (target 5U0R, a mutant of 5U15 in CDR3 loop), AlphaFold2 and H3-OPT achieved Cα-RMSDs of 6.10 Å and 4.25 Å, respectively. Additionally, the Cα-RMSDs of OmegaFold predictions were 8.05 Å and 9.84 Å, respectively. These findings suggest that both AlphaFold2 and OmegaFold effectively captured the mutation effects on conformations but achieved lower accuracy in predicting long CDR3 loops when compared to H3-OPT.

2) The proposed method lacks a confidence score or a warning to help guide the users in moderate to challenging cases.

We appreciate your suggestions and we have trained a separate module to predict confidence scores. We used the MSE loss for confidence prediction, where the label error was calculated as the Cα deviation of each residue after alignment. The inputs of this module are the same as those used for H3-OPT, and it generates a confidence score ranging from 0 to 100.

3) The fact that AF2 outperforms H3-OPT in some particular cases (e.g. Fig. 2c and Extended Data Fig. 3) raises the question: is there still room for improvements? It is not clear how sensible is H3-OPT to the defined parameters. In the same line, bench-marking against other available prediction algorithms, such as OmegaFold, could shed light on the actual accuracy limit. We totally understand your concern. Many papers have suggested that PLM-based models are computationally efficient but may have unsatisfactory accuracy when high-resolution templates and MSA are available (Fast, accurate antibody structure prediction from deep learning on massive set of natural antibodies, Ruffolo, J. A. et al, 2023). However, the accuracy of AF2 decreased substantially when the MSA information is limited. Therefore, we directly retained high-confidence structures of AF2 and introduced a PSPM to improve the accuracy of the targets with long CDR-H3 loops and few sequence homologs. The improvement in mean Cα-RMSD demonstrated the room for accurately predicting CDR-H3 loops.

We also appreciate your kind comment on defined parameters. In fact, once a benchmark dataset is established, determining an optimal cutoff value through parameter searching can indeed further improve the performance of H3-OPT in CDR3 structure prediction. However, it is important to note that this optimal cutoff value heavily depends on the testing dataset being used. Therefore, we provide a recommended cutoff value and offer a program interface for users who wish to manually define the cutoff value based on their specific requirements. Here, we showed the average Cα-RMSDs of our test set under different confidence cutoffs and the results have been added in the text accordingly.

Author response table 1.

We also appreciate your reminder, and we have conducted a benchmark against OmegaFold. The results have been included in the manuscript (Fig 4a-b).

Author response image 3.

Reviewer #1 (Recommendations For The Authors):

1) In Fig 3a, please also compare IgFold and H3-OPT (merge Fig. S2 into Fig 3a)

In Fig 3b, please separate Sub2 and Sub3, and add IgFold's performance.

Thank you very much for your professional advice. We have made revisions to the figures based on your suggestions.

Author response image 4.

2) For the three experimentally solved structures of anti-VEGF nanobodies, what are the sequence identities of the VH domain and H3 loop, compared to the best available template? What is the length of the H3 loop? Which category (Sub1/2/3) do the targets belong to? What is the performance of AF2 or AF2-Multimer on the three targets?

We feel sorry for these confusions. The sequence identities of the VH domain and H3 loop are 0.816 and 0.647, respectively, comparing with the best template. The CDR-H3 lengths of these nanobodies are both 17. According to our classification strategy, these nanobodies belong to Sub1. The confidence scores of these AlphaFold2 predicted loops were all higher than 0.8, and these loops were accepted as the outputs of H3-OPT by CBM.

3) Is AF2-Multimer better than AF2, when using the sequences of antibody VH and antigen as input?

Thanks for your suggestions. Many papers have benchmarked AlphaFold2-Multimer for protein complex modeling and demonstrated the accuracy of AlphaFold2-Multimer on predicting the protein complex is far from satisfactory (Benchmarking AlphaFold for protein complex modeling reveals accuracy determinants, Rui Yin, et al., 2022). Additionally, there is no significantly difference between AlphaFold2 and AlphaFold2-Multimer on antibody modeling (Structural Modeling of Nanobodies: A Benchmark of State-of-the-Art Artificial Intelligence Programs, Mario S. Valdés-Tresanco, et al., 2023)

From the data perspective, we employed a non-redundant dataset for training and validation. Since these structures are valuable, considering the antigen sequence would reduce the size of our dataset, potentially leading to underfitting.

4) For H3 loop grafting, I noticed that only identical target and template H3 sequences can trigger grafting (lines 348-349). How many such cases are in the test set?

We appreciate your comment from this perspective. There are thirty targets in our database with identical CDR-H3 templates.

Reviewer #2 (Recommendations For The Authors):

• It is not clear to me whether the three structures apparently used as experimental confirmation of the predictions have been determined previously in this study or not. This is a key aspect, as a retrospective validation does not have the same conceptual value as a prospective, a posteriori validation. Please note that different parts of the text suggest different things in this regard "The model was validated by experimentally solving three structures of anti-VEGF nanobodies predicted by H3-OPT" is not exactly the same as "we then sought to validate H3-OPT using three experimentally determined structures of anti-VEGF nanobodies, including a wild-type (WT) and two mutant (Mut1 and Mut2) structures, that were recently deposited in protein data bank". The authors are kindly advised to make this point clear. By the way, "protein data bank" should be in upper case letters.

We gratefully thank you for your feedback and fully understand your concerns. To validate the performance of H3-OPT, we initially solved the structures of both the wild-type and mutants of anti-VEGF nanobodies and submitted these structures to Protein Data Bank. We have corrected “that were recently deposited in protein data bank” into “that were recently deposited in Protein Data Bank” in our revised manuscript.

• It would be good to clarify the goal and importance of the binding affinity prediction, as it seems a bit disconnected from the rest of the paper. Also, it would be good to include the production MD runs as Sup, Mat.

Thanks for your valuable comment. We have added the following sentence in our manuscript to clarify the goal and importance of the molecular dynamics calculations: “Since affinity prediction plays a crucial role in antibody therapeutics engineering, we performed MD simulations to compare the differences in binding affinities between AF2-predicted complexes and H3-OPT-predicted complexes.”. The details of production runs have been described in Method section.

• Has any statistical test been performed to compare the mean Cα-RMSD values across the modeling approaches included in the benchmark exercise?

Thanks for this kind recommendation. We conducted a statistical test to assess the performance of different modeling approaches and demonstrated significant improvements with H3-OPT compared to other methods (p<0.001). Additionally, we have trained H3-OPT with five random seeds and compared mean Cα-RMSD values with all five models of AF2. Here, we showed the average Cα-RMSDs of H3-OPT and AlphaFold2.

Author response table 1.

• In Fig. 2c-f, I think it would be adequate to make the ordering criterion of the data points explicit in the caption or the graph itself.

We appreciate your comment and suggestion. We have revised the graph in the manuscript accordingly.

Author response image 5.

• Please revise Figure S2 caption and/or its content. It is not clear, in parts b and c, which is the performance of H3-OPT. Why weren´t some other antibody-specific tools such as IgFold included in this comparison?

Thanks for your comments. The performance of H3-OPT is not included in Figure S2. Prior to training H3-OPT, we conducted several preliminary studies, and the detailed results are available in the supplementary sections. We showed that AlphaFold2 outperformed other methods (including AI-based methods and TBM methods) and produced sub-angstrom predictions in framework regions. The comparison of IgFold with other methods was discussed in a previous work (Fast, accurate antibody structure prediction from deep learning on massive set of natural antibodies, Ruffolo, J. A. et al, 2023). In that study, we found that IgFold largely yielded results comparable to AlphaFold2 but with lower prediction cost. Additionally, we have also conducted a detailed comparison of CDR-H3 loops with IgFold in our main text.

• It is stated that "The relative binding affinities of the antigen-antibody complexes were evaluated using the Python script...". Which Python script?

Thank you for your comments, and I apologize for the confusion. This python script is a module of AMBER software, we have corrected “The relative binding affinities of the antigen-antibody complexes were evaluated using the python script” into “The relative binding affinities of the antigen-antibody complexes were evaluated using the MMPBSA module of AMBER software”.

Reviewer #3 (Recommendations For The Authors):

Does H3-OPT improve the AF2 score on the CDR-H3? It would be interesting to see whether grafted and PSPM loops improve the pLDDT score by using for example AF2Rank [https://doi.org/10.1103/PhysRevLett.129.238101]. That could also be a way to include a confidence score into H3-OPT.

We are so grateful for your kind question. H3-OPT could not provide a confidence score for output in current version, so we did not know whether H3-OPT improve the AF2 score or not.

We appreciate your kind recommendations and have calculated the pLDDT scores of all models predicted by H3-OPT and AF2 using AF2Rank. We showed that the average of pLDDT scores of different predicted models did not match the results of Cα-RMSD values.

Author response table 3.

Therefore, we have trained a separate module to predict the confidence score of the optimized CDR-H3 loops. We hope that this module can provide users with reliable guidance on whether to use predicted CDR-H3 loops.

The test case of Nb PDB id. 8CWU is an interesting example where AF2 outperforms H3-OPT and PLMs. The top AF2 model according to ColabFold (using default options and no template [https://doi.org/10.1038/s41592-022-01488-1]) shows a remarkably good model of the CDR-H3, explaining the low Ca-RMSD in the Extended Data Fig. 3. However, the pLDDT score of the 4 tip residues (out of 12), forming the hairpin of the CDR-H3 loop, pushes down the average value bellow the CBM cut-off of 80. I wonder if there is a lesson to learn from that test case. How sensible is H3-OPT to the CBM cut-off definition? Have the authors tried weighting the residue pLDDT score by some structural criteria before averaging? I guess AF2 may have less confidence in hydrophobic tip residues in exposed loops as the solvent context may not provide enough support for the pLDDT score.

Thanks for your valuable feedback. We showed the average Cα-RMSDs of our test set under different confidence cutoffs and the results have been added in the text accordingly.

Author response table 4.

We greatly appreciate your comment on this perspective. Inspired on your kind suggestions, we will explore the relationship between cutoff values and structural information in related work. Your feedback is highly valuable as it will contribute to the development of our approach.

A comparison against the new folding prediction method OmegaFold [https://doi.org/10.1101/2022.07.21.500999] is missed. OmegaFold seems to outperform AF2, ESM, and IgFold among others in predicting the CDR-H3 loop conformation (See [https://doi.org/10.3390/molecules28103991] and [https://doi.org/10.1101/2022.07.21.500999]). Indeed, prediction of anti-VEGF Nb structure (PDB WT_QF_0329, chain B in supplementary data) by OmegaFold as implemented in ColabFold [https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/beta/omegafold.ipynb] and setting 10 cycles, renders Ca-RMSD 1.472 Å for CDR-H3 (residues 98-115).

We appreciate your valuable suggestion. We have added the comparison against OmegaFold in our manuscript. The results have been included in the manuscript (Fig 4a-b).

Author response image 6.

In our test set, OmegaFold outperformed ESMFold in predicting the CDR-H3 loop conformation. However, it failed to match the accuracy of AF2, IgFold, and H3-OPT. We discussed the difference between MSA-based methods (such as AlphaFold2) and MSA-free methods (such as IgFold) in predicting CDR-H3 loops. Similarly, OmegaFold provided comparative results with HelixFold-Single and other MSA-free methods but still failed to match the accuracy of AlphaFold2 and H3-OPT on Sub1.

The time-consuming step in H3-OPT is the AF2 prediction. However, most of the time is spent in modeling the mAb and Nb scaffolds, which are already very well predicted by PLMs (See Fig. 4 in [https://doi.org/10.3390/molecules28103991]). Hence, why not use e.g. OmegaFold as the first step, whose score also correlates to the RMSD values [https://doi.org/10.3390/molecules28103991]? If that fails, then use AF2 or grafting. Alternatively, use a PLM model to generate a template, remove/mask the CDR loops (at least CDR-H3), and pass it as a template to AF2 to optimize the structure with or without MSA (e.g. using AF2Rank).

Thanks for your professional feedbacks. It is really true that the speed of MSA searching limited the application of high-throughput structure prediction. Previous studies have demonstrated that the deep learning methods performed well on framework residues. We once tried to directly predict the conformations of CDR-H3 loops using PLM-based methods, but this initial version of H3-OPT lacking the CBM could not replicate the accuracy of AF2 in Sub1. Similarly, we showed that IgFold and OmegaFold also provide lower accuracy in Sub1 (average Cα-RMSD is 1.71 Å and 1.83 Å, respectively, whereas AF2 predicted an average of 1.07 Å). Therefore, The predictions of AlphaFold2 not only produce scaffolds but also provide the highest quality of CDR-H3 loops when high-resolution templates and MSA are available.

Thank you once again for your kind recommendation. In the current version of H3-OPT, we have highlighted the strengths of H3-OPT in combining the AF2 and PLM models in various scenarios. AF2 can provide accurate predictions for short loops with fewer than 10 amino acids, and PLM-based models show little or no improvement in such cases. In the next version of H3-OPT, as the first step, we plan to replace the AF2 models with other methods if any accurate MSA-free method becomes available in the future.

Line 115: The statement "IgFold provided higher accuracy in Sub3" is not supported by Fig. 2a.

We are sorry for our carelessness. We have corrected “IgFold provided higher accuracy in Sub3” into “IgFold provided higher accuracy in Sub3 (Fig. 3a)”.

Lines 195-203: What is the statistical significance of results in Fig 5a and 5b?

Thank you for your kind comments. The surface residues of AF2 models are significantly higher than those of H3-OPT models (p < 0.005). In Fig. 5b, H3-OPT models predicted lower values than AF2 models in terms of various surface properties, including polarity (p <0.05) and hydrophilicity (p < 0.001).

Lines 212-213: It is not easy to compare and quantify the differences between electrostatic maps in Fig. 5d. Showing a Dmap (e.g. mapmodel - mapexperiment) would be a better option. Additionally, there is no methodological description of how the maps were generated nor the scale of the represented potential.

Thank you for pointing this out. We have modified the figure (Fig. 5d) according to your kind recommendation and added following sentences to clarify the methodological description on the surface electrostatic potential:

“Analysis of surface electrostatic potential

We generated two-dimensional projections of CDR-H3 loop’s surface electrostatic potential using SURFMAP v2.0.0 (based on GitHub from February 2023: commit: e0d51a10debc96775468912ccd8de01e239d1900) with default parameters. The 2D surface maps were calculated by subtracting the surface projection of H3-OPT or AF2 predicted H3 loops to their native structures.”

Author response image 7.

Lines 237-240 and Table 2: What is the meaning of comparing the average free energy of the whole set? Why free energies should be comparable among test cases? I think the correct way is to compare the mean pair-to-pair difference to the experimental structure. Similarly, reporting a precision in the order of 0.01 kcal/mol seems too precise for the used methodology, what is the statistical significance of the results? Were sampling issues accounted for by performing replicates or longer MDs?

Thanks for your rigorous advice and pointing out these issues. We have modified the comparisons of free energies of different predicted methods and corrected the precision of these results. The average binding free energies of H3-OPT complexes is lower than AF2 predicted complexes, but there is no significant difference between these energies (p >0.05).

Author response table 4.

Comparison of binding affinities obtained from MD simulations using AF2 and H3-OPT.

Thanks for your comments on this perspective. Longer MD simulations often achieve better convergence for the average behavior of the system, while replicates provide insights into the variability and robustness of the results. In our manuscript, each MD simulation had a length of 100 nanoseconds, with the initial 90 nanoseconds dedicated to achieving system equilibrium, which was verified by monitoring RMSD (Root Mean Square Deviation). The remaining 10 nanoseconds of each simulation were used for the calculation of free energy. This approach allowed us to balance the need for extensive sampling with the verification of system stability.

Regarding MD simulations for CDR-H3 refinement, its successful application highly depends on the starting conformation, the force field, and the sampling strategy [https://doi.org/10.1021/acs.jctc.1c00341]. In particular, the applied plan MD seems a very limited strategy (there is not much information about the simulated times in the supplementary material). Similarly, local structure optimizations with QM methods are not expected to improve a starting conformation that is far from the experimental conformation.

Thank you very much for your valuable feedback. We fully agree with your insights regarding the limitations of MD simulations. Before training H3-OPT, we showed the challenge of accurately predicting CDR-H3 structures. We then tried to optimize the CDR-H3 loops by computational tools, such as MD simulations and QM methods (detailed information of MD simulations is provided in the main text). Unfortunately, these methods were not expected to improve the accuracy of AF2 predicted CDR-H3 loops. These results showed that MD simulations and QM methods not only are time-consuming, but also failed to optimize the CDR-H3 loops. Therefore, we developed H3-OPT to tackle these issues and improve the accuracy of CDR3-H3 for the development of antibody therapeutics.

Text improvements

Relevant statistical and methodological parameters are presented in a dispersed manner throughout the text. For example, the number of structures in test, training, and validation datasets is first presented in the caption of Fig. 4. Similarly, the sequence identity % to define redundancy is defined in the caption of Fig. 1a instead of lines 87-88, where authors define "we constructed a non-redundant dataset with 1286 high-resolution (<2.5 Å)". Is the sequence redundancy for the CDR-H3 or the whole mAb/Nb?

Thank you for pointing out these issues. We have added the number of structures in each subgroup in the caption of Fig. 1a: “Clustering of the filtered, high-resolution structures yielded three datasets for training (n = 1021), validation (n = 134), and testing (n = 131).” and corrected “As data quality has large effects on prediction accuracy, we constructed a non-redundant dataset with 1286 high-resolution (<2.5 Å) antibody structures from SAbDab” into “As data quality has large effects on prediction accuracy, we constructed a non-redundant dataset (sequence identity < 0.8) with 1286 high-resolution (<2.5 Å) antibody structures from SAbDab” in the revised manuscript. The sequence redundancy applies to the whole mAb/Nb.

The description of ablation studies is not easy to follow. For example, what does removing TGM mean in practical terms (e.g. only AF2 is used, or PSPM is applied if AF2 score < 80)? Similarly, what does removing CBM mean in practical terms (e.g. all AF2 models are optimized by PSPM, and no grafting is done)? Thanks for your comments and suggestions. We have corrected “d, Differences in H3-OPT accuracy without the template module. e, Differences in H3-OPT accuracy without the CBM. f, Differences in H3-OPT accuracy without the TGM.” into “d, Differences in H3-OPT accuracy without the template module. This ablation study means only PSPM is used. e, Differences in H3-OPT accuracy without the CBM. This ablation study means input loop is optimized by TGM and PSPM. f, Differences in H3-OPT accuracy without the TGM. This ablation study means input loop is optimized by CBM and PSPM.”.

Authors should report the values in the text using the same statistical descriptor that is used in the figures to help the analysis by the reader. For example, in lines 223-224 a precision score of 0.75 for H3-OPT is reported in the text (I assume this is the average value), while the median of ~0.85 is shown in Fig. 6a.

Thank you for your careful checks. We have corrected “After identifying the contact residues of antigens by H3-OPT, we found that H3-OPT could substantially outperform AF2 (Fig. 6a), with a precision of 0.75 and accuracy of 0.94 compared to 0.66 precision and 0.92 accuracy of AF2.” into “After identifying the contact residues of antigens by H3-OPT, we found that H3-OPT could substantially outperform AF2 (Fig. 6a), with a median precision of 0.83 and accuracy of 0.97 compared to 0.64 precision and 0.95 accuracy of AF2.” in proper place of manuscript.

Minor corrections

Lines 91-94: What do length values mean? e.g. is 0-2 Å the RMSD from the experimental structure?

We appreciate your comment and apologize for any confusion. The RMSD value is actually from experimental structure. The RMSD value evaluates the deviation of predicted CDR-H3 loop from native structure and also represents the degree of prediction difficulty in AlphaFold2 predictions. We have added following sentence in the proper place of the revised manuscript: “(RMSD, a measure of the difference between the predicted structure and an experimental or reference structure)”.

Line 120: is the "AF2 confidence score" for the full-length or CDR-H3?

We gratefully appreciate for your valuable comment and have corrected “Interestingly, we observed that AF2 confidence score shared a strong negative correlation with Cα-RMSDs (Pearson correlation coefficient =-0.67 (Fig. 2b)” into “Interestingly, we observed that AF2 confidence score of CDR-H3 shared a strong negative correlation with Cα-RMSDs (Pearson correlation coefficient =-0.67 (Fig. 2b)” in the revised manuscript.

Line 166: Do authors mean "Taken" instead of "Token"?

We are really sorry for our careless mistakes. Thank you for your reminder.

Line 258: Reference to Fig. 1 seems wrong, do authors mean Fig. 4?

We sincerely thank the reviewer for careful reading. As suggested by the reviewer, we have corrected the “Fig. 1” into “Fig. 4”.

Author response image 7.

Point out which plot corresponds to AF2 and which one to H3-OPT

Thanks for pointing out this issue. We have added the legends of this figure in the proper positions in our manuscript.

Reviewer #2 (Public Review):

This work provides a new tool (H3-Opt) for the prediction of antibody and nanobody structures, based on the combination of AlphaFold2 and a pre-trained protein language model, with a focus on predicting the challenging CDR-H3 loops with enhanced accuracy than previously developed approaches. This task is of high value for the development of new therapeutic antibodies. The paper provides an external validation consisting of 131 sequences, with further analysis of the results by segregating the test sets in three subsets of varying difficulty and comparison with other available methods. Furthermore, the approach was validated by comparing three experimentally solved 3D structures of anti-VEGF nanobodies with the H3-Opt predictions

Strengths:

The experimental design to train and validate the new approach has been clearly described, including the dataset compilation and its representative sampling into training, validation and test sets, and structure preparation. The results of the in silico validation are quite convincing and support the authors' conclusions.

The datasets used to train and validate the tool and the code are made available by the authors, which ensures transparency and reproducibiity, and allows future benchmarking exercises with incoming new tools.

Compared to AlphaFold2, the authors' optimization seems to produce better results for the most challenging subsets of the test set.

Weaknesses:

The comparison of affinity predictions derived from AlphaFold2 and H3-opt models, based on molecular dynamics simulations, should have been discussed in depth. In some cases, there are huge differences between the estimations from H3-opt models and those from experimental structures. It seems that the authors obtained average differences of the real delta, instead of average differences of the absolute value of the delta. This can be misleading, because high negative differences might be compensated by high positive differences when computing the mean value. Moreover, it would have been good for the authors to disclose the trajectories from the MD simulations.

Author Response

We would like to thank the senior editor, reviewing editor and all the reviewers for taking out precious time to review our manuscript and appreciating our study. We are excited that all of you have found strength in our work and have provided comments to strengthen it further. We sincerely appreciate the valuable comments and suggestions, which we believe will help us to further improve the quality of our work.

Reviewer 1

The manuscript by Dubey et al. examines the function of the acetyltransferase Tip60. The authors show that (auto)acetylation of a lysine residue in Tip60 is important for its nuclear localization and liquid-liquid-phase-separation (LLPS). The main observations are: (i) Tip60 is localized to the nucleus, where it typically forms punctate foci. (ii) An intrinsically disordered region (IDR) within Tip60 is critical for the normal distribution of Tip60. (iii) Within the IDR the authors show that a lysine residue (K187), that is auto-acetylated, is critical. Mutation of that lysine residue to a non-acetylable arginine abolishes the behavior. (iv) biochemical experiments show that the formation of the punctate foci may be consistent with LLPS.

On balance, this is an interesting study that describes the role of acetylation of Tip60 in controlling its biochemical behavior as well as its localization and function in cells. The authors mention in their Discussion section other examples showing that acetylation can change the behavior of proteins with respect to LLPS; depending on the specific context, acetylation can promote (as here for Tip60) or impair LLPS.

Strengths:

The experiments are largely convincing and appear to be well executed.

Weaknesses:

The main concern I have is that all in vivo (i.e. in cells) experiments are done with overexpression in Cos-1 cells, in the presence of the endogenous protein. No attempt is made to use e.g. cells that would be KO for Tip60 in order to have a cleaner system or to look at the endogenous protein. It would be reassuring to know that what the authors observe with highly overexpressed proteins also takes place with endogenous proteins.

Response: The main reason to perform these experiments with overexpression system was to generate different point mutants and deletion mutants of TIP60 and analyse their effect on its properties and functions. To validate our observations with overexpression system, we also examined localization pattern of endogenous TIP60 by IFA and results depict similar kind of foci pattern within the nucleus as observed with overexpressed TIP60 protein (Figure 4A). However, we understand the reviewers concern and agree to repeat some of the overexpression experiments under endogenous TIP60 knockdown conditions using siRNA or shRNA against 3’ UTR region.

Also, it is not clear how often the experiments have been repeated and additional quantifications (e.g. of western blots) would be useful.

Response: The experiments were performed as independent biological replicates (n=3) and this is mentioned in the figure legends. Regarding the suggestion for quantifying Western blots, we want to bring into the notice that where ever required (for blots such as Figure 2F, 6H) that require quantitative estimation, graph representing quantitated value with p-value had already been added. However as suggested, in addition, quantitation for Figure 6D will be performed and added in the revised version.

In addition, regarding the LLPS description (Figure 1), it would be important to show the wetting behaviour and the temperature-dependent reversibility of the droplet formation.

Response: We appreciate the suggestion, and we will perform these assays and include the results in the revised version.

In Fig 3C the mutant (K187R) Tip60 is cytoplasmic, but still appears to form foci. Is this still reflecting phase separation, or some form of aggregation?

Response: TIP60 (K187R) mutant remains cytosolic with homogenous distribution as shown in Figure 2E. Also with TIP60 partners like PXR or p53, this mutant protein remains homogenously distributed in the cytosol. However, when co-expressed with TIP60 (Wild-type) protein, this mutant protein although still remain cytosolic some foci-like pattern is also observed at the nuclear periphery which we believe could be accumulated aggregates.

Reviewer 2

The manuscript "Autoacetylation-mediated phase separation of TIP60 is critical for its functions" by Dubey S. et al reported that the acetyltransferase TIP60 undergoes phase separation in vitro and cell nuclei. The intrinsically disordered region (IDR) of TIP60, particularly K187 within the IDR, is critical for phase separation and nuclear import. The authors showed that K187 is autoacetylated, which is important for TIP60 nuclear localization and activity on histone H4. The authors did several experiments to examine the function of K187R mutants including chromatin binding, oligomerization, phase separation, and nuclear foci formation. However, the physiological relevance of these experiments is not clear since TIP60 K187R mutants do not get into nuclei. The authors also functionally tested the cancer-derived R188P mutant, which mimics K187R in nuclear localization, disruption of wound healing, and DNA damage repair. However, similar to K187R, the R188P mutant is also deficient in nuclear import, and therefore, its defects cannot be directly attributed to the disruption of the phase separation property of TIP60. The main deficiency of the manuscript is the lack of support for the conclusion that "autoacetylation-mediated phase separation of TIP60 is critical for its functions".

This study offers some intriguing observations. However, the evidence supporting the primary conclusion, specifically regarding the necessity of the intrinsically disordered region (IDR) and K187ac of TIP60 for its phase separation and function in cells, lacks sufficient support and warrants more scrutiny. Additionally, certain aspects of the experimental design are perplexing and lack controls to exclude alternative interpretations. The manuscript can benefit from additional editing and proofreading to improve clarity.

Response: We understand the point raised by the reviewer, however we would like to draw his attention to the data where we clearly demonstrated that acetylation of lysine 187 within the IDR of TIP60 is required for its phase separation (Figure 2J). We would like to draw reviewer’s attention to other TIP60 mutants within IDR (R177H, R188H, K189R) which all enters the nucleus and make phase separated foci. Cancer-associated mutation at R188 behaves similarly because it also hampers TIP60 acetylation at the adjacent K187 residue. Our in vitro and in cellulo results clearly demonstrate that autoacetylation of TIP60 at K187 within its IDR is critical for multiple functions including its translocation inside the nucleus, its protein-protein interaction and oligomerization which are prerequisite for phase separation of TIP60.

There are two putative NLS sequences (NLS #1 from aa145; NLS #2 from aa184) in TIP60, both of which are within the IDR. Deletion of the whole IDR is therefore expected to abolish the nuclear localization of TIP60. Since K187 is within NLS #2, the cytoplasmic localization of the IDR and K187R mutants may not be related to the ability of TIP60 to phase separation.

Response: We are not disputing the presence of putative NLS within IDR region of TIP60, however our results through different mutations within IDR region (K76, K80, K148, K150, R177, R178, R188, K189) clearly demonstrate that only K187 residue acetylation is critical to shuttle TIP60 inside the nucleus while all other lysine mutants located within these putative NLS region exhibited no impact on TIP60’s nuclear shuttling. We have mentioned this in our discussion, that autoacetylation of TIP60’s K187 may induce local structural modifications in its IDR which is critical for translocating TIP60 inside the nucleus where it undergoes phase separation critical for its functions. A previous example of similar kind shows, acetylation of lysine within the NLS region of TyrRS by PCAF promote its nuclear localization (Cao X et al 2017, PNAS). IDR region (which also contains K187 site) is important for phase separation once the protein enters inside the nucleus. This could be the cell’s mechanism to prevent unwarranted action of TIP60 until it enters the nucleus and phase separate on chromatin at appropriate locations.

The chromatin-binding activity of TIP60 depends on HAT activity, but not phase-separation (Fig 1I), (Fig 2B). How do the authors reconcile the fact that the K187R mutant is able to bind to chromatin with lower activity than the HAT mutant (Fig 2F, 2I)?

Response: K187 acetylation is required for TIP60’s nuclear translocation but not critical for chromatin binding. When soluble fraction is prepared in fractionation experiment, nuclear membrane is disrupted and TIP60 (K187R) mutant has no longer hindrance in accessing the chromatin and thus can load on the chromatin (although not as efficient as Wild-type protein). For efficient chromatin binding auto-acetylation of other lysine residues in TIP60 is required which might be hampered due to reduced catalytic activity or not sufficient enough to maintain equilibrium with HDAC’s activity inside the nucleus. In case of K187R, the reduced auto-acetylation is captured when protein is the cytosol. During fractionation, once this mutant has access to chromatin, it might auto-acetylate other lysine residues critical for chromatin loading (remember catalytic domain is intact in this mutant). This is evident due to hyper auto-acetylation of Wild-type protein compared to K187R or HAT mutant proteins. We want to bring into notice that phase-separation occurs only after efficient chromatin loading of TIP60 that is the reason that under in-cellulo conditions, both K187R (which cannot enter the nucleus) and HAT mutant (which enters the nucleus but fails to efficiently binds onto the chromatin) fails to form phase separated nuclear punctate foci.

The DIC images of phase separation in Fig 2I need to be improved. The image for K187R showed the irregular shape of the condensates, which suggests particles in solution or on the slide. The authors may need to use fluorescent-tagged TIP60 in the in vitro LLPS experiments.

Response: We believe this comment is for figure 2J. The irregularly shaped condensates observed for TIP60 K187R are unique to the mutant protein and are not caused by particles on the slide. We would like to draw reviewer’s attention to supplementary figure S2A, where DIC images for TIP60 (Wild-type) protein tested under different protein and PEG8000 conditions are completely clear where protein did not made phase separated droplets ruling out the probability of particles in solution or slides.

The authors mentioned that the HAT mutant of TIP60 does not phase separate, which needs to be included.

Response: We have already added the image of RFP-TIP60 (HAT mutant) in supplementary Fig S4A (panel 2) in the manuscript.

Related to Point 3, the HAT mutant that doesn't form punctate foci by itself, can incorporate into WT TIP60 (Fig 5A). In vitro LLPS assay for WT, HAT, and K187R mutants with or without acetylation should be included. WT and mutant TIP can be labelled with GFP and RFP, respectively.

Response: We would like to draw reviewer’s attention towards our co-expression experiments performed in Figure 5 where Wild-type protein (both tagged and untagged condition) is able to phase separate and make punctate foci with co-expressed HAT mutant protein (with depleted autoacetylation capacity). We believe these in cellulo experiments are already able to answer the queries what reviewer is suggesting to acheive by in vitro experiments.

Fig 3A and 3B showed that neither K187 mutant nor HAT mutant could oligomerize. If both experiments were conducted in the absence of in vitro acetylation, how do the authors reconcile these results?

Response: We thank the reviewer for highlighting our oversight in omitting the mention of acetyl coenzyme A here. To induce acetylation under in vitro conditions, we have added 10 µM acetyl CoA into the reactions depicted in Figure 3A and 3B. The information for acetyl CoA for Figure 3B was already included in the GST-pull down assay (material and methods section). We will add the same in the oligomerization assay of material and methods in the revised manuscript.

In Fig 4, the colocalization images showed little overlap between TIP60 and nuclear speckle (NS) marker SC35, indicating that the majority of TIP60 localized in the nuclear structure other than NS. Have the authors tried to perturbate the NS by depleting the NS scaffold protein and examining TIP60 foci formation? Do PXR and TP53 localize to NS?

Response: Under normal conditions majority of TIP60 is not localized in nuclear speckles (NS) so we believe that perturbing NS will not have significant effect on TIP60 foci formation. Interestingly, recently a study by Shelly Burger group (Alexander KA et al Mol Cell. 2021 15;81(8):1666-1681) had shown that p53 localizes to NS to regulate subset of its targeted genes. We have mentioned about it in our discussion section. No information is available about localization of PXR in NS.

Were TIP60 substrates, H4 (or NCP), PXR, TP53, present inTIP60 condensates in vitro? It's interesting to see both PXR and TP53 had homogenous nuclear signals when expressed together with K187R, R188P (Fig 6E, 6G), or HAT (Suppl Fig S4A) mutants. Are PXR or TP53 nuclear foci dependent on their acetylation by TIP60? This can and should be tested.

Response: Both p53 and PXR are known to be acetylated by TIP60. In case of PXR, TIP60 acetylate PXR at lysine 170 and this TIP60-mediated acetylation of PXR at K170 is important for TIP60-PXR foci which now we know are formed by phase separation (Bakshi K et al Sci Rep. 2017 Jun 16;7(1):3635).

Since R188P mutant, like K187R, does not get into the nuclei, it is not suitable to use this mutant to examine the functional relevance of phase separation for TIP60. The authors need to find another mutant in IDR that retains nuclear localization and overall HAT activity but specifically disrupts phase separation. Otherwise, the conclusion needs to be restated. All cancer-derived mutants need to be tested for LLPS in vitro.

Response: We appreciate the reviewer’s point here, but it is important to note that the objective of these experiments is to understand the impact of K187R (critical in multiple aspects of TIP60 including phase separation) and R188P (a naturally occurring cancer-associated mutation and behaving similarly to K187R) on TIP60’s activities to determine their functional relevance. As suggested by the reviewer to test and find IDR mutant that fails to phase separate however retains nuclear localization and catalytic activity can be examined in future studies.

For all cellular experiments, it is not mentioned whether endogenous TIP60 was removed and absent in the cell lines used in this study. It's important to clarify this point because the localization and function of mutant TIP60 are affected by WT TIP60 (Fig 5).

Response: Endogenous TIP60 was present in in cellulo experiments, however as suggested by reviewer 1 we will perform some of the in cellulo experiments under endogenous TIP60 knockdown condition to validate our findings.

It is troubling that H4 peptide is used for in vitro HAT assay since TIP60 has much higher activity on nucleosomes and its preferred substrates include H2A.

Response: The purpose of using H4 peptide in the HAT assay is to determine the impact of mutations of TIP60’s catalytic activity. As H4 is one of the major histone substrate for TIP60, we believe it satisfy the objective of experiments.

Reviewer 3

This study presents results arguing that the mammalian acetyltransferase Tip60/KAT5 auto-acetylates itself on one specific lysine residue before the MYST domain, which in turn favors not only nuclear localization but also condensate formation on chromatin through LLPS. The authors further argue that this modification is responsible for the bulk of Tip60 autoacetylation and acetyltransferase activity towards histone H4. Finally, they suggest that it is required for association with txn factors and in vivo function in gene regulation and DNA damage response.

These are very wide and important claims and, while some results are interesting and intriguing, there is not really close to enough work performed/data presented to support them. In addition, some results are redundant between them, lack consistency in the mutants analyzed, and show contradiction between them. The most important shortcoming of the study is the fact that every single experiment in cells was done in over-expressed conditions, from transiently transfected cells. It is well known that these conditions can lead to non-specific mass effects, cellular localization not reflecting native conditions, and disruption of native interactome. On that topic, it is quite striking that the authors completely ignore the fact that Tip60 is exclusively found as part of a stable large multi-subunit complex in vivo, with more than 15 different proteins. Thus, arguing for a single residue acetylation regulating condensate formation and most Tip60 functions while ignoring native conditions (and the fact that Tip60 cannot function outside its native complex) does not allow me to support this study.

Response: We appreciate the reviewer’s point here, but it is important to note that the main purpose to use overexpression system in the study is to analyse the effect of different generated point/deletion mutations on TIP60. We have overexpressed proteins with different tags (GFP or RFP) or without tags (Figure 3C, Figure 5) to confirm the behaviour of protein which remains unperturbed due to presence of tags. To validate we have also examined localization of endogenous TIP60 protein which also depict similar localization behaviour as overexpressed protein. We would like to draw attention that there are several reports in literature where similar kind of overexpression system are used to determine functions of TIP60 and its mutants. Also nuclear foci pattern observed for TIP60 in our studies is also reported by several other groups.

Sun, Y., et. al. (2005) A role for the Tip60 histone acetyltransferase in the acetylation and activation of ATM. Proc Natl Acad Sci U S A, 102(37):13182-7.

Kim, C.-H. et al. (2015) ‘The chromodomain-containing histone acetyltransferase TIP60 acts as a code reader, recognizing the epigenetic codes for initiating transcription’, Bioscience, Biotechnology, and Biochemistry, 79(4), pp. 532–538.

Wee, C. L. et al. (2014) ‘Nuclear Arc Interacts with the Histone Acetyltransferase Tip60 to Modify H4K12 Acetylation(1,2,3).’, eNeuro, 1(1). doi: 10.1523/ENEURO.0019-14.2014.

However, as a caution and suggested by other reviewers also we will perform some of these overexpression experiments in absence of endogenous TIP60 by using 3’ UTR specific siRNA/shRNA.

We thank the reviewer for his comment on muti-subunit complex proteins and we would like to expand our study by determining the interaction of some of the complex subunits with TIP60 ((Wild-type) that forms nuclear condensates), TIP60 ((HAT mutant) that enters the nucleus but do not form condensates) and TIP60 ((K187R) that do not enter the nucleus and do not form condensates). We will include the result of these experiments in the revised manuscript.

• It is known that over-expression after transient transfection can lead to non-specific acetylation of lysines on the proteins, likely in part to protect from proteasome-mediated degradation. It is not clear whether the Kac sites targeted in the experiments are based on published/public data. In that sense, it is surprising that the K327R mutant does not behave like a HAT-dead mutant (which is what exactly?) or the K187R mutant as this site needs to be auto-acetylated to free the catalytic pocket, so essential for acetyltransferase activity like in all MYST-family HATs. In addition, the effect of K187R on the total acetyl-lysine signal of Tip60 is very surprising as this site does not seem to be a dominant one in public databases.

Response: We have chosen autoacetylation sites based on previously published studies where LC-MS/MS and in vitro acetylation assays were used to identified autoacetylation sites in TIP60 which includes K187. We have already mentioned about it in the manuscript and have quoted the references (1. Yang, C., et al (2012). Function of the active site lysine autoacetylation in Tip60 catalysis. PloS one 7, e32886. 10.1371/journal.pone.0032886. 2. Yi, J., et al (2014). Regulation of histone acetyltransferase TIP60 function by histone deacetylase 3. The Journal of biological chemistry 289, 33878–33886. 10.1074/jbc.M114.575266.). We would like to emphasize that both these studies have identified K187 as autoacetylation site in TIP60. Since TIP60 HAT mutant (with significantly reduced catalytic activity) can also enter nucleus, it is not surprising that K327 could also enter the nucleus.

• As the physiological relevance of the results is not clear, the mutants need to be analyzed at the native level of expression to study real functional effects on transcription and localization (ChIP/IF). It is not clear the claim that Tip60 forms nuclear foci/punctate signals at physiological levels is based on what. This is certainly debated because in part of the poor choice of antibodies available for IF analysis. In that sense, it is not clear which Ab is used in the Westerns. Endogenous Tip60 is known to be expressed in multiple isoforms from splice variants, the most dominant one being isoform 2 (PLIP) which lacks a big part (aa96-147) of the so-called IDR domain presented in the study. Does this major isoform behave the same?

Response: TIP60 antibody used in the study is from Santa Cruz (Cat. No.- sc-166323). This antibody is widely used for TIP60 detection by several methods and has been cited in numerous publications. Cat. No. will be mentioned in the manuscript. Regarding isoforms, three isoforms are known for TIP60 among which isoform 2 is majorly expressed and used in our study. Isoform and 1 and 2 have same length of IDR (150 amino acids) while isoform 3 has IDR of 97 amino acids. Interestingly, the K187 is present in all the isoforms (already mentioned in the manuscript) and missing region (96-147 amino acid) in isoform 3 has less propensity for disordered region (marked in blue circle). This clearly shows that all the isoforms of TIP60 has the tendency to phase separate.

Author response image 1.

• It is extremely strange to show that the K187R mutant fails to get in the nuclei by cell imaging but remains chromatin-bound by fractionation... If K187 is auto-acetylated and required to enter the nucleus, why would a HAT-dead mutant not behave the same?

Response: We would like to draw attention that both HAT mutant and K187R mutant are not completely catalytically dead. As our data shows both these mutants have catalytic activity although at significantly decreased levels. We believe that K187 acetylation is critical for TIP60 to enter the nucleus and once TIP60 shuttles inside the nucleus autoacetylation of other sites is required for efficient chromatin binding of TIP60. In fractionation assay, nuclear membrane is dissolved while preparing the soluble fraction so there is no hindrance for K187R mutant in accessing the chromatin. While in the case of HAT mutant, it can acetylate the K187 site and thus is able to enter the nucleus however this residual catalytic activity is either not able to autoacetylate other residues required for its efficient chromatin binding or to counter activities of HDAC’s deacetylating the TIP60.

• If K187 acetylation is key to Tip60 function, it would be most logical (and classical) to test a K187Q acetyl-mimic substitution. In that sense, what happens with the R188Q mutant? That all goes back to the fact that this cluster of basic residues looks quite like an NLS.

Response: As suggested we will generate acetylation mimicking mutant for K187 site and examine it. Result will be added in the revised manuscript.

• The effect of the mutant on the TIP60 complex itself needs to be analyzed, e.g. for associated subunits like p400, ING3, TRRAP, Brd8...

Response: As suggested we will examine the effect of mutations on TIP60 complex

Author Response:

We take the liberty to thank all of you for your constructive and inspiring comments, which will help us substantially improve the final version of the paper. Before our final revision with details, I am writing this provisional letter to have a quick response to our reviewers’ comments.

I first give a quick and short summary for your public reviews, then respond point-by-point.

Editors:

1. More discussion is needed.

2. More discussion about eye fixation during adaptation. Discuss why increasing visual uncertainty by blurring the cursor in the present study produces the opposite findings of previous studies (Tsay et al., 2021; Makino et al., 2023).

3. Discuss the broad impact of the current model.

4. Share the codes and the metadata (instead of the current data format).

Response: This is a concise summary of the major concerns listed in the public review. Given these concerns are easy to address, we are giving a quick but point-to-point response for now. The elaborate version will be put into our formal revision.

**Reviewer 1: **

1) More credit should be given to the PReMo model: a) The PReMo model also proposes that perceptual error drives implicit adaptation, as in a new publication in Tsay et al., 2023, which was not public at the time of the current writing; and b) The PReMo model can account for some dataset, e.g. Fig 4A.

Response: We will add this new citation and point out that the new paper also uses the term perceptual error. We will also point out that the PReMo model has the potential to explain Fig 4A, though for now, it assumes an additional visual shift to explain the positive proprioceptive changes relative to the target. We would expand the discussion about the comparison between the two models.

2) The present study produced an opposite finding of a previous finding, i.e., upregulating visual uncertainty (by cursor blurring here) decreases adaptation for large perturbations but less so for small perturbations, while previous studies have shown the opposite (by using a cursor cloud; Tsay et al., 2021; Makino et al., 2023). This needs explanation.

Response: Using the cursor cloud (Tsay et al., 2021, Makino et al., 2023) to modulate visual uncertainty has inherent drawbacks that make it unsuitable for testing the sensory uncertainty effect for visuomotor rotation. For the error clamp paradigm, the error is defined as angular deviation. The cursor cloud consists of multiple cursors spanning over a range of angles, which affects both the sensory uncertainty (the intended outcome) AND the sensory estimate of angles (the error itself, the undesired outcome). In Bayesian terms, the cursor cloud aims to modulate the sigma of a distribution (sigma_v in our model), but it additionally affects the mean of the distribution (mu). This unnecessary confound is avoided by using cursor blurring, which is still a cursor with its center (mu) unchanged from an un-blurred cursor. Furthermore, as correctly pointed out in the original paper by Tsay et al., 2021, the cursor cloud often overlaps with the visual target. This “target hit” would affect adaptation, possibly via a reward learning mechanism (See Kim et al., 2019 eLife). This is a second confound that accompanies the cursor cloud. We will expand our discussion to explain the discrepancy between our findings and previous findings.

3) The estimation of visual uncertainty (our exp1) required people to fixate on the target, while this might not reflect the actual scenario during adaptation where people are free to look wherever they want.

Response: Our data shows otherwise: in a typical error-clamp setting, people fixate on the target for the majority of the time. For our Exp1, the fixation on the straight line between the starting position and the target is 86%-95% (as shown in Figure S1). We also collected eye-tracking data in our Exp4, which is a typical error-clamp experiment. More than 95% of gaze falls with +/- 50 pixels around the center of the screen, even slightly higher than Exp1. We will provide this part of the data in the revision. In fact, we designed our Exp1 to mimic the eye-tracking pattern as in typical error-clamp learning with carefully executed pilot experiments.

This high percentage of fixating on the target is not surprising: the error-clamp task requires participants to use their hands to move towards the target and to ignore the cursor. In fact, we would also like to point out that the high percentage of fixation on the aiming target is also true for conventional visuomotor rotation, which involves strategic re-aiming (shown in de Brouwer et al. 2018; Bromberg et al. 2019; we have an upcoming paper to show this). This is one reason that our new theory would also apply to other types of motor adaptation.

4) More methodology details are needed. E.g., a figure showing the visual blurring, a figure showing individual data, a table showing data from individual sessions, code sharing, and a possible new correlational analysis.

Response: All these additional methodological/analysis information will be provided. We were self-limited by writing a short paper, but the revision would be extended for all these details.

Reviewer 2:

1) More discussions are needed since the focus of this study is narrowly confined to visuomotor rotation. “A general computational principle, and its contributions to other motor learning paradigms remain to be explored”.

Response: This is a great suggestion since we also think our original Discussion has not elaborated on the possible broad impact of our theory. Our model is not limited to the error-clamp adaptation, where the participants were explicitly told to ignore the rotated cursor. The error-clamp paradigm is one rare example that implicit motor learning can be isolated in a nearly idealistic way. Our findings thus imply two key aspects of implicit adaptation: 1) localizing one’s effector is implicitly processed and continuously used to update the motor plan; 2) Bayesian cue combination is at the core of integrating multimodal feedback and motor-related cues (motor prediction cue in our model) when forming procedural knowledge for action control.

We will propose that the same two principles should be applied to various kinds of motor adaptation and motor skill learning, which constitutes motor learning in general. Most of our knowledge about motor adaptation is from visuomotor rotation, prism adaptation, force field adaptation, and saccadic adaptation. The first three types all involve localizing one’s effector under the influence of perturbed sensory feedback, and they also have implicit learning. We believe they can be modeled by variants of our model, or at least we should consider using the two principles above to think of their computational nature. For skill learning, especially for de novo learning, the area still lacks a fundamental computational model that accounts for the skill acquisition process on the level of relevant movement cues. Our model suggests a promising route, i.e., repetitive movements with a Bayesian cue combination of movement-related cues might underlie the implicit process of motor skills.

We will add more discussion on the possible broad implications of our model in the revision.

Reviewer 3:

1) Similar to Reviewer 1, raised the concern about whether people’s fixation in typical motor adaptation settings is similar to the fixation that we instructed in our Exp1.

Response: see above.

2) Similar to Reviewer 2, the concern was raised about whether our new theory is applicable to a broad context. Especially, error clamp appears to be a strange experimental manipulation that has no real-life appeal, “(i)Ignoring errors and suppressing adaptation would also be a disastrous strategy to use in the real world”.

Response: about the broad impact of our model, please see responses to Reviewer 2 above. We agree that ignoring errors (and thus “trying” to suppress adaptation) should not be a movement strategy for real-world intentional tasks. However, even in real life, we constantly attend to one thing and do the other thing; that’s when implicit motor processes are in charge. Furthermore, it is this exact “ignoring” instruction that elicits the implicit adaptation that we can work on. In this sense, the error-clamp paradigm is a great vehicle to isolate implicit adaptation and allows us to unpack its cognitive mechanism.

3) In Exp1, the 1s delay between the movement end and the presentation of the reference cursor might inflate the actual visual uncertainty.

Response: The 1s delay of the reference cursor would not inflate the estimate of visual uncertainty. Our Exp1 used a similar paradigm by visual science (e.g., White, Levi, and Aitsebaomo, Vision Research, 1992), which shows that delay does not lead to an obvious increase in visual uncertainty over a broad range of values (from 0.2s to >1s, see their Figure 5-6). We will add more methodology justifications in our revision.

4) Our Fig4A used Tsay et al., 2021 data, which, in the reviewer’s view, is not an appropriate measure of proprioceptive bias. The reason is that in this dataset, “participants actively move to a visual target, the reported hand positions do not reflect proprioception, but mostly the remembered position of the target participants were trying to move to.”

Response: We agree that Tsay et al., 2021 study used an unconventional way to measure the influence of implicit adaptation on proprioception. And, their observed “proprioceptive changes” should not be called “proprioceptive bias” which is conventionally a reserved term for measuring the difference between the estimated hand location relative to the actual hand location (and better to be a passively moved hand). However, we think their dataset is still subject to the same Bayesian cue combination principle and thus can be modeled. Our modeling of this dataset includes all relevant cues: the implicitly perceived hand position and the proprioceptive cue (given that the hand stays at the movement end). Both cues are in the extrinsic coordinates, which happened to set the target position as zero. But where to set the zero (whether it is the target or the actual hand location) does not matter for the model fitting. Note that our Exp4 is also based on PEA modeling of proprioceptive bias, and this time the data is presented relative to the actual location.

In the revision, we would keep the current Fig4A and start to call the data as proprioceptive change as opposed to proprioceptive bias to follow the convention.

Reviewer #1 (Public Review):

This valuable study demonstrates a novel mechanism by which implicit motor adaptation saturates for large visual errors in a principled normative Bayesian manner. Additionally, the study revealed two notable empirical findings: visual uncertainty increases for larger visual errors in the periphery, and proprioceptive shifts/implicit motor adaptation are non-monotonic, rather than ramp-like. This study is highly relevant for researchers in sensory cue integration and motor learning. However, I find some areas where statistical quantification is incomplete, and the contextualization of previous studies to be puzzling.

Issue #1: Contextualization of past studies.

While I agree that previous studies have focused on how sensory errors drive motor adaptation (e.g., Burge et al., 2008; Wei and Kording, 2009), I don't think the PReMo model was contextualized properly. Indeed, while PReMo should have adopted clearer language - given that proprioception (sensory) and kinaesthesia (perception) have been used interchangeably, something we now make clear in our new study (Tsay, Chandy, et al. 2023) - PReMo's central contribution is that a perceptual error drives implicit adaptation (see Abstract): the mismatch between the felt (perceived) and desired hand position. The current paper overlooks this contribution. I encourage the authors to contextualize PReMo's contribution more clearly throughout. Not mentioned in the current study, for example, PReMo accounts for the continuous changes in perceived hand position in Figure 4 (Figure 7 in the PReMo study).

There is no doubt that the current study provides important additional constraints on what determines perceived hand position: Firstly, it offers a normative Bayesian perspective in determining perceived hand position. PReMo suggests that perceived hand position is determined by integrating motor predictions with proprioception, then adding a proprioceptive shift; PEA formulates this as the optimal integration of these three inputs. Secondly, PReMo assumed visual uncertainty to remain constant for different visual errors; PEA suggests that visual uncertainty ought to increase (but see Issue #2).

Issue #2: Failed replication of previous results on the effect of visual uncertainty.

2a. A key finding of this paper is that visual uncertainty linearly increases in the periphery; a constraint crucial for explaining the non-monotonicity in implicit adaptation. One notable methodological deviation from previous studies is the requirement to fixate on the target: Notably, in the current experiments, participants were asked to fixate on the target, a constraint not imposed in previous studies. In a free-viewing environment, visual uncertainty may not attenuate as fast, and hence, implicit adaptation does not attenuate as quickly as that revealed in the current design with larger visual errors. Seems like this current fixation design, while important, needs to be properly contextualized considering how it may not represent most implicit adaptation experiments.

2b. Moreover, the current results - visual uncertainty attenuates implicit adaptation in response to large, but not small, visual errors - deviates from several past studies that have shown that visual uncertainty attenuates implicit adaptation to small, but not large, visual errors (Tsay, Avraham, et al. 2021; Makino, Hayashi, and Nozaki, n.d.; Shyr and Joshi 2023). What do the authors attribute this empirical difference to? Would this free-viewing environment also result in the opposite pattern in the effect of visual uncertainty on implicit adaptation for small and large visual errors?

2c. In the current study, the measure of visual uncertainty might be inflated by brief presentation times of comparison and referent visual stimuli (only 150 ms; our previous study allowed for a 500 ms viewing time to make sure participants see the comparison stimuli). Relatedly, there are some individuals whose visual uncertainty is greater than 20 degrees standard deviation. This seems very large, and less likely in a free-viewing environment.

2d. One important confound between clear and uncertain (blurred) visual conditions is the number of cursors on the screen. The number of cursors may have an attenuating effect on implicit adaptation simply due to task-irrelevant attentional demands (Parvin et al. 2022), rather than that of visual uncertainty. Could the authors provide a figure showing these blurred stimuli (gaussian clouds) in the context of the experimental paradigm? Note that we addressed this confound in the past by comparing participants with and without low vision, where only one visual cursor is provided for both groups (Tsay, Tan, et al. 2023).

Issue #3: More methodological details are needed.

3a. It's unclear why, in Figure 4, PEA predicts an overshoot in terms of perceived hand position from the target. In PReMo, we specified a visual shift in the perceived target position, shifted towards the adapted hand position, which may result in overshooting of the perceived hand position with this target position. This visual shift phenomenon has been discovered in previous studies (e.g., (Simani, McGuire, and Sabes 2007)).

3b. The extent of implicit adaptation in Experiment 2, especially with smaller errors, is unclear. The implicit adaptation function seems to be still increasing, at least by visual inspection. Can the authors comment on this trend, and relatedly, show individual data points that help the reader appreciate the variability inherent to these data?

3c. The same participants were asked to return for multiple days/experiments. Given that the authors acknowledge potential session effects, with attenuation upon re-exposure to the same rotation (Avraham et al. 2021), how does re-exposure affect the current results? Could the authors provide clarity, perhaps a table, to show shared participants between experiments and provide evidence showing how session order may not be impacting results?

3d. The number of trials per experiment should be detailed more clearly in the Methods section (e.g., Exp 4). Moreover, could the authors please provide relevant code on how they implemented their computational models? This would aid in future implementation of these models in future work. I, for one, am enthusiastic to build on PEA.

3f. In addition to predicting a correlation between proprioceptive shift and implicit adaptation on a group level, both PReMo and PEA (but not causal inference) predict a correlation between individual differences in proprioceptive shift and proprioceptive uncertainty with the extent of implicit adaptation (Tsay, Kim, et al. 2021). Interestingly, shift and uncertainty are independent (see Figures 4F and 6C in Tsay et al, 2021). Does PEA also predict independence between shift and uncertainty? It seems like PEA does predict a correlation.

References:

Avraham, Guy, Ryan Morehead, Hyosub E. Kim, and Richard B. Ivry. 2021. "Reexposure to a Sensorimotor Perturbation Produces Opposite Effects on Explicit and Implicit Learning Processes." PLoS Biology 19 (3): e3001147.<br /> Makino, Yuto, Takuji Hayashi, and Daichi Nozaki. n.d. "Divisively Normalized Neuronal Processing of Uncertain Visual Feedback for Visuomotor Learning."<br /> Parvin, Darius E., Kristy V. Dang, Alissa R. Stover, Richard B. Ivry, and J. Ryan Morehead. 2022. "Implicit Adaptation Is Modulated by the Relevance of Feedback." BioRxiv. https://doi.org/10.1101/2022.01.19.476924.<br /> Shyr, Megan C., and Sanjay S. Joshi. 2023. "A Case Study of the Validity of Web-Based Visuomotor Rotation Experiments." Journal of Cognitive Neuroscience, October, 1-24.<br /> Simani, M. C., L. M. M. McGuire, and P. N. Sabes. 2007. "Visual-Shift Adaptation Is Composed of Separable Sensory and Task-Dependent Effects." Journal of Neurophysiology 98 (5): 2827-41.<br /> Tsay, Jonathan S., Guy Avraham, Hyosub E. Kim, Darius E. Parvin, Zixuan Wang, and Richard B. Ivry. 2021. "The Effect of Visual Uncertainty on Implicit Motor Adaptation." Journal of Neurophysiology 125 (1): 12-22.<br /> Tsay, Jonathan S., Anisha M. Chandy, Romeo Chua, R. Chris Miall, Jonathan Cole, Alessandro Farnè, Richard B. Ivry, and Fabrice R. Sarlegna. 2023. "Implicit Motor Adaptation and Perceived Hand Position without Proprioception: A Kinesthetic Error May Be Derived from Efferent Signals." BioRxiv. https://doi.org/10.1101/2023.01.19.524726.<br /> Tsay, Jonathan S., Hyosub E. Kim, Darius E. Parvin, Alissa R. Stover, and Richard B. Ivry. 2021. "Individual Differences in Proprioception Predict the Extent of Implicit Sensorimotor Adaptation." Journal of Neurophysiology, March. https://doi.org/10.1152/jn.00585.2020.<br /> Tsay, Jonathan S., Steven Tan, Marlena Chu, Richard B. Ivry, and Emily A. Cooper. 2023. "Low Vision Impairs Implicit Sensorimotor Adaptation in Response to Small Errors, but Not Large Errors." Journal of Cognitive Neuroscience, January, 1-13.

NLS linkage conventions

for - hyper link - hyperlink - what's in a link? - keep the links outside the file

link

the original hyperlink that was active and allowed dynamic exploration of linked context with appropriate viewspecs

Now in Plex or Trail Mark.s the identity of a high resolution addressable content (the addressed is derived from the plexmark trailmark being present in an outline

whether it is in its own document or a clueon or plexon or a clue structure or plex structure outline within the context of a plexon clueon page virtual document

is not couple with a view spec but the very identity of a document carries marks that indicate - intentional - semantic - symmathetic - self-describing

qualifier marks tht in turn are amenable to interpretation by humans and machines such that "viewspec" or rahter "miorphic interpretations" can be associated with them

becuase of the immanent structuring no linear listing were ever used in Meta-Lisp in practice, dynamic navigation

although a complete listing via depth first traversal was always possible and told the complete story

parse rule

only contain parse information and node-building

just like MetaLISP!

figure out supposed to do

Certainly! Let's go through the possible outcomes and what the function would return in each case:

Case 1: Document Found and Deleted

If the document with the specified _id is found and successfully deleted, the function will return the deleted document. In the example:

```javascript const mongoose = require('mongoose'); const User = mongoose.model('User');

const userIdToDelete = "5f4fbf7d94bf551063d84924";

User.findByIdAndDelete(userIdToDelete) .then(deletedUser => { if (deletedUser) { console.log(User with _id ${userIdToDelete} deleted successfully.); console.log("Deleted User:", deletedUser); } else { console.log(No user found with _id ${userIdToDelete}.); } }) .catch(error => { console.error(Error deleting user: ${error}); }); ```

If the user with _id "5f4fbf7d94bf551063d84924" exists, the output might look like:

User with _id 5f4fbf7d94bf551063d84924 deleted successfully. Deleted User: { "_id": ObjectId("5f4fbf7d94bf551063d84924"), "name": "John Doe", "age": 30, "email": "john@example.com" }

Case 2: Document Not Found

If no document with the specified _id is found, the function will return null. In the example:

```javascript const mongoose = require('mongoose'); const User = mongoose.model('User');

const userIdToDelete = "nonexistent_id";

User.findByIdAndDelete(userIdToDelete) .then(deletedUser => { if (deletedUser) { console.log(User with _id ${userIdToDelete} deleted successfully.); console.log("Deleted User:", deletedUser); } else { console.log(No user found with _id ${userIdToDelete}.); } }) .catch(error => { console.error(Error deleting user: ${error}); }); ```

If there's no user with the specified _id, the output might look like:

No user found with _id nonexistent_id.

Case 3: Error Occurs

If an error occurs during the operation (e.g., database connection issues), the function will reject the promise, and the .catch block will be executed to handle the error. In the example:

```javascript const mongoose = require('mongoose'); const User = mongoose.model('User');

const userIdToDelete = "5f4fbf7d94bf551063d84924";

User.findByIdAndDelete(userIdToDelete) .then(deletedUser => { if (deletedUser) { console.log(User with _id ${userIdToDelete} deleted successfully.); console.log("Deleted User:", deletedUser); } else { console.log(No user found with _id ${userIdToDelete}.); } }) .catch(error => { console.error(Error deleting user: ${error}); }); ```

If there's an error, the output might look like:

Error deleting user: [Error message describing the issue]Certainly! Let's break down the provided information into simpler terms with an example:

Purpose:

The goal of this function is to find a document in a MongoDB database using its _id field and then delete that document.

Parameters:

• id: This is the unique identifier of the document you want to find and delete. It can be an object, number, or string representing the _id value.
• options: An optional object that allows you to customize the behavior of the operation.

Returns:

The function returns a Query object, which is a way to interact with MongoDB queries.

Example:

Suppose you have a MongoDB collection named "users" with documents like this:

json { "_id": ObjectId("5f4fbf7d94bf551063d84924"), "name": "John Doe", "age": 30, "email": "john@example.com" }

Now, if you want to find and delete the user with the _id "5f4fbf7d94bf551063d84924," you can use the function like this:

```javascript const mongoose = require('mongoose'); const User = mongoose.model('User'); // Assume you have a model named 'User'

const userIdToDelete = "5f4fbf7d94bf551063d84924";

// Using findByIdAndDelete function User.findByIdAndDelete(userIdToDelete) .then(deletedUser => { if (deletedUser) { console.log(User with _id ${userIdToDelete} deleted successfully.); } else { console.log(No user found with _id ${userIdToDelete}.); } }) .catch(error => { console.error(Error deleting user: ${error}); }); ```

This code finds the user with the specified _id and deletes it using the findByIdAndDelete function. The function returns a promise, so we use .then to handle success and .catch to handle errors. The deletedUser variable contains the deleted user document if found, or it is null if no user was found with the specified _id.

Certainly! Let's break down the explanation with examples in simple words:

Resetting State with a Key:

In React, you can reset a component's state by changing its key. This is demonstrated in the example:

```jsx import { useState } from 'react';

export default function App() { const [version, setVersion] = useState(0);

function handleReset() { setVersion(version + 1); }

return ( <> <button onClick={handleReset}>Reset</button> {/ Form component with a key based on the version state /} <Form key={version} /> <br /> ); }

function Form() { const [name, setName] = useState('Taylor');

return ( <> <input value={name} onChange={e => setName(e.target.value)} />

Hello, {name}.

<br /> ); } ```

In this example, the Form component's key is tied to the version state. When you click the "Reset" button, it increments the version, causing React to recreate the Form component from scratch. This results in the state of the Form being reset.

Storing Information from Previous Renders:

Sometimes, you might want to adjust state in response to rendering. The example introduces the CountLabel component:

```jsx import { useState } from 'react';

export default function CountLabel({ count }) { const [prevCount, setPrevCount] = useState(count); const [trend, setTrend] = useState(null);

if (prevCount !== count) { setPrevCount(count); setTrend(count > prevCount ? 'increasing' : 'decreasing'); }

return ( <>

{count}

{trend &&

The count is {trend}

} <br /> ); } ```

Here, the CountLabel component tracks the previous count and determines the trend (increasing or decreasing) when the count prop changes during rendering.

Troubleshooting Tips:

The examples provide troubleshooting tips for common issues, such as avoiding infinite re-renders, handling state updates, and understanding the behavior of initializer and updater functions.

Summary:

• Changing a component's key can reset its state.
• Storing information from previous renders may require additional state variables.
• Troubleshooting tips help address common issues related to state updates.

These concepts are essential for effective state management in React components.

Certainly! Let's explore the difference between passing an updater function and passing the next state directly in simple words with examples:

Example 1: Passing the Updater Function

```jsx import { useState } from 'react';

export default function Counter() { const [age, setAge] = useState(42);

function increment() { setAge(a => a + 1); }

return ( <>

Your age: {age}

<button onClick={() => { increment(); increment(); increment(); }}>+3</button> <button onClick={() => { increment(); }}>+1</button> <br /> ); } ```

In this example:

• The increment function uses an updater function to increase the age by 1.
• The "+3" button calls increment three times. Because of the updater function, each call gets the latest state, resulting in the age being incremented by 3.

Example 2: Passing the Next State Directly

```jsx import { useState } from 'react';

export default function Counter() { const [age, setAge] = useState(42);

function increment() { setAge(age + 1); }

return ( <>

Your age: {age}

<button onClick={() => { increment(); increment(); increment(); }}>+3</button> <button onClick={() => { increment(); }}>+1</button> <br /> ); } ```

In this example:

• The increment function directly passes the next state (age + 1) to setAge.
• The "+3" button calls increment three times. However, because the state updates are asynchronous, the value of age may not be what you expect due to the batching of updates.

Why Use Updater Functions?

Using updater functions is often preferred, especially when you need to calculate the next state based on the current state. It ensures that each call gets the most recent state, even if there are multiple state updates in quick succession.

Example 3: Updating an Object in State

```jsx import { useState } from 'react';

export default function Form() { const [form, setForm] = useState({ firstName: 'Barbara', lastName: 'Hepworth', email: 'bhepworth@sculpture.com', });

return ( <> <label> First name: <input value={form.firstName} onChange={e => { setForm({ ...form, firstName: e.target.value }); }} /> </label> <label> Last name: <input value={form.lastName} onChange={e => { setForm({ ...form, lastName: e.target.value }); }} /> </label> <label> Email: <input value={form.email} onChange={e => { setForm({ ...form, email: e.target.value }); }} /> </label>

{form.firstName}{' '} {form.lastName}{' '} ({form.email})

<br /> ); } ```

In this example, we use the spread syntax and an updater function to update an object (form) in state.

Simple Explanation:

• Updater Function: Use it when you want to ensure that the state you're working with is the most recent one, especially in scenarios with quick and multiple updates.

• Directly Passing Next State: This approach may not guarantee the most up-to-date state due to batching, and it's typically used when the next state doesn't depend on the current state.

In summary, while both approaches work, using updater functions is often preferred when the next state depends on the current state, providing a more reliable and expected behavior in your React components.

Certainly! Let's dive into this concept with more detail and simple language:

Updating State Based on Previous State:

In React, when you want to update the state based on its current value, you need to be mindful of the asynchronous nature of the setState function. If you repeatedly call setState with the same state value in a synchronous block of code, React might not have updated the state in between calls.

Example of the Issue:

Consider the following code:

```jsx function Counter() { const [age, setAge] = useState(42);

function handleClick() { setAge(age + 1); // age is 42, setAge(42 + 1) setAge(age + 1); // age is still 42, setAge(42 + 1) setAge(age + 1); // age is still 42, setAge(42 + 1) }

return ( <div>

Age: {age}

<button onClick={handleClick}>Increment Age</button> </div> ); } ```

Here, even though setAge is called three times in a row, after the first call, age remains 42 in the subsequent calls because the state updates are asynchronous.

Solving the Problem with Updater Function:

To ensure that you're working with the most recent state, you can pass an updater function to setAge instead of the next state directly:

```jsx function Counter() { const [age, setAge] = useState(42);

function handleClick() { setAge((prevAge) => prevAge + 1); // prevAge is 42, setAge(42 => 43) setAge((prevAge) => prevAge + 1); // prevAge is now 43, setAge(43 => 44) setAge((prevAge) => prevAge + 1); // prevAge is now 44, setAge(44 => 45) }

return ( <div>

Age: {age}

<button onClick={handleClick}>Increment Age</button> </div> ); } ```

Now, by using an updater function with setAge, React ensures that each call gets the most up-to-date state (prevAge), and you get the correct sequence of state updates.

How Updater Function Works:

• The updater function, in this case, takes the previous state (prevAge) and calculates the next state by adding 1.
• React puts these updater functions in a queue.
• During the next render, React calls each updater function in the order they were queued, ensuring the correct sequence of state updates.

By convention, the argument to the updater function is often named after the state variable, like prevAge. This makes it clear that it represents the previous state of the age variable.

Strict Mode Warnings:

In development mode, React might call your updater functions twice to check for accidental impurities. However, this doesn't affect the production behavior.

In simpler terms, using updater functions with setState ensures that you work with the latest state when updating based on the previous state, preventing issues caused by the asynchronous nature of state updates in React.

Certainly! Let's break down the concept of batching state updates in React in simple words with an example:

Batching State Updates in React:

When you make multiple state updates in a React component, React doesn't immediately apply each update. Instead, it batches them together for efficiency. This means that several state updates are grouped and processed together to avoid unnecessary renders.

Example - Without Batching:

Consider this example without batching:

```jsx function Counter() { const [count, setCount] = useState(0);

function handleClick() { setCount(count + 1); // Update 1 setCount(count + 1); // Update 2 setCount(count + 1); // Update 3 }

return ( <div>

Count: {count}

<button onClick={handleClick}>Increment Count</button> </div> ); } ```

In this example:

• Initially, count is 0.
• When you click the button, three consecutive state updates are triggered.

Without batching, React would process each update immediately. After the first update, the subsequent updates would still see the old count value.

Example - With Batching:

React batches these updates together, ensuring they are processed in a more efficient way. Here's how React would handle it:

```jsx function Counter() { const [count, setCount] = useState(0);

function handleClick() { // React batches these updates setCount(count + 1); // Update 1 setCount(count + 1); // Update 2 setCount(count + 1); // Update 3 // React processes them together, avoiding unnecessary renders }

return ( <div>

Count: {count}

<button onClick={handleClick}>Increment Count</button> </div> ); } ```

With batching, React processes these state updates together. It calculates the new state based on the current state at the time of the batch, ensuring that the updates are applied more efficiently.

Simple Explanation:

Batching is like collecting multiple change requests and applying them all at once, rather than one by one. It's similar to a waiter taking multiple orders from a table and bringing all the dishes together to save time.

In the context of React, batching state updates helps prevent unnecessary renders and makes the application more efficient.

In summary, React batches state updates to optimize performance by processing multiple updates together, resulting in a more responsive and efficient user interface.

Absolutely, let's simplify the concepts with examples:

1. Declaring State with useState:

When using useState in React, you declare state variables at the top level of your component. The convention is to name state variables using array destructuring, like [something, setSomething].

```jsx import { useState } from 'react';

function MyComponent() { // Declaring state variables: age and name const [age, setAge] = useState(42); const [name, setName] = useState('Taylor'); // ... } ```

Here, age and name are state variables initialized with default values, and setAge and setName are the corresponding functions to update these state variables.

2. Updating State with set Functions:

To update the state and trigger a re-render, you use the set functions:

jsx function handleClick() { // Updating the state variables setName('Robin'); setAge((prevAge) => prevAge + 1); // Using a function to calculate the next state based on the previous state }

• setName('Robin'): Updates the name state variable directly.
• setAge((prevAge) => prevAge + 1): Uses a function to calculate the next state of age based on the previous state.

3. Pitfall: Asynchronous Nature:

One pitfall to be aware of is that calling the set function doesn't immediately change the state in the current code execution:

jsx function handleClick() { setName('Robin'); console.log(name); // Still "Taylor"! }

Here, the console.log will still output the old value of name. The state update takes effect on the next render, not immediately.

4. Avoiding Unnecessary Renders:

React has optimizations in place to avoid unnecessary renders. If the new state provided to the set function is identical to the current state, React skips re-rendering:

jsx setName('Taylor'); // No re-render because the new value is the same as the current value

This is an optimization to make React more efficient.

5. Special Behavior for Functions:

If you pass a function to setState, it's treated as an updater function, and it's called during the next render:

jsx setAge((prevAge) => prevAge + 1);

Here, the function receives the previous state (prevAge) and returns the next state, allowing more dynamic state updates.

6. Strict Mode Warnings:

In Strict Mode, React might call your updater function twice during development to help identify accidental impurities. However, this doesn't affect production.

In simple terms, useState is a powerful hook that allows functional components to have and manage state, enabling dynamic and interactive user interfaces in React.

Certainly! Let's break it down in simple terms:

1. Props Changing Over Time:

In React, a component can receive different pieces of information, called "props," from its parent component. Props can change over time, reflecting the changing state of the application.

2. Example: Clock Component:

Imagine a Clock component that displays the current time and has a specified color. The Clock component is given two props: color and time.

jsx // Clock.js export default function Clock({ color, time }) { return ( <h1 style={{ color: color }}> {time} </h1> ); }

Here, the Clock component uses these props to display the time in a specified color.

3. Props Are Immutable:

Props are like snapshots of data at a specific moment. They represent the state of the component at a particular time. However, once set, props are immutable, meaning they cannot be directly changed.

4. Changing Props:

If you want to change the information shown by a component in response to user actions (like selecting a different color), you don't directly change the props. Instead, you ask the parent component to provide new props. The component then receives these new props, and it re-renders with the updated information.

5. Setting State for Interactivity:

To handle user interactions or dynamic changes, you use "state." If you need to update information dynamically (like changing the selected color in the example), you use state. Changing state triggers a re-render of the component with the updated information.

6. Recap:

• Passing Props: Props are like information passed down to a component. You add them in JSX, just like HTML attributes.

• Reading Props: Use destructuring syntax to read props inside the component.

• Immutable Nature: Props are immutable, meaning you can't directly change them. If something needs to change, ask the parent component for new props.

• Setting Default Values: You can set default values for props, ensuring they have a value even if not explicitly provided.

• Interactivity and State: For dynamic changes and user interactivity, use "state" instead of trying to change props directly.

By understanding and following these principles, you ensure that your React components stay predictable and can handle changes effectively.

wrong code

• The historian will have some puzzles to solve --> interesting way of stating that their logic is not clear? (3)
• Eugenics always had an evaluative logic at its core. Some human life was of more value -- to the state, the nation, the race, future generations -- than other life, and thus its advocates sought to implement these practices differentially ( 4) ==> did we ever examine who was making these policies and procedures and place a value on their lives
• Peak in the 1920s --> im assuming that this is referring to the US
• Contributing factors = nationalism, tech changes, changes in ideas about citizenry, labor force, future fitness (4) ABOUT MODERNITY
• Darwins work that highlighted that we are a part of nature and that we follow the same natural laws as everyone else critically undermined the idea that humans were made specially (4)
• Humans could and should replace natural selection by other processes that are more merciful and not less effective (4) huh --> he thought eugenics is merciful and more effective than natural selection (I wonder why this became so popular, especially when this time period is known for being religious
• Galton understood eugenics to be rational planning of and intervention into human breeding --> thought that the state should intervene into peoples own choices and such (5)
• Positive and negative eugenics (5)
• Oneida, NY the women professed: "we do not belong to ourselves", disrespectfully what in the world is this cult (5)
• American eugenics and ideas about eugenics shaping NS Germany
• 1926 Soviet Civic Code (5)
• aiming to restrict the reproduction of those with conditions and diseases considered heritable (CONSIDERED) --> did they know exactly (6)
• eugenic intervention directed at those who already belonged (6)
• Different priorities to the varying eugenic movements across the world --> southern US v NS Germany v Australian English (6)
• "Polluting" was not racial outsiders but often marginalized insiders
• Geographic insight --> more concentrated on the urban problem populations of industrialization (6) --> why
• America --> poor, rural whites: southern European immigrants; African Americans (7)
• Anxieties about interracial marriage were frequently linked to colonial rule (7)
• Race crossing --> Erst Rodenwalt (7)
• Australia --> half-caste problem (7) biological and cultural assimilation influenced by eugenic ideas ==> boarding school type beat
• Prospect of extinction (8)
• primitive societies where weak offspring were not nursed were admired as naturally eugenic (8) ==> looking at primitive examples to propel an ide of modernity
• relationship between eugenic and birth control
• In Eastern Europe --> more public health spending (8)
• into the management of infectious disease (9)
• Kenya example --> uneducable making their education irrelevant to the state (9)
• linked to the proliferation of asylums from the 19th century
• Dr. Harry Haselden withheld treatment for newborns --> what about the Hippocratic oath (9)
• designations of fit and unfit applied to both populations and to individuals
• one eye on the past generations and one eye on the future (9)
• pedigree chart (10)
• both research and propaganda
• Darwin knew about the principles of genetics, yet married his first cousin (10)
• insurance used information from Biometrika (10)
• Issue with the pedigree system bc it thinks that things like intelligence and other "good" characteristics are associated with inheritance but not with access to social support and resources (10)
• Eastern Europe --> eugenics could match and enshrine the aspirations of new nation-states anxious to establish their legitimacy (12)
• Japanese modernization manifested itself as nationalism (quality over quantity) (12)
• homogeneity over heterogeneity (12)
• strong connection between eugenics and the left and to progressive reform and politics (13)
• sterilization has informed the concept of "rights" in reproductive arenas (14)
• Consent --> Nuremberg trials (14)
• States that were looking to modernize were those who took up eugenics enthusiastically
• NS Germany (14/15)
• mythical concern --> where did this originate from (15)
• similarities across so many countries and cultures is interesting in the eugenics movements (15)
• Eugenics in immigration regulations (16)
• Central America and Southern Europe distinguished itself (16)
• Cross cultural ideas and exchange allowed eugenics to move so fast and so far but then they all wanted to be independent and create their own identity (16)
• Hygiene was also linked to eugenics --> "Rassenhygiene" in DE (17)
• Interesting to see how they distanced themselves from forms of eugenics that didn't come from their own home country
• sterilization procedures were antithetical to catholic doctrine and were highly questionable (18)
• British sought the legalization of sterilization but voluntary sterilization was always their aim (18)
• US is a contradiction --> commitment to liberty also passed compulsory sterilization laws (18)
• Voluntary v coerced (18)
• eugenics has always had opposition (19)
• Jennings essentially conceptualized pre-natal testing (19)
• 1970s drew large opposition (20)
• Tuskegee syphilis experiment highlighted conversations about races and medical ethics (20)
• This was a period of strongly left-oriented intellectual critique of science (20)

This is incredibly sad but visible in our BIPOC communities.

While exploring the NASW Code of Ethics, I found an important aspect related to power and inequality in the section on Social and Political Action (6.04). This section emphasizes the duty of social workers to engage in action against social injustice. However, it raises questions about the depth of guidance provided regarding power dynamics in the profession. I've noted this section to express my curiosity about how the Code of Ethics could offer clearer guidance on addressing power imbalances within social work organizations and in broader society. This encourages a deeper exploration of strategies to challenge oppressive systems.

Instead of keeping track of a num_reviews variable, we can also loop over the index and review of each movie simultaneously by using enumerate(movies). This gives us fewer variables to think about and makes the code a bit more elegant and readable.

The length count includes all the spaces, period(,) and exclamation mark.

Dans le code HTML de à-propos il y à une erreur : - vous avez rajouter class="lien-icone" au niveau du logo de la page a propos , ce qui lui applique le changement d'opacité au survole identique au lien hypertexte Instagram et twitter qui possède eu aussi la classe déclarer .lien-icone.

Escape sequences in JavaScript are special combinations of characters that are used to represent characters that would otherwise be difficult or impossible to include directly in a string. These sequences start with a backslash \ followed by another character or characters.

Here are some common escape sequences in JavaScript:

1. \n - Newline:
2. Represents a line break.

javascript console.log("Hello\nWorld"); // Output: // Hello // World

1. \t - Tab:
2. Represents a horizontal tab.

javascript console.log("This\tis\tTab"); // Output: This is Tab

1. \ - Backslash:
2. Represents a literal backslash.

javascript console.log("This is a backslash: \\"); // Output: This is a backslash: \

1. \' - Single Quote:
2. Represents a single quote within a string declared with single quotes.

javascript console.log('It\'s a sunny day.'); // Output: It's a sunny day.

1. \" - Double Quote:
2. Represents a double quote within a string declared with double quotes.

javascript console.log("She said, \"Hello!\""); // Output: She said, "Hello!"

1. \uXXXX - Unicode Escape:
2. Represents a Unicode character, where XXXX is the Unicode code point in hexadecimal.

javascript console.log("\u0041"); // Output: A

These escape sequences allow you to include special characters in your strings without causing syntax errors. For example, if you want to include a quote character within a string that is already enclosed in quotes, you can use the escape sequence to prevent confusion and errors.

Certainly! Let's break it down in simpler terms:

String Primitives and String Objects:

• In JavaScript, there are two types of strings: string primitives and String objects.
• String literals (created using double or single quotes) and strings returned from non-constructor String calls are primitive strings.
• String objects are created using the new String() syntax.

Examples:

```javascript const strPrim = "foo"; // A string primitive const strPrim2 = String(1); // Coerced into the string primitive "1" const strPrim3 = String(true); // Coerced into the string primitive "true" const strObj = new String(strPrim); // String with new returns a string wrapper object.

console.log(typeof strPrim); // "string" console.log(typeof strPrim2); // "string" console.log(typeof strPrim3); // "string" console.log(typeof strObj); // "object" ```

• strPrim, strPrim2, and strPrim3 are string primitives, while strObj is a String object.

Using eval():

• When using eval() with string primitives, the string is treated as source code, and the result is evaluated.
• When using eval() with String objects, it treats the object like any other object and returns the object itself.

Examples:

```javascript const s1 = "2 + 2"; // creates a string primitive const s2 = new String("2 + 2"); // creates a String object

console.log(eval(s1)); // returns the number 4 console.log(eval(s2)); // returns the string "2 + 2" ```

• eval(s1) evaluates the string primitive as code and returns the result (number 4).
• eval(s2) returns the String object itself (string "2 + 2"), not the evaluated result.

Conversion to Primitive:

• To convert a String object to its primitive counterpart, you can use the valueOf() method.

Example:

javascript console.log(eval(s2.valueOf())); // returns the number 4

• valueOf() converts the String object (s2) to its primitive form before being evaluated, resulting in the number 4.

Conclusion:

• In general, it's rare to use String as a constructor (using new String()), and working with string primitives is more common. Understanding the differences is important when dealing with certain situations like using eval() or expecting a specific type of string.

This section of the NASW code of ethics raises important questions regarding power and structural inequality because sometimes it may be hard for social workers to find the clear boundary of dual-relationships. For example, if a client invites you to an event they are holding that is unrelated to your professional relationship like a retirement party or another life milestone event, how do you decipher which type of event is appropriate to attend and which is not? Due to your professional influence on this person's life, what if your personal bias for this client exceeds the level that is appropriate for a standard social worker/client relationship but you don't see it that way because they are "inviting you in" or consenting to it. I feel it's important for social workers to build strong rapport with their clients to facilitate more effective practice however, ones judgement on when this rapport building may be crossing the line into unprofessional territory may vary on a case by case basis.

This part of the NASW code of ethics pertains to my field work because in my role as the housing navigation specialist at the shelter, I not only help clients secure permanent housing through government subsidies but, I also advocate on their behalf when they are being faced with blatant discrimination. This advocacy is not only reflected in my efforts to educate my clients on their own housing rights but also by educating landlords, relators, and property managers on the policy of housing subsidies like state wide section 8 and local municipality housing voucher programs. Recently, I was working with a client who has a section 8 voucher through the state. This client was about to sign their request for tenancy with the landlord but once the landlord looked over the DCA (department of community affairs) terms of agreement they stated in writing that they "did not want to move forward with a voucher holder." Luckily, New Jersey is one of the 49 states that have fair housing legislature in place so I was able to report this correspondence to my clients DCA worker as discrimination and they were granted more time to secure permanent housing. There is a lot of stigma tied to the homeless community, especially those who obtain housing subsidies through local and federal government entities. This is why legislature like The Fair Housing Act is so important in the work that I do and the vulnerable community I serve.

React Hooks are functions provided by React that allow you to use state and lifecycle features in functional components, making them more powerful and expressive. They were introduced in React version 16.8 to let developers use state and other React features without writing a class.

useState Hook:

useState is a Hook that allows you to add state to functional components. It returns an array with two elements: the current state value and a function that lets you update it. Here's a simple example:

```jsx import React, { useState } from 'react';

function Counter() { // Declare a state variable named "count" with an initial value of 0 const [count, setCount] = useState(0);

return ( <div>

Count: {count}

<button onClick={() => setCount(count + 1)}>Increment</button> </div> ); } ```

In this example, count is the state variable, and setCount is the function to update it. When the "Increment" button is clicked, it updates the count state, and React automatically re-renders the component.

useEffect Hook:

useEffect is a Hook that enables you to perform side effects in functional components. It's similar to lifecycle methods in class components. Here's a simple example fetching data:

```jsx import React, { useState, useEffect } from 'react';

function DataFetcher() { const [data, setData] = useState(null);

useEffect(() => { // Fetch data when the component mounts fetch('https://api.example.com/data') .then(response => response.json()) .then(data => setData(data));

// Clean up any resources when the component unmounts
return () => {
  // Cleanup code here
};

}, []); // The empty array ensures this effect runs only once on mount

return ( <div>

Data: {data ? data.value : 'Loading...'}

</div> ); } ```

In this example, useEffect runs when the component mounts. It fetches data and updates the state. The empty dependency array ([]) means the effect runs only once when the component mounts.

useContext Hook:

useContext is a Hook that allows you to subscribe to React context without introducing nesting. It lets you access the value of a context directly.

```jsx import React, { useContext } from 'react';

const ThemeContext = React.createContext('light');

function ThemedComponent() { const theme = useContext(ThemeContext);

return

Current Theme: {theme}

; } ```

In this example, ThemedComponent can directly access the current theme value from the ThemeContext.

These are just a few examples of React Hooks. There are others like useReducer, useCallback, useMemo, etc., each serving a specific purpose to enhance the functionality of functional components. Hooks allow you to manage state and side effects more effectively in functional components, making your code cleaner and more maintainable. Absolutely! Let's break down the statement in simpler terms:

useRef Explanation:

useRef is like a tool in React that helps you create a special reference to something, and this reference won't force your component to re-render when the referenced thing changes.

Simple Explanation:

1. Creating a Reference:
2. With useRef, you can create a reference to a value, like a number, an object, or even a DOM element.

```jsx import { useRef } from 'react';

function MyComponent() { const myRef = useRef(0); // Creating a reference to the number 0 return <div>{myRef.current}</div>; } ```

In this example, myRef is a reference to the number 0.

1. No Re-rendering:
2. When the value of the reference changes, your component won't automatically re-render. This is useful when you want to keep track of something without affecting the UI.

```jsx import { useRef, useState, useEffect } from 'react';

function Counter() { const count = useRef(0); // Creating a reference to the number 0 const [renderCount, setRenderCount] = useState(0);

 useEffect(() => {
   // This effect will run when the component renders
   setRenderCount(renderCount + 1);

   // But changing the count won't cause a re-render
   count.current = count.current + 1;
 }, [count, renderCount]);

 return (
   <div>
     <p>Render Count: {renderCount}</p>
     <p>Count (no re-render): {count.current}</p>
   </div>
 );

} ```

In this example, changing the count won't trigger a re-render of the component.

When to Use useRef:

• Use useRef when you want to keep track of a value that doesn't affect your component's display directly and shouldn't cause re-renders.

• Common use cases include interacting with the DOM directly or storing mutable values that shouldn't trigger updates.

In summary, useRef is a handy tool in React for creating references that won't cause your component to re-render when the referenced value changes. It's useful for scenarios where you need to keep track of something without impacting the UI. Certainly! Let's break down the statement in very simple terms with multiple examples:

Explanation:

1. Memoized Object:
2. When you use useRef to create a reference, React keeps track of it. Instead of creating a new reference object every time your component re-renders, React gives you the same reference object.

```jsx import { useRef } from 'react';

function MyComponent() { const myRef = useRef(0);

 // On each render, myRef is the same object
 console.log(myRef === myRef); // true

} ```

In this example, myRef remains the same object across renders.

1. Avoiding Recreation:
2. This memoization is useful when you don't want to recreate the reference object, especially when dealing with functions. It helps to maintain consistency and avoids unnecessary re-initialization.

```jsx import { useRef, useEffect } from 'react';

function EffectComponent() { const handleClick = useRef(() => { console.log('Button clicked!'); });

 useEffect(() => {
   // Access the function without causing a re-render
   handleClick.current();
 }, []);

 return <button onClick={handleClick.current}>Click me</button>;

} ```

In this example, handleClick remains the same function reference across renders.

1. Ensuring Same Object:
2. The memoized object ensures that the reference remains consistent, even when the component re-renders. This is essential when dealing with external functions or references that you want to persist.

```jsx import { useRef } from 'react';

function ReferenceComponent() { const externalFunction = () => { console.log('External function'); };

 const externalFunctionRef = useRef(externalFunction);

 // externalFunctionRef.current is the same function across renders
 console.log(externalFunctionRef.current === externalFunction); // true

} ```

In this example, externalFunctionRef.current is the same function as externalFunction across renders.

Simplified Summary:

• useRef gives you the same reference object across renders.
• This is useful to avoid recreating the reference, especially when dealing with functions.
• It ensures consistency and stability, preventing unnecessary re-initialization.

In essence, useRef helps you maintain the same reference object, ensuring stability and preventing unnecessary re-creation, which is particularly handy when working with functions or external references.Sure, let's break down useRef in simple terms with examples:

What is useRef?

useRef is a React Hook that allows you to create a reference to a value that won't trigger a re-render when it changes. It's often used for things that are not directly related to rendering, like accessing or manipulating the DOM.

How to Use useRef:

1. Referencing a Value: You can use useRef to create a reference to a value, such as a number or an object. This value can be accessed and modified without causing your component to re-render.

```jsx import { useRef } from 'react';

function MyComponent() { const intervalRef = useRef(0); // Reference to a number const inputRef = useRef(null); // Reference to an element (initially null) // ... ```

1. Manipulating the DOM: useRef is commonly used for interacting with the DOM directly. For example, if you want to focus on an input element or keep track of some DOM-related state without triggering a re-render.

```jsx import { useRef, useEffect } from 'react';

function MyComponent() { const inputRef = useRef(null);

 useEffect(() => {
   // Focus on the input element when the component mounts
   inputRef.current.focus();
 }, []);

 return <input ref={inputRef} />;

} ```

1. Avoiding Recreating the Ref Contents: useRef is memoized, meaning it returns the same object on every render. This is useful when you want to avoid recreating the ref object, especially when dealing with functions.

```jsx import { useRef, useEffect } from 'react';

function MyComponent() { const handleClick = useRef(() => { console.log('Button clicked!'); });

 useEffect(() => {
   // Access the function without causing a re-render
   handleClick.current();
 }, []);

 return <button onClick={handleClick.current}>Click me</button>;

} ```

Parameters and Returns:

• Parameters:

• initialValue: The value you want the ref object’s current property to be initially. It can be a value of any type. This argument is ignored after the initial render.

• Returns:

• useRef returns an object with a single property:
  • current: Initially set to the initialValue you have passed. You can later set it to something else. If you pass the ref object to React as a ref attribute to a JSX node, React will set its current property.

In simple terms, useRef is a tool to keep track of values or elements that won't cause your component to re-render every time they change. It's commonly used for interacting with the DOM and handling mutable values in a React component.

This passage reveals the importance of academic integrity and how academic dishonesty is not tolerated at DVC.

Trolling can be driven by a nihilistic mindset where empathy is disregarded, aiming solely to provoke reactions, often manifesting as hostility towards specific groups. This form of trolling is widely condemned as it inflicts harm, failing to adhere to basic principles of respect and integrity.

The section provides a profound exploration of the complexities involved in understanding and evaluating disruptive behaviors in social media contexts. It compellingly illustrates how the formation of groups, the use of stereotypes, and the enforcement of norms are all deeply intertwined with our cognitive processes and societal structures. The examination of trolling as a form of disruption that can be deployed for both just and unjust ends invites readers to reflect on the multifaceted nature of these actions and their ethical implications.

Author Response

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

eLife assessment

This study presents potentially valuable results on glutamine-rich motifs in relation to protein expression and alternative genetic codes. The author's interpretation of the results is so far only supported by incomplete evidence, due to a lack of acknowledgment of alternative explanations, missing controls and statistical analysis and writing unclear to non experts in the field. These shortcomings could be at least partially overcome by additional experiments, thorough rewriting, or both.

We thank both the Reviewing Editor and Senior Editor for handling this manuscript.

Based on your suggestions, we have provided controls, performed statistical analysis, and rewrote our manuscript. The revised manuscript is significantly improved and more accessible to non-experts in the field.

Reviewer #1 (Public Review):

Summary

This work contains 3 sections. The first section describes how protein domains with SQ motifs can increase the abundance of a lacZ reporter in yeast. The authors call this phenomenon autonomous protein expression-enhancing activity, and this finding is well supported. The authors show evidence that this increase in protein abundance and enzymatic activity is not due to changes in plasmid copy number or mRNA abundance, and that this phenomenon is not affected by mutants in translational quality control. It was not completely clear whether the increased protein abundance is due to increased translation or to increased protein stability.

In section 2, the authors performed mutagenesis of three N-terminal domains to study how protein sequence changes protein stability and enzymatic activity of the fusions. These data are very interesting, but this section needs more interpretation. It is not clear if the effect is due to the number of S/T/Q/N amino acids or due to the number of phosphorylation sites.

In section 3, the authors undertake an extensive computational analysis of amino acid runs in 27 species. Many aspects of this section are fascinating to an expert reader. They identify regions with poly-X tracks. These data were not normalized correctly: I think that a null expectation for how often poly-X track occur should be built for each species based on the underlying prevalence of amino acids in that species. As a result, I believe that the claim is not well supported by the data.

Strengths

This work is about an interesting topic and contains stimulating bioinformatics analysis. The first two sections, where the authors investigate how S/T/Q/N abundance modulates protein expression level, is well supported by the data. The bioinformatics analysis of Q abundance in ciliate proteomes is fascinating. There are some ciliates that have repurposed stop codons to code for Q. The authors find that in these proteomes, Q-runs are greatly expanded. They offer interesting speculations on how this expansion might impact protein function.

Weakness

At this time, the manuscript is disorganized and difficult to read. An expert in the field, who will not be distracted by the disorganization, will find some very interesting results included. In particular, the order of the introduction does not match the rest of the paper.

In the first and second sections, where the authors investigate how S/T/Q/N abundance modulates protein expression levels, it is unclear if the effect is due to the number of phosphorylation sites or the number of S/T/Q/N residues.

There are three reasons why the number of phosphorylation sites in the Q-rich motifs is not relevant to their autonomous protein expression-enhancing (PEE) activities:

First, we have reported previously that phosphorylation-defective Rad51-NTD (Rad51-3SA) and wild-type Rad51-NTD exhibit similar autonomous PEE activity. Mec1/Tel1-dependent phosphorylation of Rad51-NTD antagonizes the proteasomal degradation pathway, increasing the half-life of Rad51 from ∼30 min to ≥180 min (1). (page 1, lines 11-14)

Second, in our preprint manuscript, we have already shown that phosphorylation-defective Rad53-SCD1 (Rad51-SCD1-5STA) also exhibits autonomous PEE activity similar to that of wild-type Rad53-SCD (Figure 2D, Figure 4A and Figure 4C). We have highlighted this point in our revised manuscript (page 9, lines 19-21).

Third, as revealed by the results of Figure 4, it is the percentages, and not the numbers, of S/T/Q/N residues that are correlated with the PEE activities of Q-rich motifs.

The authors also do not discuss if the N-end rule for protein stability applies to the lacZ reporter or the fusion proteins.

The autonomous PEE function of S/T/Q-rich NTDs is unlikely to be relevant to the N-end rule. The N-end rule links the in vivo half-life of a protein to the identity of its N-terminal residues. In S. cerevisiae, the N-end rule operates as part of the ubiquitin system and comprises two pathways. First, the Arg/N-end rule pathway, involving a single N-terminal amidohydrolase Nta1, mediates deamidation of N-terminal asparagine (N) and glutamine (Q) into aspartate (D) and glutamate (E), which in turn are arginylated by a single Ate1 R-transferase, generating the Arg/N degron. N-terminal R and other primary degrons are recognized by a single N-recognin Ubr1 in concert with ubiquitin-conjugating Ubc2/Rad6. Ubr1 can also recognize several other N-terminal residues, including lysine (K), histidine (H), phenylalanine (F), tryptophan (W), leucine (L) and isoleucine (I) (68-70). Second, the Ac/N-end rule pathway targets proteins containing N-terminally acetylated (Ac) residues. Prior to acetylation, the first amino acid methionine (M) is catalytically removed by Met-aminopeptidases (MetAPs), unless a residue at position 2 is non-permissive (too large) for MetAPs. If a retained N-terminal M or otherwise a valine (V), cysteine (C), alanine (A), serine (S) or threonine (T) residue is followed by residues that allow N-terminal acetylation, the proteins containing these AcN degrons are targeted for ubiquitylation and proteasome-mediated degradation by the Doa10 E3 ligase (71).

The PEE activities of these S/T/Q-rich domains are unlikely to arise from counteracting the N-end rule for two reasons. First, the first two amino acid residues of Rad51-NTD, Hop1-SCD, Rad53-SCD1, Sup35-PND, Rad51-ΔN, and LacZ-NVH are MS, ME, ME, MS, ME, and MI, respectively, where M is methionine, S is serine, E is glutamic acid and I is isoleucine. Second, Sml1-NTD behaves similarly to these N-terminal fusion tags, despite its methionine and glutamine (MQ) amino acid signature at the N-terminus. (Page 12, line 3 to page 13, line 2)

The most interesting part of the paper is an exploration of S/T/Q/N-rich regions and other repetitive AA runs in 27 proteomes, particularly ciliates. However, this analysis is missing a critical control that makes it nearly impossible to evaluate the importance of the findings. The authors find the abundance of different amino acid runs in various proteomes. They also report the background abundance of each amino acid. They do not use this background abundance to normalize the runs of amino acids to create a null expectation from each proteome. For example, it has been clear for some time (Ruff, 2017; Ruff et al., 2016) that Drosophila contains a very high background of Q's in the proteome and it is necessary to control for this background abundance when finding runs of Q's.

We apologize for not explaining sufficiently well the topic eliciting this reviewer’s concern in our preprint manuscript. In the second paragraph of page 14, we cite six references to highlight that SCDs are overrepresented in yeast and human proteins involved in several biological processes (5, 43) and that polyX prevalence differs among species (79-82).

We will cite a reference by Kiersten M. Ruff in our revised manuscript (38).

K. M. Ruff, J. B. Warner, A. Posey and P. S. Tan (2017) Polyglutamine length dependent structural properties and phase behavior of huntingtin exon1. Biophysical Journal 112, 511a.

The authors could easily address this problem with the data and analysis they have already collected. However, at this time, without this normalization, I am hesitant to trust the lists of proteins with long runs of amino acid and the ensuing GO enrichment analysis. Ruff KM. 2017. Washington University in St.

Ruff KM, Holehouse AS, Richardson MGO, Pappu RV. 2016. Proteomic and Biophysical Analysis of Polar Tracts. Biophys J 110:556a.

We thank Reviewer #1 for this helpful suggestion and now address this issue by means of a different approach described below.

Based on a previous study (43), we applied seven different thresholds to seek both short and long, as well as pure and impure, polyX strings in 20 different representative near-complete proteomes, including 4X (4/4), 5X (4/5-5/5), 6X (4/6-6/6), 7X (4/7-7/7), 8-10X (≥50%X), 11-10X (≥50%X) and ≥21X (≥50%X).

To normalize the runs of amino acids and create a null expectation from each proteome, we determined the ratios of the overall number of X residues for each of the seven polyX motifs relative to those in the entire proteome of each species, respectively. The results of four different polyX motifs are shown in our revised manuscript, i.e., polyQ (Figure 7), polyN (Figure 8), polyS (Figure 9) and polyT (Figure 10). Thus, polyX prevalence differs among species and the overall X contents of polyX motifs often but not always correlate with the X usage frequency in entire proteomes (43).

Most importantly, our results reveal that, compared to Stentor coeruleus or several non-ciliate eukaryotic organisms (e.g., Plasmodium falciparum, Caenorhabditis elegans, Danio rerio, Mus musculus and Homo sapiens), the five ciliates with reassigned TAAQ and TAGQ codons not only have higher Q usage frequencies, but also more polyQ motifs in their proteomes (Figure 7). In contrast, polyQ motifs prevail in Candida albicans, Candida tropicalis, Dictyostelium discoideum, Chlamydomonas reinhardtii, Drosophila melanogaster and Aedes aegypti, though the Q usage frequencies in their entire proteomes are not significantly higher than those of other eukaryotes (Figure 1). Due to their higher N usage frequencies, Dictyostelium discoideum, Plasmodium falciparum and Pseudocohnilembus persalinus have more polyN motifs than the other 23 eukaryotes we examined here (Figure 8). Generally speaking, all 26 eukaryotes we assessed have similar S usage frequencies and percentages of S contents in polyS motifs (Figure 9). Among these 26 eukaryotes, Dictyostelium discoideum possesses many more polyT motifs, though its T usage frequency is similar to that of the other 25 eukaryotes (Figure 10).

In conclusion, these new normalized results confirm that the reassignment of stop codons to Q indeed results in both higher Q usage frequencies and more polyQ motifs in ciliates.  

Reviewer #2 (Public Review):

Summary:

This study seeks to understand the connection between protein sequence and function in disordered regions enriched in polar amino acids (specifically Q, N, S and T). While the authors suggest that specific motifs facilitate protein-enhancing activities, their findings are correlative, and the evidence is incomplete. Similarly, the authors propose that the re-assignment of stop codons to glutamine-encoding codons underlies the greater user of glutamine in a subset of ciliates, but again, the conclusions here are, at best, correlative. The authors perform extensive bioinformatic analysis, with detailed (albeit somewhat ad hoc) discussion on a number of proteins. Overall, the results presented here are interesting, but are unable to exclude competing hypotheses.

Strengths:

Following up on previous work, the authors wish to uncover a mechanism associated with poly-Q and SCD motifs explaining proposed protein expression-enhancing activities. They note that these motifs often occur IDRs and hypothesize that structural plasticity could be capitalized upon as a mechanism of diversification in evolution. To investigate this further, they employ bioinformatics to investigate the sequence features of proteomes of 27 eukaryotes. They deepen their sequence space exploration uncovering sub-phylum-specific features associated with species in which a stop-codon substitution has occurred. The authors propose this stop-codon substitution underlies an expansion of ploy-Q repeats and increased glutamine distribution.

Weaknesses:

The preprint provides extensive, detailed, and entirely unnecessary background information throughout, hampering reading and making it difficult to understand the ideas being proposed.

The introduction provides a large amount of detailed background that appears entirely irrelevant for the paper. Many places detailed discussions on specific proteins that are likely of interest to the authors occur, yet without context, this does not enhance the paper for the reader.

The paper uses many unnecessary, new, or redefined acronyms which makes reading difficult. As examples:

1) Prion forming domains (PFDs). Do the authors mean prion-like domains (PLDs), an established term with an empirical definition from the PLAAC algorithm? If yes, they should say this. If not, they must define what a prion-forming domain is formally.

The N-terminal domain (1-123 amino acids) of S. cerevisiae Sup35 was already referred to as a “prion forming domain (PFD)” in 2006 (48). Since then, PFD has also been employed as an acronym in other yeast prion papers (Cox, B.S. et al. 2007; Toombs, T. et al. 2011).

B. S. Cox, L. Byrne, M. F., Tuite, Protein Stability. Prion 1, 170-178 (2007). J. A. Toombs, N. M. Liss, K. R. Cobble, Z. Ben-Musa, E. D. Ross, [PSI+] maintenance is dependent on the composition, not primary sequence, of the oligopeptide repeat domain. PLoS One 6, e21953 (2011).

2) SCD is already an acronym in the IDP field (meaning sequence charge decoration) - the authors should avoid this as their chosen acronym for Serine(S) / threonine (T)-glutamine (Q) cluster domains. Moreover, do we really need another acronym here (we do not).

SCD was first used in 2005 as an acronym for the Serine (S)/threonine (T)-glutamine (Q) cluster domain in the DNA damage checkpoint field (4). Almost a decade later, SCD became an acronym for “sequence charge decoration” (Sawle, L. et al. 2015; Firman, T. et al. 2018).

L. Sawle and K, Ghosh, A theoretical method to compute sequence dependent configurational properties in charged polymers and proteins. J. Chem Phys. 143, 085101(2015).

T. Firman and Ghosh, K. Sequence charge decoration dictates coil-globule transition in intrinsically disordered proteins. J. Chem Phys. 148, 123305 (2018).

3) Protein expression-enhancing (PEE) - just say expression-enhancing, there is no need for an acronym here.

Thank you. Since we have shown that the addition of Q-rich motifs to LacZ affects protein expression rather than transcription, we think it is better to use the “PEE” acronym.

The results suggest autonomous protein expression-enhancing activities of regions of multiple proteins containing Q-rich and SCD motifs. Their definition of expression-enhancing activities is vague and the evidence they provide to support the claim is weak. While their previous work may support their claim with more evidence, it should be explained in more detail. The assay they choose is a fusion reporter measuring beta-galactosidase activity and tracking expression levels. Given the presented data they have shown that they can drive the expression of their reporters and that beta gal remains active, in addition to the increase in expression of fusion reporter during the stress response. They have not detailed what their control and mock treatment is, which makes complete understanding of their experimental approach difficult. Furthermore, their nuclear localization signal on the tag could be influencing the degradation kinetics or sequestering the reporter, leading to its accumulation and the appearance of enhanced expression. Their evidence refuting ubiquitin-mediated degradation does not have a convincing control.

Although this reviewer’s concern regarding our use of a nuclear localization signal on the tag is understandable, we are confident that this signal does not bias our findings for two reasons. First, the negative control LacZ-NV also possesses the same nuclear localization signal (Figure 1A, lane 2). Second, another fusion target, Rad51-ΔN, does not harbor the NVH tag (Figure 1D, lanes 3-4). Compared to wild-type Rad51, Rad51-ΔN is highly labile. In our previous study, removal of the NTD from Rad51 reduced by ~97% the protein levels of corresponding Rad51-ΔN proteins relative to wild-type (1).

Based on the experimental results, the authors then go on to perform bioinformatic analysis of SCD proteins and polyX proteins. Unfortunately, there is no clear hypothesis for what is being tested; there is a vague sense of investigating polyX/SCD regions, but I did not find the connection between the first and section compelling (especially given polar-rich regions have been shown to engage in many different functions). As such, this bioinformatic analysis largely presents as many lists of percentages without any meaningful interpretation. The bioinformatics analysis lacks any kind of rigorous statistical tests, making it difficult to evaluate the conclusions drawn. The methods section is severely lacking. Specifically, many of the methods require the reader to read many other papers. While referencing prior work is of course, important, the authors should ensure the methods in this paper provide the details needed to allow a reader to evaluate the work being presented. As it stands, this is not the case.

Thank you. As described in detail below, we have now performed rigorous statistical testing using the GofuncR package (Figure 11, Figure 12 and DS7-DS32).

Overall, my major concern with this work is that the authors make two central claims in this paper (as per the Discussion). The authors claim that Q-rich motifs enhance protein expression. The implication here is that Q-rich motif IDRs are special, but this is not tested. As such, they cannot exclude the competing hypothesis ("N-terminal disordered regions enhance expression").

In fact, “N-terminal disordered regions enhance expression” exactly summarizes our hypothesis.

On pages 12-13 and Figure 4 of our preprint manuscript, we explained our hypothesis in the paragraph entitled “The relationship between PEE function, amino acid contents, and structural flexibility”.

The authors also do not explore the possibility that this effect is in part/entirely driven by mRNA-level effects (see Verma Na Comms 2019).

As pointed out by the first reviewer, we present evidence that the increase in protein abundance and enzymatic activity is not due to changes in plasmid copy number or mRNA abundance (Figure 2), and that this phenomenon is not affected in translational quality control mutants (Figure 3).

As such, while these observations are interesting, they feel preliminary and, in my opinion, cannot be used to draw hard conclusions on how N-terminal IDR sequence features influence protein expression. This does not mean the authors are necessarily wrong, but from the data presented here, I do not believe strong conclusions can be drawn. That re-assignment of stop codons to Q increases proteome-wide Q usage. I was unable to understand what result led the authors to this conclusion.

My reading of the results is that a subset of ciliates has re-assigned UAA and UAG from the stop codon to Q. Those ciliates have more polyQ-containing proteins. However, they also have more polyN-containing proteins and proteins enriched in S/T-Q clusters. Surely if this were a stop-codon-dependent effect, we'd ONLY see an enhancement in Q-richness, not a corresponding enhancement in all polar-rich IDR frequencies? It seems the better working hypothesis is that free-floating climate proteomes are enriched in polar amino acids compared to sessile ciliates.

We thank this reviewer for raising this point, however her/his comments are not supported by the results in Figure 7.

Regardless, the absence of any kind of statistical analysis makes it hard to draw strong conclusions here.

We apologize for not explaining more clearly the results of Tables 5-7 in our preprint manuscript.

To address the concerns about our GO enrichment analysis by both reviewers, we have now performed rigorous statistical testing for SCD and polyQ protein overrepresentation using the GOfuncR package (https://bioconductor.org/packages/release/bioc/html/GOfuncR.html). GOfuncR is an R package program that conducts standard candidate vs. background enrichment analysis by means of the hypergeometric test. We then adjusted the raw p-values according to the Family-wise error rate (FWER). The same method had been applied to GO enrichment analysis of human genomes (89).

The results presented in Figure 11 and Figure 12 (DS7-DS32) support our hypothesis that Q-rich motifs prevail in proteins involved in specialized biological processes, including Saccharomyces cerevisiae RNA-mediated transposition, Candida albicans filamentous growth, peptidyl-glutamic acid modification in ciliates with reassigned stop codons (TAAQ and TAGQ), Tetrahymena thermophila xylan catabolism, Dictyostelium discoideum sexual reproduction, Plasmodium falciparum infection, as well as the nervous systems of Drosophila melanogaster, Mus musculus, and Homo sapiens (78). In contrast, peptidyl-glutamic acid modification and microtubule-based movement are not overrepresented with Q-rich proteins in Stentor coeruleus, a ciliate with standard stop codons.

Recommendations for the authors:

Please note that you control which revisions to undertake from the public reviews and recommendations for the authors.

Reviewer #1 (Recommendations For The Authors):

The order of paragraphs in the introduction was very difficult to follow. Each paragraph was clear and easy to understand, but the order of paragraphs did not make sense to this reader. The order of events in the abstract matches the order of events in the results section. However, the order of paragraphs in the introduction is completely different and this was very confusing. This disordered list of facts might make sense to an expert reader but makes it hard for a non-expert reader to understand.

Apologies. We endeavored to improve the flow of our revised manuscript to make it more readable.

The section beginning on pg 12 focused on figures 4 and 5 was very interesting and highly promising. However, it was initially hard for me to tell from the main text what the experiment was. Please add to the text an explanation of the experiment, because it is hard to figure out what was going on from the figures alone. Figure 4 is fantastic, but would be improved by adding error bars and scaling the x-axis to be the same in panels B,C,D.

Thank you for this recommendation. We have now scaled both the x-axis and y-axis equivalently in panels B, C and D of Figure 4. Error bars are too small to be included.

It is hard to tell if the key variable is the number of S/T/Q/N residues or the number of phosphosites. I think a good control would be to add a regression against the number of putative phosphosites. The sequences are well designed. I loved this part but as a reader, I need more interpretation about why it matters and how it explains the PEE.

As described above, we have shown that the number of phosphorylation sites in the Q-rich motifs is not relevant to their autonomous protein expression-enhancing (PEE) activities.

I believe that the prevalence of polyX runs is not meaningful without normalizing for the background abundance of each amino acid. The proteome-wide abundance and the assumption that amino acids occur independently can be used to form a baseline expectation for which runs are longer than expected by chance. I think Figures 6 and 7 should go into the supplement and be replaced in the main text with a figure where Figure 6 is normalized by Figure 7. For example in P. falciparum, there are many N-runs (Figure 6), but the proteome has the highest fraction of N’s (Figure 7).

Thank you for these suggestions. The three figures in our preprint manuscript (Figures 6-8) have been moved into the supplementary information (Figures S1-S3). For normalization, we have provided four new figures (Figures 7-10) in our revised manuscript.

The analysis of ciliate proteomes was fascinating. I am particularly interested in the GO enrichment for “peptidyl-glutamic acid modification” (pg 20) because these enzymes might be modifying some of Q’s in the Q-runs. I might be wrong about this idea or confused about the chemistry. Do these ciliates live in Q-rich environments? Or nitrogen rich environments?

Polymeric modifications (polymodifications) are a hallmark of C-terminal tubulin tails, whereas secondary peptide chains of glutamic acids (polyglutamylation) and glycines (polyglycylation) are catalyzed from the γ-carboxyl group of primary chain glutamic acids. It is not clear if these enzymes can modify some of the Q’s in the Q-runs.

To our knowledge, ciliates are abundant in almost every liquid water environment, i.e., oceans/seas, marine sediments, lakes, ponds, and rivers, and even soils.

I think you should include more discussion about how the codons that code for Q’s are prone to slippage during DNA replication, and thus many Q-runs are unstable and expand (e.g. Huntington’s Disease). The end of pg 24 or pg 25 would be good places.

We thank the reviewer for these comments.

PolyQ motifs have a particular length-dependent codon usage that relates to strand slippage in CAG/CTG trinucleotide repeat regions during DNA replication. In most organisms having standard genetic codons, Q is encoded by CAGQ and CAAQ. Here, we have determined and compared proteome-wide Q contents, as well as the CAGQ usage frequencies (i.e., the ratio between CAGQ and the sum of CAGQ, CAGQ, TAAQ, and TAGQ).

Our results reveal that the likelihood of forming long CAG/CTG trinucleotide repeats are higher in five eukaryotes due to their higher CAGQ usage frequencies, including Drosophila melanogaster (86.6% Q), Danio rerio (74.0% Q), Mus musculus (74.0% Q), Homo sapiens (73.5% Q), and Chlamydomonas reinhardtii (87.3% Q) (orange background, Table 2). In contrast, another five eukaryotes that possess high numbers of polyQ motifs (i.e., Dictyostelium discoideum, Candida albicans, Candida tropicalis, Plasmodium falciparum and Stentor coeruleus) (Figure 1) utilize more CAAQ (96.2%, 84.6%, 84.5%, 86.7% and 75.7%) than CAAQ (3.8%, 15.4%, 15.5%, 13.3% and 24.3%), respectively, to avoid the formation of long CAG/CTG trinucleotide repeats (green background, Table 2). Similarly, all five ciliates with reassigned stop codons (TAAQ and TAGQ) have low CAGQ usage frequencies (i.e., from 3.8% Q in Pseudocohnilembus persalinus to 12.6% Q in Oxytricha trifallax) (red font, Table 2). Accordingly, the CAG-slippage mechanism might operate more frequently in Chlamydomonas reinhardtii, Drosophila melanogaster, Danio rerio, Mus musculus and Homo sapiens than in Dictyostelium discoideum, Candida albicans, Candida tropicalis, Plasmodium falciparum, Stentor coeruleus and the five ciliates with reassigned stop codons (TAAQ and TAGQ).

Author response table 1.

Usage frequencies of TAA, TAG, TAAQ, TAGQ, CAAQ and CAGQ codons in the entire proteomes of 20 different organisms.

Pg 7, paragraph 2 has no direction. Please add the conclusion of the paragraph to the first sentence.

This paragraph has been moved to the “Introduction” section” of the revised manuscript.

Pg 8, I suggest only mentioning the PFDs used in the experiments. The rest are distracting.

We have addressed this concern above.

Pg 12. Please revise the "The relationship...." text to explain the experiment.

We apologize for not explaining this topic sufficiently well in our preprint manuscript.

SCDs are often structurally flexible sequences (4) or even IDRs. Using IUPred2A (https://iupred2a.elte.hu/plot_new), a web-server for identifying disordered protein regions (88), we found that Rad51-NTD (1-66 a.a.) (1), Rad53-SCD1 (1-29 a.a.) and Sup35-NPD (1-39 a.a.) are highly structurally flexible. Since a high content of serine (S), threonine (T), glutamine (Q), asparanine (N) is a common feature of IDRs (17-20), we applied alanine scanning mutagenesis approach to reduce the percentages of S, T, Q or N in Rad51-NTD, Rad53-SCD1 or Sup35-NPD, respectively. As shown in Figure 4 and Figure 5, there is a very strong positive relationship between STQ and STQN amino acid percentages and β-galactosidase activities. (Page 13, lines 5-10)

Pg 13, first full paragraph, "Futionally, IDRs..." I think this paragraph belongs in the Discussion.

This paragraph is now in the “Introduction” section (Page 5, Lines 11-15).

Pg. 15, I think the order of paragraphs should be swapped.

These paragraphs have been removed or rewritten in the “Introduction section” of our revised manuscript.

Pg 17 (and other parts) I found the lists of numbers and percentages hard to read and I think you should refer readers to the tables.

Thank you. In the revised manuscript, we have avoided using lists of numbers and percentages, unless we feel they are absolutely essential.

Pg. 19 please add more interpretation to the last paragraph. It is very cool but I need help understanding the result. Are these proteins diverging rapidly? Perhaps this is a place to include the idea of codon slippage during DNA replication.

Thank you. The new results in Table 2 indicate that the CAG-slippage mechanism is unlikely to operate in ciliates with reassigned stop codons (TAAQ and TAGQ).

Pg 24. "Based on our findings from this study, we suggest that Q-rich motifs are useful toolkits for generating novel diversity during protein evolution, including by enabling greater protein expression, protein-protein interactions, posttranslational modifications, increased solubility, and tunable stability, among other important traits." This idea needs to be cited. Keith Dunker has written extensively about this idea as have others. Perhaps also discuss why Poly Q rich regions are different from other IDRs and different from other IDRs that phase-separate.

Agreed, we have cited two of Keith Dunker’s papers in our revised manuscript (73, 74).

Minor notes:

Please define Borg genomes (pg 25).

Borgs are long extrachromosomal DNA sequences in methane-oxidizing Methanoperedens archaea, which display the potential to augment methane oxidation (101). They are now described in our revised manuscript. (Page 15, lines 12-14)

Reviewer #2 (Recommendations For The Authors):

The authors dance around disorder but never really quantify or show data. This seems like a strange blindspot.

We apologize for not explaining this topic sufficiently well in our preprint manuscript. We have endeavored to do so in our revised manuscript.

The authors claim the expression enhancement is "autonomous," but they have not ruled things out that would make it not autonomous.

Evidence of the “autonomous” nature of expression enhancement is presented in Figure 1, Figure 4, and Figure 5 of the preprint manuscript.

Recommendations for improving the writing and presentation.

The title does not recapitulate the entire body of work. The first 5 figures are not represented by the title in any way, and indeed, I have serious misgivings as to whether the conclusion stated in the title is supported by the work. I would strongly suggest the authors change the title.

Figure 2 could be supplemental.

Thank you. We think it is important to keep Figure 2 in the text.

Figures 4 and 5 are not discussed much or particularly well.

This reviewer’s opinion of Figure 4 and Figure 5 is in stark contrast to those of the first reviewer.

The introduction, while very thorough, takes away from the main findings of the paper. It is more suited to a review and not a tailored set of minimal information necessary to set up the question and findings of the paper. The question that the authors are after is also not very clear.

Thank you. The entire “Introduction” section has been extensively rewritten in the revised manuscript.

Schematics of their fusion constructs and changes to the sequence would be nice, even if supplemental.

Schematics of the fusion constructs are provided in Figure 1A.

The methods section should be substantially expanded.

The method section in the revised manuscript has been rewritten and expanded. The six Javascript programs used in this work are listed in Table S4.

The text is not always suited to the general audience and readership of eLife.

We have now rewritten parts of our manuscript to make it more accessible to the broad readership of eLife.

In some cases, section headers really don't match what is presented, or there is no evidence to back the claim.

The section headers in the revised manuscript have been corrected.

A lot of the listed results in the back half of the paper could be a supplemental table, listing %s in a paragraph (several of them in a row) is never nice

Acknowledged. In the revised manuscript, we have removed almost all sentences listing %s.

Minor corrections to the text and figures.

There is a reference to table 1 multiple times, and it seems that there is a missing table. The current table 1 does not seem to be the same table referred to in some places throughout the text.

Apologies for this mistake, which we have now corrected in our revised manuscript.

In some places its not clear where new work is and where previous work is mentioned. It would help if the authors clearly stated "In previous work...."

Acknowledged. We have corrected this oversight in our revised manuscript.

Not all strains are listed in the strain table (KO's in figure 3 are not included)

Apologies, we have now corrected Table S2, as suggested by this reviewer.

Author response table 2.

S. cerevisiae strains used in this study

Certainly! Let's break down the provided information into simple terms:

1. Guard in Headers:
2. Headers are like additional notes attached to an HTTP request or response.
3. They have a property called "guard" that determines which operations are allowed on the headers object.
4. Guard values include:
   • none (default): No specific restrictions.
   • request: Applied to headers obtained from a request.
   • request-no-cors: Applied to headers from a no-cors request.
   • response: Applied to headers obtained from a response.
   • immutable: Renders headers read-only, commonly used in ServiceWorkers.

Example: javascript const myHeaders = new Headers(); // Applying guard to headers obtained from a request const requestHeaders = new Headers(myHeaders, { guard: "request" });

1. Response Objects:
2. When you make a request using fetch(), it returns a Response object.
3. Key properties include:
   • Response.status: An integer indicating the response status code (e.g., 200 for success).
   • Response.statusText: A string corresponding to the HTTP status code message.
   • Response.ok: A boolean indicating if the status is in the range 200-299.

Example: javascript const response = await fetch("https://example.com/api/data"); console.log(response.status); // 200 console.log(response.statusText); // OK console.log(response.ok); // true

1. Creating Response Object Programmatically:
2. You can create a Response object programmatically in a ServiceWorker.
3. This is useful when providing a custom response to a received request using respondWith().

Example: ```javascript const myBody = new Blob();

addEventListener("fetch", (event) => { // ServiceWorker intercepting a fetch event.respondWith( new Response(myBody, { headers: { "Content-Type": "text/plain" }, }), ); }); ```

1. Body Content in Requests and Responses:
2. Both requests and responses may have body content, which can be of various types (e.g., ArrayBuffer, Blob, JSON).
3. Methods like arrayBuffer(), blob(), formData(), json(), and text() help extract the body content.

Example: javascript const responseData = await response.json(); // Or for request body const formData = new FormData(document.getElementById("myForm")); fetch("/api/data", { method: "POST", body: formData, });

1. Feature Detection:
2. You can check if the Fetch API is supported in a browser by looking for the existence of fetch on the window or Worker scope.

Example: javascript if (window.fetch) { // Fetch is supported, proceed with fetch requests } else { // Use alternative like XMLHttpRequest }

1. Differences from jQuery.ajax():
2. The Fetch API differs from jQuery.ajax() in terms of error handling and handling of credentials in cross-origin requests.

Example: javascript fetch("https://example.com/api/data") .then((response) => { if (!response.ok) { throw new Error("Network response was not OK"); } return response.json(); }) .then((data) => { console.log(data); }) .catch((error) => { console.error("Error:", error); });

These explanations and examples aim to simplify the concepts introduced in the provided information about Headers, Response objects, Body content, and feature detection in the Fetch API.

Certainly! Let's break down each section with simple explanations and examples:

1. Uploading a File:
2. To upload a file, you can use an HTML <input type="file" /> element to let users choose a file.
3. Combine this with FormData() to gather form data, and then use fetch() to send it to a server.

Example: ```javascript async function upload(formData) { try { const response = await fetch("https://example.com/profile/avatar", { method: "PUT", body: formData, }); const result = await response.json(); console.log("Success:", result); } catch (error) { console.error("Error:", error); } }

const formData = new FormData(); const fileField = document.querySelector('input[type="file"]'); formData.append("username", "abc123"); formData.append("avatar", fileField.files[0]);

upload(formData); ```

1. Uploading Multiple Files:
2. If you want to upload multiple files, use the multiple attribute in the <input type="file" /> element.
3. Similar to the single-file example, use FormData() and fetch().

Example: ```javascript async function uploadMultiple(formData) { try { const response = await fetch("https://example.com/posts", { method: "POST", body: formData, }); const result = await response.json(); console.log("Success:", result); } catch (error) { console.error("Error:", error); } }

const photos = document.querySelector('input[type="file"][multiple]'); const formData = new FormData();

formData.append("title", "My Vegas Vacation"); for (const [i, photo] of Array.from(photos.files).entries()) { formData.append(photos_${i}, photo); }

uploadMultiple(formData); ```

1. Processing a Text File Line by Line:
2. If you need to process a text file line by line, you can create an iterator.
3. This example assumes UTF-8 encoding and uses a TextDecoder.

Example: ```javascript async function* makeTextFileLineIterator(fileURL) { // ... (see provided code) }

async function run() { for await (const line of makeTextFileLineIterator(urlOfFile)) { processLine(line); } }

run(); ```

1. Checking Fetch Success:
2. It's essential to check if a fetch operation was successful.
3. You can do this by checking response.ok to ensure the network response was okay.

Example: javascript async function fetchImage() { try { const response = await fetch("flowers.jpg"); if (!response.ok) { throw new Error("Network response was not OK"); } const myBlob = await response.blob(); myImage.src = URL.createObjectURL(myBlob); } catch (error) { console.error("There has been a problem with your fetch operation:", error); } }

These examples cover uploading files, handling multiple file uploads, processing text files line by line, and checking the success of a fetch operation. They demonstrate practical uses of the Fetch API in various scenarios.

Certainly! Let's break down the code and concepts in simpler terms:

1. Fetch API Overview:
2. The Fetch API is a way for JavaScript to make network requests (like fetching data from a server) and handle the responses.

3. It's an improvement over the older XMLHttpRequest, providing a cleaner, promise-based syntax.

4. Basic Fetch Request:

5. The fetch() function is used to initiate a network request.
6. It takes a URL as an argument, specifying where to fetch data from.

7. The fetch() function returns a Promise, which represents the result of the request.

8. Asynchronous Code with await:

9. The async keyword in the function declaration indicates that the function contains asynchronous code.

10. The await keyword is used to wait for the completion of a Promise before moving on to the next line of code.

11. Fetching JSON Data:

12. In the example, we're fetching data from "http://example.com/movies.json."

13. The response from the server is stored in the response variable.

14. Parsing JSON Response:

15. The response.json() method is used to parse the response body as JSON.

16. It returns another Promise that resolves to the actual JSON data.

17. Logging the Result:

18. The movies variable holds the parsed JSON data.

19. In this example, it's logged to the console. You could do other things with the data, like updating a webpage or performing calculations.

20. Summary:

21. fetch() initiates a network request and returns a Promise.
22. await is used to wait for the Promise to resolve, making asynchronous code easier to read and write.
23. response.json() parses the JSON content of the response.
24. The whole process is wrapped in an async function for cleaner asynchronous handling.

Example in Simpler Terms: ```javascript async function logMovies() { // Fetch data from a server const response = await fetch("http://example.com/movies.json");

// Parse the data as JSON const movies = await response.json();

// Print the movies to the console console.log(movies); }

// Call the function to see it in action logMovies(); ```

This function fetches movie data, waits for the response, parses the JSON, and then logs the movies to the console. It's a convenient way to handle asynchronous operations in JavaScript.

Any of the text can be annotated

This section particularly the exploration of utilitarianism in the context of social media, provides a thought-provoking perspective on ethical decision-making. The concept of the utility calculus as a method of predicting the outcomes and moral implications of our actions highlights the importance of comprehensive data collection and the potential pitfalls of biased or incomplete data. The discussion cleverly highlighted the challenges of navigating social media in an ethical manner, which must consider

I think that utilitarianism is not fit for every conditions and I don't actually agree with this concept. I think that personal benefits and feelings is also very important. People do not need to sacrifice in order to make everyone happy.

I expereinced what is context collapse unkowingly recently and learning about the dramaturgical analysis it made me feel alot better about it. I hosted a birthday party and decided I wanted who I wanted around me for my birthday and without much thought, invited a bunch of people. To my surprise I hadn't thought about how all these people wold mesh together and had a mini identity crisis because of it. The code switching the room was crazy.

This statement emphasises the relativity of behaviour, i.e. the same behaviour may present different expressions in different contexts. This quote emphasises that authenticity is a relative concept that depends on the specific environment and social context in which an individual finds themselves. For example, through the example of sharing an experience with a close friend, it emphasises that authenticity in an intimate relationship may be expressed in a more relaxed and natural way.

We act "code switch" in different places (being serious at work but relaxed with friends) and both ways can be the real us. On social media, when we post, all our different groups see the same posts, it can make us wonder if we're being true to ourselves in each situation.

I know this label of "code switching" means an extreme of code switching, But it does come from the need to inherit the same attributes and beliefs of those around you to fit in. This can be related to a certain work environment "etc" where it is easier to agree to your boss than to contribute your own opinion. This can be an issue however when you are in a position with some power where your acts have possible influences compared to a boss employee scenario where the employee has very little to no power in their situation.

I use code-switching on a daily basis. The way in which I speak and act at work or in class is completely different than the way in which I act at home with my family and friends. Even my online self tends to showcase my more formal side as if I am at work or another professional setting. This is honestly why I have multiple accounts; One of my accounts is more business and a representation of how I want to be perceived when in a formal setting whereas my other account highlights more of the ways I act at home with friends or family. Code-switching makes you feel safe and accepted in an environment that you feel would not be as accepting of your true self.

The prevalence of multiple personas on social media makes this intriguing to me. People frequently behave differently when they are in front of a screen. Furthermore, a lot of people identify quite differently.The concept of a public persona and code-switching also reflects the dynamic nature of human behavior and communication.

I feel that it takes practice to make it seem more authentic. Although it can be useful for certain situations, it can be draining by the end of it.

In my opinion, I think public personas and code-switching are intricate yet crucial facets of interpersonal communication. They enable people to successfully negotiate various social environments, showcasing various aspects of their true selves in polite and situation-appropriate ways. I also really like the example of Obama of how he used different type of movement to different audience. This is one of his way to show his social intelligence and respect to others.

to

Certainly! Let's break down the information in simpler terms with examples:

Under the Hood - MongoDB Update Queries with Timestamps:

1. Properties Added by Mongoose:

2. When you perform update queries with timestamps enabled, Mongoose adds two special properties to the MongoDB update operation:

   • updatedAt: Added to the $set operator. It represents the last update time.
   • createdAt: Added to the $setOnInsert operator. It represents the creation time and is applied only when a new document is upserted (inserted if not found).

3. Example - Update Query:

4. Consider the following code:

```javascript const userSchema = new Schema({ name: String }, { timestamps: true }); const User = mongoose.model('User', userSchema);

await User.findOneAndUpdate({}, { name: 'test' }); ```

• In the debug output, you'll see MongoDB update operators like $setOnInsert and $set:

plaintext Mongoose: users.findOneAndUpdate({}, { '$setOnInsert': { createdAt: new Date("Sun, 27 Feb 2022 00:26:27 GMT") }, '$set': { updatedAt: new Date("Sun, 27 Feb 2022 00:26:27 GMT"), name: 'test' }}, {...})

1. Explanation of $setOnInsert and $set:
2. $setOnInsert: It sets the specified values only if a new document is inserted during an upsert operation. In the example, it sets createdAt only if a new document is created.

3. $set: It sets the specified values regardless of whether the document is new or existing. In the example, it sets updatedAt and updates the name.

4. Disabling updatedAt Timestamp and Setting Manually:

5. If you want to handle updatedAt manually and disable automatic updates, you can do so:

javascript await User.findOneAndUpdate({}, { $setOnInsert: { updatedAt: new Date() } }, { timestamps: { createdAt: true, updatedAt: false } });

• This way, you can control when updatedAt is set, and it won't be automatically managed by Mongoose.

In simple terms, Mongoose adds special properties like updatedAt and createdAt to MongoDB update queries when timestamps are enabled. These properties are essential for tracking update and creation times. You can customize the behavior by manually handling timestamps or adjusting the update options.

Certainly! Let's simplify the information and examples provided:

Timestamps and Property Names:

1. Custom Property Names:
2. By default, Mongoose uses createdAt and updatedAt as timestamp properties. However, you can customize these names:

javascript const userSchema = new Schema({ name: String }, { timestamps: { createdAt: 'created_at', updatedAt: 'updated_at' } });

Now, instead of createdAt and updatedAt, your properties will be named created_at and updated_at.

1. Disabling Timestamps:
2. You can choose to skip updating timestamps for specific operations by setting timestamps: false.

```javascript // Example with save() await doc.save({ timestamps: false });

// Example with findOneAndUpdate() doc = await User.findOneAndUpdate({ _id: doc._id }, { name: 'test3' }, { new: true, timestamps: false }); ```

This prevents the updatedAt from being automatically updated during those operations.

1. Custom Configuration:
2. You can configure createdAt and updatedAt separately using an object:

javascript await doc.save({ timestamps: { createdAt: true, updatedAt: false } });

This example sets createdAt on save but skips updating updatedAt.

1. Disabling Timestamps and Setting Manually:
2. If you need to correct or set timestamps manually:

``javascript // Example with updatingupdatedAt` doc = await User.findOneAndUpdate({ _id: doc._id }, { updatedAt: new Date(0) }, { new: true, timestamps: false });

// Example with updating createdAt (requires setting strict: false because createdAt is immutable) doc = await User.findOneAndUpdate({ _id: doc._id }, { createdAt: new Date(0) }, { new: true, timestamps: false, strict: false }); ```

Timestamps on Subdocuments:

1. Setting Timestamps on Subdocuments:
2. You can also have timestamps on subdocuments:

javascript const roleSchema = new Schema({ value: String }, { timestamps: true }); const userSchema = new Schema({ name: String, roles: [roleSchema] });

Here, each role in the roles array will have its own createdAt and updatedAt.

1. Overwriting Subdocuments and Timestamps:
2. Overwriting a subdocument will also overwrite its createdAt and updatedAt.

javascript doc.roles[0] = { value: 'root' }; await doc.save();

1. Updating Subdocuments and Timestamps:
2. Updating a subdocument preserves createdAt and updates updatedAt.

javascript doc.roles[0].value = 'admin'; await doc.save();

In simple terms, these features allow you to customize how timestamps are handled in your Mongoose models, including changing property names, disabling automatic updates for specific operations, and managing timestamps on subdocuments.

la requête plutôt que le code ?

c'est valable pour les autres images montrant des requêtes.

en markdown tu peux utiliser un balisage particulier pour encapsuler un bloc de code :

nom-du-langage query tousMesarticles { user { blablabla } }

Cette notation évite d'avoir à gérer des images plutôt lourdes à la fois en terme de poids qu'en terme de maintenance (si la requête venait à changer, il faudrait générer une nouvelle image ...)

Lorsqu'on est sur la page Contact ou Menu du site, le lien Accueil ne fonctionne pas...

Certainly! Let's break down the code and explanations in simpler terms:

1. Schema Definition:
2. You have defined a Mongoose schema for a "Cat" with a property called "name." The "name" field is of type String and is marked as required, meaning every cat must have a name.

javascript const schema = new Schema({ name: { type: String, required: true } });

1. Creating a Cat Model:
2. You use the schema to create a Mongoose model named "Cat."

javascript const Cat = db.model('Cat', schema);

1. Creating a Cat Instance without a Name:
2. You create a new cat instance without providing a name.

javascript const cat = new Cat();

Now, this cat doesn't have a name.

1. Attempting to Save the Cat:
2. You try to save the cat using cat.save().

javascript let error; try { await cat.save(); } catch (err) { error = err; }

Since the "name" is marked as required in the schema, and the cat doesn't have a name, saving it should result in an error.

1. Handling the Error:
2. You catch the error that occurs during the save operation.

javascript assert.equal(error.errors['name'].message, 'Path `name` is required.');

The error message indicates that the "name" field is required, and it points out that the "name" path is missing.

1. Validating Sync:
2. You also perform synchronous validation using cat.validateSync().

javascript error = cat.validateSync();

This is another way to trigger validation without saving the document. The error object will contain information about validation errors.

1. Asserting the Validation Error:
2. You assert that the error received from validation has the expected message indicating that the "name" path is required.

javascript assert.equal(error.errors['name'].message, 'Path `name` is required.');

This assertion confirms that the validation is working as expected, and it identifies the issue with the missing "name" field.

In summary, the code demonstrates how Mongoose enforces validation rules specified in the schema, ensuring that essential fields, like "name" in this case, are present before saving a document.

It's always interesting, how you can just import a package and then write a few lines of code that feeds info to things from the package, and then the package does all the work for you after. I feel this must be a powerful thing in programming? Imagine the increase of productivity compared to when this is not a thing. I wonder if this was always a thing in programming? Maybe local imports were normal? How about one's like this from elsewhere? (Were they just downloaded manually and placed somewhere that can be access?)

This is like an OD diagnostic code telling you where the problem is. Got an XYZ receptor AA? something is fucking up your XYZ receptors

Author Response

eLife assessment

This useful study uses a mouse model of pancreatic cancer to examine mitochondrial mass and structure in atrophying muscle along with aspects of mitochondrial metabolism in the same tissue. Most relevant are the solid transcriptomics and proteomics approaches to map out related changes in gene expression networks in muscle during cancer cachexia.

Response: We very much appreciate the positive feedback from the editors on our article and are delighted to have it published in eLife. Our sincere thanks to the Reviewers for their positive feedback on our work, and for their insightful and constructive comments.

Reviewer #1 (Public Review):

Summary:

This important study provides a comprehensive evaluation of skeletal muscle mitochondrial function and remodeling in a genetically engineered mouse model of pancreatic cancer cachexia. The study builds upon and extends previous findings that implicate mitochondrial defects in the pathophysiology of cancer cachexia. The authors demonstrate that while the total quantity of mitochondria from skeletal muscles of mice with pancreatic cancer cachexia is similar to controls, mitochondria were elongated with disorganized cristae, and had reduced oxidative capacity. The mitochondrial dysfunction was not associated with exercise-induced metabolic stress (insufficient ATP production), suggesting compensation by glycolysis or other metabolic pathways. However, mitochondrial dysfunction can lead to increased production of ROS/oxidative stress and would be expected to interfere with carbohydrate and lipid metabolism, events that are linked to cancer-induced muscle loss. The data are convincing and were collected and analyzed using state-of-the-art techniques, with unbiased proteomics and transcriptomics analyses supporting most of their conclusions.

Additional Strengths:

The authors utilize a genetically engineered mouse model of pancreatic cancer which recapitulates key aspects of human PDAC including the development of cachexia, making the model highly appropriate and translational.

The authors perform transcriptomic and proteomics analyses on the same tissue, providing a comprehensive analysis of the transcriptional networks and protein networks changed in the context of PDAC cachexia.

Weaknesses:

The authors refer to skeletal muscle wasting induced by PDAC as sarcopenia. However, the term sarcopenia is typically reserved for the loss of skeletal muscle mass associated with aging.

Response: We agree that the term sarcopenia initially refers to aged muscle, but its use has spread to other fields, including oncology (for example, in this article, which we quote: Mintziras I et al. Sarcopenia and sarcopenic obesity are significantly associated with poorer overall survival in patients with pancreatic cancer: Systematic review and meta-analysis. Int J Surg 2018;59:19-26). Actually, the term sarcopenia is now widely used in the literature and in the clinic to describe the loss of muscle mass and strength in cancer patients (see for example, this recent review: Papadopetraki A. et al. The Role of Exercise in Cancer-Related Sarcopenia and Sarcopenic Obesity. Cancers 2023;15;5856).

In Figure 2, the MuRF1 IHC staining appears localized to the extracellular space surrounding blood vessels and myofibers-which causes concern as to the specificity of the antibody staining. MuRF1, as a muscle-specific E3 ubiquitin ligase that degrades myofibrillar proteins, would be expected to be expressed in the cytosol of muscle fibers.

Response: We agree that MuRF1 IHC staining was also observed in the extracellular space, which was a surprise, for which we have no explanation to date.

Disruptions to skeletal muscle metabolism in PDAC mice are predicted based on mitochondrial dysfunction and the transcriptomic and proteomics data. The manuscript could therefore be strengthened by additional measures looking at skeletal muscle metabolites, or linking the findings to previous work that has looked at the skeletal muscle metabolome in related models of PDAC cachexia (Neyroud et al., 2023).

Response: We agree that our omics data could be strengthened by additional measures looking at skeletal muscle metabolites. It's an excellent suggestion to parallel the transcriptomic and proteomic data we obtained on the gastrocnemius muscle with the metabolomic data obtained by Neyroud et al. on the same muscle. These authors used another mouse model of PDAC than our KIC GEMM model, namely the allograft model implanting KPC cells (derived from the pancreatic tumor of KPC mice, another PDAC GEMM model) into syngeneic recipient mice. They carried out a proteomic study on the tibialis anterior muscle and a metabolomic study on the gastrocnemius muscle. Proteomics data identified in particular a KPC-induced reduction in the relative abundance of proteins annotating to oxidative phosphorylation, consistently with our data showing reduced mitochondrial activity pathways. Metabolomic data showed reduced abundance of many amino acids as expected, and of intermediates of the mitochondrial TCA cycle (malate and fumarate) in KPC-atrophied muscle consistently with reduced mitochondrial metabolic pathways that we illustrated. In contrast, metabolites that were increased in abundance included those related to oxidative stress and redox homeostasis, which is not surprising regarding the profound oxidative stress affecting atrophied muscle. Finally, we noted in Neyroud's metabolomic data the dysregulation of certain lipids and nucleotides in atrophied muscle, which is very interesting to relate to our study describing alterations in lipid and nucleotide metabolic pathways.

Reviewer #2 (Public Review):

The present work analyzed the mitochondrial function and bioenergetics in the context of cancer cachexia induced by pancreatic cancer (PDAC). The authors used the KIC transgenic mice that spontaneously develop PDAC within 9-11 weeks of age. They deeply characterize bioenergetics in living mice by magnetic resonance (MR) and mitochondrial function/morphology mainly by oxygraphy and imaging on ex vivo muscles. By MR they found that phosphocreatine resynthesis and maximal oxidative capacity were reduced in the gastrocnemius muscle of tumor-bearing mice during the recovery phase after 6 minutes of 1 Hz electrical stimulation while pH was reduced in muscle during the stimulation time. By oxygraphy, the authors showed a decrease in basal respiration, proton leak, and maximal respiration in tumor-bearing mice that was associated with the decrease of complex I, II, and IV activity, a reduction of OXPHOS proteins, mitochondrial mass, mtDNA, and to several morphological alterations of mitochondrial shape. The authors performed transcriptomic and proteomic analyses to get insights into mitochondrial defects in the muscles of PDAC mice. By IPA analyses on transcriptomics, they found an increase in the signature of protein degradation, atrophy, and glycolysis and a downregulation of muscle function. Focusing on mitochondria they showed a downregulation mainly in OXPHOS, TCA cycle, and mitochondrial dynamics genes and upregulation of glycolysis, ROS defense, mitophagy, and amino acid metabolism. IPA analysis on proteomics revealed major changes in muscle contraction and metabolic pathways related to lipids, protein, nucleotide, and DNA metabolism. Focusing on mitochondria, the protein changes mainly were related to OXPHOS, TCA cycle, translation, and amino acid metabolism.

The major strength of the paper is the bioenergetics and mitochondrial characterization associated with the transcriptomic and proteomic analyses in PDAC mice that confirmed some published data of mitochondrial dysfunction but underlined some novel metabolic insights such as nucleotide metabolism.

There are minor weaknesses related to some analyses on mitochondrial proteins and to the fact that proteomic and transcriptomic comparison may be problematic in catabolic conditions because some gene expression is required to maintain or re-establish enzymes/proteins that are destroyed by the proteolytic systems (including the autophagy proteins and ubiquitin ligases). The authors should consider the following points.

Point 1. The authors used the name sarcopenia as synonymous with muscle atrophy. However, sarcopenia clearly defines the disease state (disease code: ICD-10-CM (M62.84)) of excessive muscle loss and force drop during ageing (Ref: Anker SD et al. J Cachexia Sarcopenia Muscle 2016 Dec;7(5):512-514.). Therefore, the word sarcopenia must be used only when pathological age-related muscle loss is the subject of study. Sarcopenia can be present in cancer patients who also experience cachexia, however since the age of tumor-bearing mice in this study is 7-9 weeks old, the authors should refrain from using sarcopenia and instead replace it with the words muscle atrophy/ muscle wasting/muscle loss.

Response: This issue has also been raised by the Reviewer #1. We agree that the term sarcopenia historically refers to aged muscle, but it is also used in oncology (for example, in this article, which we quote: Mintziras I et al. Sarcopenia and sarcopenic obesity are significantly associated with poorer overall survival in patients with pancreatic cancer: Systematic review and meta-analysis. Int J Surg 2018;59:19-26). Actually, the term sarcopenia is now widely used in the literature and in the clinic to describe the loss of muscle mass and strength in cancer patients (see for example, this recent review: Papadopetraki A. et al. The Role of Exercise in Cancer-Related Sarcopenia and Sarcopenic Obesity. Cancers 2023;15;5856).

Point 2. Most of the analyses of mitochondrial function are appropriate. However, the methodological approach to determining mitochondrial fusion and fission machinery shown in Fig. 5F is wrong. The correct way is to normalize the OPA1, MFn1/2 on mitochondrial proteins such as VDAC/porin. In fact, by loading the same amount of total protein (see actin in panel 5F) the difference between a normal and a muscle with enhanced protein breakdown is lost. In fact, we should expect a decrease in actin level in tumor-bearing mice with muscle atrophy while the blots clearly show the same level due to the normalization of protein content. Moreover, by loading the same amount of proteins in the gel, the atrophying muscle lysates become enriched in the proteins/organelles that are less affected by the proteolysis resulting in an artefactual increase. The correct way should be to lyse the whole muscle of control and tumor-bearing mice in an identical volume and to load in western blot the same volume between control cachectic muscles. Alternatively, the relative abundance of mitochondrial shaping proteins related to mitochondrial transmembrane or matrix proteins (mito mass) should compensate for the loading normalization. Because the authors showed elongated mitochondria despite mitophagy genes being up, fragmentation may be altered. Moreover, DNM1l gene is suppressed and therefore DRP1 protein must be analyzed. Finally, OPA 1 protein has different isoforms due to the action of proteases like OMA1, and YME1L that elicit different functions being the long one pro-fusion while the short ones do not. The authors must quantify the long and short isoforms of OPA1.

Response: We acknowledge that our analysis of a minor set of proteins involved in mitochondrial dynamics by Western blotting (Figure 5F) is basic and could have been improved. We thank the Reviewer for all the suggestions, which will be very useful in future projects studying the subject in greater depth and according to the molecular characteristics of each player in mitochondrial fusion, fission, mitophagy and biogenesis.

Point 3. The comparison of proteomic and transcriptomic profiles to identify concordance or not is problematic when atrophy programs are induced. In fact, most of the transcriptional-dependent upregulation is to preserve/maintain/reestablish enzymes that are consumed during enhanced protein breakdown. For instance, the ubiquitin ligases when activated undergo autoubiquitination and proteasome degradation. The same happens for several autophagy-related genes belonging to the conjugation system (LC3, Gabarap), the cargo recognition pathways (e.g. Ubiquitin, p62/SQSTM1) and the selective autophagy system (e.g. BNIP3, PINK/PARKIN) and metabolic enzymes (e.g. GAPDH, lipin). Finally, in case identical amounts of proteins have been loaded in mass spec the issues rise in point 2 of selective enrichment should be considered. Therefore, when comparing proteomic and transcriptomic these issues should be considered in discussion.

Response: We fully agree with the Reviewer that seeking concordance between transcriptomic and proteomic data in the case of an organ affected by a high level of proteolysis is a difficult business. Another major difficulty we discussed in the Discussion section of the article is the fact that there is no concordance between RNA and protein level for a good proportion of proteins, for multiple reasons, so each level of omics has to be interpreted independently to give information on the pathophysiology of the organ studied.

Reviewer #2 (Public Review):

The present work analyzed the mitochondrial function and bioenergetics in the context of cancer cachexia induced by pancreatic cancer (PDAC). The authors used the KIC transgenic mice that spontaneously develop PDAC within 9-11 weeks of age. They deeply characterize bioenergetics in living mice by magnetic resonance (MR) and mitochondrial function/morphology mainly by oxygraphy and imaging on ex vivo muscles. By MR they found that phosphocreatine resynthesis and maximal oxidative capacity were reduced in the gastrocnemius muscle of tumor-bearing mice during the recovery phase after 6 minutes of 1 Hz electrical stimulation while pH was reduced in muscle during the stimulation time. By oxygraphy, the authors showed a decrease in basal respiration, proton leak, and maximal respiration in tumor-bearing mice that was associated with the decrease of complex I, II, and IV activity, a reduction of OXPHOS proteins, mitochondrial mass, mtDNA, and to several morphological alterations of mitochondrial shape. The authors performed transcriptomic and proteomic analyses to get insights into mitochondrial defects in the muscles of PDAC mice. By IPA analyses on transcriptomics, they found an increase in the signature of protein degradation, atrophy, and glycolysis and a downregulation of muscle function. Focusing on mitochondria they showed a downregulation mainly in OXPHOS, TCA cycle, and mitochondrial dynamics genes and upregulation of glycolysis, ROS defense, mitophagy, and amino acid metabolism. IPA analysis on proteomics revealed major changes in muscle contraction and metabolic pathways related to lipids, protein, nucleotide, and DNA metabolism. Focusing on mitochondria, the protein changes mainly were related to OXPHOS, TCA cycle, translation, and amino acid metabolism.

The major strength of the paper is the bioenergetics and mitochondrial characterization associated with the transcriptomic and proteomic analyses in PDAC mice that confirmed some published data of mitochondrial dysfunction but underlined some novel metabolic insights such as nucleotide metabolism.

There are minor weaknesses related to some analyses on mitochondrial proteins and to the fact that proteomic and transcriptomic comparison may be problematic in catabolic conditions because some gene expression is required to maintain or re-establish enzymes/proteins that are destroyed by the proteolytic systems (including the autophagy proteins and ubiquitin ligases). The authors should consider the following points.

Point1. The authors used the name sarcopenia as synonymous with muscle atrophy. However, sarcopenia clearly defines the disease state (disease code: ICD-10-CM (M62.84)) of excessive muscle loss and force drop during ageing (Ref: Anker SD et al. J Cachexia Sarcopenia Muscle 2016 Dec;7(5):512-514.). Therefore, the word sarcopenia must be used only when pathological age-related muscle loss is the subject of study. Sarcopenia can be present in cancer patients who also experience cachexia, however since the age of tumor-bearing mice in this study is 7-9 weeks old, the authors should refrain from using sarcopenia and instead replace it with the words muscle atrophy/ muscle wasting/muscle loss.

Point2. Most of the analyses of mitochondrial function are appropriate. However, the methodological approach to determining mitochondrial fusion and fission machinery shown in Fig. 5F is wrong. The correct way is to normalize the OPA1, MFn1/2 on mitochondrial proteins such as VDAC/porin. In fact, by loading the same amount of total protein (see actin in panel 5F) the difference between a normal and a muscle with enhanced protein breakdown is lost. In fact, we should expect a decrease in actin level in tumor-bearing mice with muscle atrophy while the blots clearly show the same level due to the normalization of protein content. Moreover, by loading the same amount of proteins in the gel, the atrophying muscle lysates become enriched in the proteins/organelles that are less affected by the proteolysis resulting in an artefactual increase. The correct way should be to lyse the whole muscle of control and tumor-bearing mice in an identical volume and to load in western blot the same volume between control cachectic muscles. Alternatively, the relative abundance of mitochondrial shaping proteins related to mitochondrial transmembrane or matrix proteins (mito mass) should compensate for the loading normalization. Because the authors showed elongated mitochondria despite mitophagy genes being up, fragmentation may be altered. Moreover, DNM1l gene is suppressed and therefore DRP1 protein must be analyzed. Finally, OPA 1 protein has different isoforms due to the action of proteases like OMA1, and YME1L that elicit different functions being the long one pro-fusion while the short ones do not. The authors must quantify the long and short isoforms of OPA1.

Point3. The comparison of proteomic and transcriptomic profiles to identify concordance or not is problematic when atrophy programs are induced. In fact, most of the transcriptional-dependent upregulation is to preserve/maintain/reestablish enzymes that are consumed during enhanced protein breakdown. For instance, the ubiquitin ligases when activated undergo autoubiquitination and proteasome degradation. The same happens for several autophagy-related genes belonging to the conjugation system (LC3, Gabarap), the cargo recognition pathways (e.g. Ubiquitin, p62/SQSTM1) and the selective autophagy system (e.g. BNIP3, PINK/PARKIN) and metabolic enzymes (e.g. GAPDH, lipin). Finally, in case identical amounts of proteins have been loaded in mass spec the issues rise in point 2 of selective enrichment should be considered. Therefore, when comparing proteomic and transcriptomic these issues should be considered in discussion.

Author Response

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

Reviewer #1 (Recommendations for The Authors):

Major comments:

1) The immunolabeling data in Figure S4 shows no change in puncta number but reduced puncta size in Kit KO. sIPSC data show reduced frequency but little change in amplitude. These data would seem contradictory in that one suggests reduced synaptic strength, but not number, and the other suggests reduced synapse number, but not strength. How do the authors reconcile these results?

Regarding the synaptic puncta, In Kit KO (or KL KO), we have not detected an overt reduction in the average VGAT/Gephyrin/Calbindin positive puncta density or puncta size per animal. With respect to puncta size, only in the Kit KO condition, and only when individual puncta are assessed does this modest (~10%) difference in size become statistically significant. In the revision, we eliminate this figure and focus on the per animal averages.

We interpret that the reduction in sIPSC and mIPSC frequency likely stems from a decreased proportion of functional synapse sites. The number of MLIs, their action potential generation, the density of synaptic puncta, and the ability of direct stimulation to evoke release and equivalent postsynaptic currents, are all similar in Control vs Kit KO. It is therefore feasible that a reduced frequency of postsynaptic inhibitory events is due to a reduced ability of MLI action potentials to invade the axon terminal, and/or an impaired ability for depolarization to drive (e.g. coordinated calcium flux) transmitter release. That is, while the number of MLIs and their synapses appear similar, the reduced mIPSC frequency suggests that there is a reduced proportion of, or probability that, Kit KO synapse sites that function properly.

2) Related to point 1, it would be helpful to see immunolabeling data from Kit ligand KO mice? Do these show the same pattern of reduced puncta size but no change in number?

Although we have not added a figure, we have now added experiments and a corresponding analysis in the manuscript. As we had previously for Kit KO, we now for KL KO conducted IHC for VGAT, Gephyrin, and Calbindin, and we analyzed triple-positive synaptic puncta in the molecular layer of Pcp2 Cre KL KO mice and Control (Pcp2 Cre negative, KL floxed homozygous) mice. We did not find a gross reduction in the average synaptic puncta size or density, or in the PSD-95 pinceau size. From this initial analysis, it appears that the presynaptic hypotrophy is more notable in the receptor than in the ligand knockout. We speculate that this is perhaps because the Kit receptor may have basal activity in the absence of Kit ligand, that Kit may serve a presynaptic scaffolding role that is lost in the receptor (but not the ligand) knockout, or simply that the embryonic timing of the Pax2 Cre vs Pcp2 Cre recombination events is more relevant to pinceaux development, especially as basket cells are born primarily prenatally.

3) The data using KL overexpression in PC (figure 4E,F) are intriguing, but puzzling. The reduction in sIPSC frequency and amplitude in the control PC is much greater than seen in the Kit or KL KO. The interpretation of these data, "Thus, KL-Kit levels may not set the number of MLI:PC release sites, but may instead influence the proportion of synapses that are functional for neurotransmission (Figure 4G)" is not clear and the reasoning here should be explained in more detail, perhaps in the discussion.

We have attempted to clarify this portion of the manuscript by eliminating the cartoon of the proposed model, and by revising and adding to the discussion. Either MLI Kit KO or PC KL KO seems to preserve the absolute number of MLI:PC anatomical synapse sites (IHC) but to reduce the proportion of those synapse that are contributing to neurotransmission (mIPSC). We speculate that sparse PC KL overexpression (OX) may either 1) weaken inhibition to surrounding control PCs by either diminishing KL OX PC to KL Control PC inhibition, and/or 2) act retrogradely through MLI Kit to potentiate MLI:MLI inhibition, reducing the MLI:PC inhibition at neighboring Control PCs.

Minor comments:

1) In the first sentence of the results, should "Figure 1A, B" be "Figure C, D"?

Yes, corrected.

2) The top of page 6 states "the mean mIPSC amplitude was ~10% greater in PC KL KO than in control", this does not appear to be the case in Figure 3E. control and KL KO look very similar here.

In this portion of the text citing the modest 10% increase in mIPSC amplitude, we are referring to the average amplitude of all individual mIPSC events in the PC KL KO condition; in the figure referred to by the reviewer (3E), we are instead referring to the average of all mIPSC event amplitudes per KL KO PC. Because of the dramatic difference in sample size for individual events vs cells, this modest difference rises to statistical, if not biological, significance. We include this individual event analysis only to suggest that, since we in fact saw a slightly higher event amplitude in the KL KO condition, it is unlikely that a reduced amplitude would have been a technical reason that we detected a lower event frequency.

3) Figure 3 D, duration, y-axis should be labelled "ms"

Event duration is no longer graphed or referenced. This has been replaced with total inhibitory charge.

Reviewer #2 (Recommendations For The Authors):

Methods:

• Pax2-Cre line: embryonal Cre lines sometimes suffer from germline recombination. Was this evaluated, and if yes, how?

The global loss of Kit signaling is incompatible with life, as seen from perinatal lethality in other Kit Ligand or Kit mutant mouse lines or other conditional approaches. Furthermore, a loss of Kit signaling in germ cells impedes fertility. Thus, while not explicitly ruled out, since conditional Pax2 Cre mediated Kit KO animals were born, survived, and produced offspring in normal ratios, we do not suspect that germline recombination was a major issue in this specific study.

• Include rationale for using different virus types in different studies (AAV vs. Lenti).

This rationale is now included and reflects the intention to achieve infection sparsity in the smaller and less dense tissue of perinatal mouse brains.

• How, if at all, was blinding performed for histological and electrophysiological experiments?

It was not possible for electrophysiology to be conducted blinded for the Kit KO experiments, owing to the subjects’ hypopigmentation. However, whenever feasible, resultant microscopy images or electrophysiological data sets were analyzed by Transnetyx Animal ID, and the genotypes unmasked after analysis.

• Provide justification for limiting electrophysiology recordings to lobule IV/V and why MLIs in the middle third of the molecular layer were prioritized when inhibition of PCs is dominated by large IPSCs from basket cells. Why were 2 different internals used for recording IPSCs and EPSCs in PCs and MLIs? While that choice is justified for action potential recordings, it provides poor voltage control in PC voltage clamp. Both IPSCs and EPSCs could have been isolated pharmacologically using a CsCl internal.

The rationale for regional focus has been added to the text. For MLI action potential recordings, we opted to sample the middle third of the molecular layer so that we would not be completely biased to either classic distal stellate vs proximal basket subtypes. It is our hope, in future optogenetic interrogations, to simultaneously record the dynamics of all MLI subtypes in a more unbiased way. With respect to internal solutions, we initially utilized a cesium chloride internal to maximize our ability to resolve differences in GABAA mediated currents, which was the hypothesis-driven focus of our study. While we agree that utilizing a single internal and changing the voltage clamp to arrive at per-cell analysis of Excitatory/Inhibitory input would have been most informative, our decision to utilize pharmacological methods was driven by our experience that achieving adequate voltage clamp across large Purkinje cells was often problematic, particularly in adult animals.

Introduction:

In the introduction, the authors state that inactivating Kit contributes to neurological dysfunction - their examples highlight neurological, psychiatric, and neurodevelopmental conditions.

The language has been changed.

General:

Using violin plots illustrates the data distribution better than bar graphs/SEM.

We have included violin plots throughout, and we have changed p values to numeric values, both in the interest of presenting the totality of the data more clearly.

Synapses 'onto' PCs sounds more common than 'upon' PCs.

We have changed the wording throughout.

Figure 1:

1F - there seems to be an antero-posterior gradient of Kit expression.

Though not explicitly pursued in the manuscript, it is possible that such a gradient may reflect differences in the timing of the genesis and maturation of the cerebellum along the AP axis. Regional variability is however now briefly addressed as a motivator for focused studies within lobules IV/V.

E doesn't show male/female ratios but only hypopigmentation.

This language has been corrected.

Figure 2 and associated supplementary figures:

2A/B: The frequency of sIPSCs is very high in PCs, making the detection of single events challenging. How was this accomplished? Please add strategy to the methods.

We have added methodological detail for electrophysiology analysis.

How were multi-peak events detected and analyzed? 'Duration' is not specified - do the authors refer to kinetics? If so, report rise and decay. It is likely impossible to show individual aligned sIPSCs with averages superimposed, given that sIPSCs strongly overlap. Alternatively, since no clear baseline can be determined in between events, and therefore frequency, amplitude, and kinetics quantification is near-impossible, consider plotting inhibitory charge.

Given the heterogeneity of events, we now do not refer to individual event kinetics. As suggested, we have now included an analysis of the total inhibitory charge transferred by all events during the recording epoch.

S2: Specify how density, distribution, and ML thickness were determined in methods. How many animals/cells/lobules?

For consistency with viral injections and electrophysiology, the immunohistochemical analysis was restricted to lobule IV/V. This is clearer in the revision and detail is added in the methods.

S3:

S3B: the labels of Capacitance and Input resistance are switched.

This has been corrected.

How were these parameters determined? Add to methods.

Added

In the previous figure the authors refer to 'frequency', in this figure to 'rate' - make consistent

This has been corrected.

D: example does not seem representative. Add amplitude of current pulse underneath traces.

We added new traces from nearer the group means and we now include the current trace.

F/G example traces (aligned individual events + average) are necessary.

We added example traces near the relevant group means for each condition.

Statement based on evoked IPCSs that 'synapses function normally' is a bit sweeping and can only be fully justified with paired recordings. Closer to the data would be the release probability of individual synapses is similar between control and Kit KO.

Paired recordings in both Kit Ligand and Kit receptor conditional knockout conditions is indeed an informative aim of future studies should support permit. For now, we have clarified the language to be more in line with the reviewer’s welcome suggestion.

S4:

Histological strategy cannot unambiguously distinguish MLI-PC and PC-PC synapses. Consider adding this confound to the text.

We have added this confound to the discussion.

The observation that the pinceau is decreased in size could have important implications for ephaptic coupling of MLI and PC and could be mentioned.

We agree and have added this notion to the discussion.

Y-label is missing in B.

Corrected.

Figure 3 and associated supplementary figures:

In the text, change PC-Cre to L7-Cre or Pcp2-Cre.

Changed

How do the authors explain a reduction in frequency, amplitude, and duration of sIPSCs in the KL KO but not in the Kit KO? Add to the discussion

We now address this apparent discordance in the discussion. Pax2 Cre mediates recombination weeks ahead of Pcp2 Cre. We therefore suspect that postnatal PC KL KO may be more phenotypic than embryonic MLI Kit KO because there is less time for developmental compensation. A future evaluation of the impact of postnatal Kit KO would be informative to this end.

As in Figure 2, plotting the charge might be more accurate.

We now plot total charge transfer.

Are the intrinsic properties in KL KO PCs altered? (Spontaneous firing, capacitance, input resistance).

We have added to the text that we found no difference in capacitance or input resistance between Purkinje cells from KL floxed homozygous Control animals versus those from KL floxed homozygous, PCP2 Cre positive KL KO animals. We plan to characterize both basal and MLI modulated PC firing in a future manuscript, especially since Pcp2 Cre mediated KL KO seems more phenotypic than Pax2 Cre mediated Kit KO, we agree that this seems a better testbed for investigating differences in both the basal, and the MLI-mediated modulations in, PC firing.

3D-F - Example traces would be desirable (see above, analogous to Fig. 2).

More example traces have been added.

Figure 4: 'In vivo mixtures' sounds unusual. Consider revision (e.g., 'to sparsely delete KL').

Changed

The observation that control PC sIPSC frequency is lower in KL OX PCs than in sham is interesting. This observation would be consistent with overall inhibitory synapse density being preserved. This could be evaluated with immunohistochemistry. For how far away from the injection area does this observation hold true?

Because we have now analyzed and failed to find an overt (per animal average) change in synaptic puncta size or density in the whole animal Control vs PCP2 Cre mediated KL KO conditions, we do not have confidence that it is feasible to pursue this IHC strategy in the sparse viral-mediated KL KO or OX conditions. To the reviewer’s valid point however, we intend to probe the spatial extent/specificity of the sparse phenomenon when we are resourced to complement the KL/Kit manipulations with transgenic methods for evaluating MLI-PC synapses specifically, potentially by GRASP or related methods that would not be confounded by PC-PC synapses. Transgenic MLI access would also facilitate determining the spatial extent to which opto-genetically activated MLIs evoke equivalent responses in Control vs KL manipulated PCs.

Y-legend in D clipped.

Corrected

Existing literature suggests that MLI inhibition regulates the regularity of PC firing - this could be tested in Kit and KL mutants.

For now, based upon transgenic animal availability, we have now included an evaluation of PC firing in the (Pax2 Cre mediated) Kit KO condition. PC average firing frequency, mean ISI, and ISI CV2 were not significantly different across genotypes. A KS test of individual ISI durations for Control vs Kit KO did reveal a difference (p<0.0001). We have added a supplementary figure (S6) with this data. It is possible that in the more phenotypic PC KL KO condition that we may find a difference in these PC spiking patterns of PC firing, however, we are also eager to test in future studies whether postnatal KL or Kit KO impairs the ability of MLI activation to produce pauses or other alterations in PC firing or in PF-PC mediated plasticity.

Reviewer #3 (Recommendations For The Authors):

Reference to Figure 1A in the Results section is slightly inaccurate. Kit gene modifications are illustrated in Figures 1A, B. Where Figure 1A shows Kit distribution. Please rephrase. Relatedly, the reference to Figs 1B - D are shifted in the results section, and 1E is skipped.

We have changed the text.

Please show cumulative histograms for frequency too for consistency with amplitude (e.g. Fig 2).

We have instead, for reasons outlined by other reviewers, documented total charge transfer for both Kit KO and KL KO experiments where sIPSC events were analyzed.

Fig S3: include example traces of PPR.

This is now included.

Include quantifications of GABAergic synapse density in Fig S4.

This is now included.

Include inset examples of KO in Fig S4A.

This is now included.

Add average puncta size graphs along Figure S4B. The effect apparent in the histogram of S4B is small and statistics using individual puncta as n values (in the 20,000s) therefore misleading.

Per animal analysis is now instead included in the figure and text.

Figure S4B y axis label blocked.

Corrected

Include quantification referenced in "As PSD95 immunoreactivity faithfully follows multiple markers of pinceaux size 40, we quantified PSD95 immunoreactive pinceau area and determined that pinceaux area was decreased by ~50% in Kit KO (n 26 Control vs 43 Kit KO, p<0.0001, two-tailed t-test)."

We added a graph of per animal averages, instead of in text individual pinceau areas.

Include antibody dilutions in the methods.

Added.

It's unclear from the text where the Mirow lab code comes from.

Detail has now been added in text.

Typo in methods "The Kit tm1c alle was bred...".

Corrected

Typo in Figure S4 legend "POSD-95 immuno-reactivity".

Corrected

Certainly! In simple terms, the cookie-parser is a module in Node.js (specifically for Express applications) that helps with handling cookies, which are small pieces of data stored on a user's browser.

Here's a breakdown:

1. Installation: You need to install the cookie-parser module using npm.

bash npm install cookie-parser

1. Creating a Middleware:
2. You create a middleware function using cookie-parser.
3. You can optionally provide a "secret" (a string or an array of strings) for extra security. This secret is used to sign cookies, making them tamper-proof.

```javascript const cookieParser = require('cookie-parser'); const app = express();

// Use the cookie-parser middleware app.use(cookieParser('yourSecret')); ```

1. Parsing Cookies:
2. The middleware parses the cookies sent by the user's browser and makes them available in req.cookies.

3. If a secret is provided, it also validates and unsigns any signed cookies, making them available in req.signedCookies.

4. Working with Cookies:

5. You can set cookies in the response using res.cookie().
6. You can read cookies from the request in your route handlers from req.cookies and req.signedCookies.

```javascript app.get('/set-cookie', (req, res) => { // Set a cookie named "myCookie" with value "Hello, Cookie!" res.cookie('myCookie', 'Hello, Cookie!'); res.send('Cookie has been set!'); });

app.get('/read-cookie', (req, res) => { // Access the value of the "myCookie" cookie const myCookieValue = req.cookies.myCookie; res.send(Value of myCookie: ${myCookieValue}); }); ```

1. Additional Features:
2. You can work with signed cookies for added security.
3. It supports special "JSON cookies" for storing JSON data.

Remember, cookies are often used to store small pieces of information on the user's browser, and cookie-parser helps you handle them easily in your Express.js applications.

In an Express.js application, you can set cookies in the browser using the res.cookie() method. This method is provided by the cookie-parser middleware. Here's a simple example:

1. Install cookie-parser if you haven't already:

bash npm install cookie-parser

1. Use cookie-parser in your Express app:

```javascript const express = require('express'); const cookieParser = require('cookie-parser');

const app = express();

// Use the cookie-parser middleware app.use(cookieParser()); ```

1. Set a cookie in a route handler:

```javascript app.get('/set-cookie', (req, res) => { // Set a cookie named "myCookie" with value "Hello, Cookie!" res.cookie('myCookie', 'Hello, Cookie!');

 // Send a response
 res.send('Cookie has been set!');

}); ```

In the above example, when the user accesses the /set-cookie endpoint, a cookie named "myCookie" with the value "Hello, Cookie!" will be set in their browser.

1. Set additional options (optional):

You can set additional options for cookies, such as maxAge, expires, path, domain, and others. For example:

javascript res.cookie('myCookie', 'Hello, Cookie!', { maxAge: 900000, httpOnly: true });

This sets the maxAge option to 900,000 milliseconds (15 minutes) and makes the cookie accessible only through HTTP (not through JavaScript).

Remember to adapt the code to your specific use case and requirements. The client's browser will now store the cookie, and subsequent requests from that client to your server will include the cookie information in the request headers.

Certainly! The way it works is when you create a new note and associate it with a user, you will typically provide the user's unique identifier (ID) as the value for the user field. In a typical scenario, this user ID would come from the "user" collection in your database.

Here's a brief example in code:

```javascript // Assume you have a User model and you have obtained a user's ID const userId = "someUserId"; // This would be the actual ID of the user in your database

// Creating a new note and associating it with the user by providing the user ID const newNote = new Notes({ user: userId, title: "Sample Note", description: "This is a sample note.", tag: "General", deadline: new Date(), deadlinetime: "12:00 PM", });

// Save the new note to the database newNote.save() .then((savedNote) => { console.log("Note saved successfully:", savedNote); }) .catch((error) => { console.error("Error saving note:", error); }); ```

In this example, the user field of the new note is set to the userId obtained from the "user" collection. When you save this note to the database, the user field will store the provided user ID. Later, when you retrieve this note, you can use this user ID to identify which user the note is associated with.

It's important to ensure that the user ID you provide for the user field actually corresponds to an existing user in the "user" collection. This way, you maintain the relationship between the "notes" and "user" collections in your MongoDB database.

接收有两种方式，直接返回和被调用时返回

I thought this was very interesting, how the tweets from an iPhone and Android could have such big difference. Also how they could tell the difference between the campaign's tweets which to be an iPhone, and to Trump's own android. I never really paid attention to Trumps election tweets, but I thought this is very interesting.

Author Response

Author responses to the original review:

The data we produce are not criticized as such and thus, do not require revision; the criticisms concern our interpretation of them. General themes of the reviews are that i) genetic signatures do not matter for defining neuronal types (here sympathetic versus parasympathetic); ii) that a cholinergic postganglionic autonomic neuron must be parasympathetic; and iii) that some physiology of the pelvic region would deserve the label “parasympathetic”. We answered the latter argument in (Espinosa-Medina et al., 2018) to which we refer the interested reader; and we fully disagree with the first two. Of note, part of the last sentence of the eLife assessment is misleading and does not reflect the referees’ comments. Our paper analyses genetic differences between the cranial and sacral outflow and uses them to argue that they cannot be both parasympathetic. The eLife assessment acknowledges the “genetic differences” but concludes that, somehow, they don’t detract from a common parasympathetic identity. We take issue with this paradox, of course, but it is coherent with the referee’s comments. On the other hand, the eLife assessment alone pushes the paradox one step further by stating that “functional differences” between the cranial and sacral outflows can’t either prevent them from being both parasympathetic. We would also object to this, but the only “functional differences” used by the referees to dismiss our diagnostic of a sympathetic-like character (rather than parasympathetic) for the sacral outflow are between noradrenergic and cholinergic, and between sympathetic and parasympathetic (and we also disagree with those, see above, and below) —not between cranial and sacral.

We will thus use the opportunity offered by eLife to keep the paper as it is (with a few minor stylistic changes). We respond below to the referees’ detailed remarks and hope that the publication, as per eLife new model, of the paper, the referees’ comments and our response will help move the field forward.

Public review by Referee #1

“Consistently, the P3 cluster of neurons is located close to sympathetic neuron clusters on the map, echoing the conventional understanding that the pelvic ganglia are mixed, containing both sympathetic and parasympathetic neurons”.

The greater closeness of P3 than of P1/2/4 to the sympathetic cluster can be used to judge P1/2/4 less sympathetic than P3 (and more… something else), but not more parasympathetic. There is no echo of the “conventional understanding” here.

“A closer look at the expression showed that some genes are expressed at higher levels in sympathetic neurons and in P2 cluster neurons ” [We assume that the referee means “in sympathetic neurons and in P3 cluster neurons”] but much weaker in P1, P2, and P4 neurons such as Islet1 and GATA2, and the opposite is true for SST. Another set of genes is expressed weakly across clusters, like HoxC6, HoxD4, GM30648, SHISA9, and TBX20.

These statements are inaccurate; On the one hand, the classification is not based on impression by visual inspection of the heatmap, but by calculations, using thresholds. Admittedly, the thresholds have an arbitrary aspect, but the referee can verify (by eye inspection of heatmap) that genes which we calculate as being at “higher levels in sympathetic neurons and in P3 cluster neurons, but much weaker in P1, P2, and P4 neurons” or vice versa, i.e. noradrenergic or cholinergic neurons (genes from groups V and VI, respectively), have a much bigger difference than those cited by the referee, indeed are quasi-absent from the weaker clusters or ganglia. In addition, even by subjective eye inspection:

Islet is equally expressed in P4 and sympathetics.

SST is equally expressed in P1 and sympathetics.

Tbx20 is equally expressed in P2 and sympathetics.

HoxC6, HoxD4, GM30648, SHISA9 are equally expressed in all clusters and all sympathetic ganglia.

“Since the pelvic ganglia are in a caudal body part, it is not surprising to have genes expressed in pelvic ganglia, but not in rostral sphenopalatine ganglia, and vice versa (to have genes expressed in sphenopalatine ganglia, but not in pelvic ganglia), according to well recognized rostro-caudal body patterning, such as nested expression of hox genes.”

We do not simply show “genes expressed in pelvic ganglia, but not in rostral sphenopalatine ganglia, and vice versa”, i.e. a genetic distance between pelvic and sphenopalatine, but many genes expressed in all pelvic cells and sympathetic ones, i.e. a genetic proximity between pelvic and sympathetic. This situation can be deemed “unsurprising”, but it can only be used to question the parasympathetic nature of pelvic cells (as we do), or considered irrelevant (as the referee does, because genes would not define cell types, see our response to an equivalent stance by Referee#2). Concerning Hox genes, we do take them into account, and speculate in the discussion that their nested expression is key to the structure of the autonomic nervous system, including its division into sympathetic and parasympathetic outflows.

It is much simpler and easier to divide the autonomic nervous system into sympathetic neurons that release noradrenaline versus parasympathetic neurons that release acetylcholine, and these two systems often act in antagonistic manners, though in some cases, these two systems can work synergistically. It also does not matter whether or not pelvic cholinergic neurons could receive inputs from thoracic-lumbar preganglionic neurons (PGNs), not just sacral PGNs; such occurrence only represents a minor revision of the anatomy. In fact, it makes much more sense to call those cholinergic neurons located in the sympathetic chain ganglia parasympathetic.

This “minor revision of the anatomy” would make spinal preganglionic neurons which are universally considered sympathetic (in the thoraco-lumbar chord), synapse onto large numbers of parasympathetic neurons (in the paravertebral chains for sweat glands and periosteum, and in the pelvic ganglion), robbing these terms of any meaning.

Thus, from the functionality point of view, it is not justified to claim that "pelvic organs receive no parasympathetic innervation".

There never was any general or rigorous functional definition of the sympathetic and parasympathetic nervous systems — it is striking, almost ironic, that Langley, creator of the term parasympathetic and the ultimate physiologist, provides an exclusively anatomic definition in his Autonomic Nervous System, Part I. Hence, our definition cannot clash with any “functionality point of view”. In fact, as we briefly say in the discussion and explore in (Espinosa-Medina et al., 2018), it is the “sacral parasympathetic” paradigm which is unjustified from a functionality point of view, for implying a functional antagonism across the lumbo-sacral gap, which has been disproven repeatedly. It remains to be determined which neurons are antagonistic to which on the blood vessels of the external genitals; antagonism within one division of the autonomic nervous system would not be without precedent (e.g. there exist both vasoconstrictor and vasodilator sympathetic neurons, and both, inhibitor and activator enteric motoneurons). The way to this question is finally open to research, and as referee#2 says “it is early days”.

Public review by Referee #2

This work further documents differences between the cranial and sacral parasympathetic outflows that have been known since the time of Langley - 100 years ago.

We assume that the referee means that it is the “cranial and sacral parasympathetic outflows” which “have been known since the time of Langley”, not their differences (that we would “further document”): the differences were explicitly negated by Langley. As a matter of fact, the sacral and cranial outflows were first likened to each other by Gaskell, 140 years ago (Gaskell, 1886). This anatomic parallel (which is deeply flawed (Espinosa-Medina et al., 2018)) was inherited wholesale by Langley, who added one physiological argument (Langley and Anderson, 1895) (which has been contested many times (Espinosa-Medina et al., 2018) and references within).

In addition, the sphenopalatine and other cranial ganglia develop from placodes and the neural crest, while sympathetic and sacral ganglia develop from the neural crest alone.

Contrary to what the referee says, the sphenopalatine has no placodal contribution. There is no placodal contribution to any autonomic ganglion, sympathetic or parasympathetic (except an isolated claim concerning the ciliary ganglion (Lee et al., 2003)). All autonomic ganglia derive from the neural crest as determined a long time ago in chicken. For the sphenopalatine in mouse, see our own work (Espinosa-Medina et al., 2016).

One feature that seems to set the pelvic ganglion apart is […] the convergence of preganglionic sympathetic and parasympathetic synapses on individual ganglion cells (Figure 3). This unusual organization has been reported before using microelectrode recordings (see Crowcroft and Szurszewski, J Physiol (1971) and Janig and McLachlan, Physiol Rev (1987)). Anatomical evidence of convergence in the pelvic ganglion has been reported by Keast, Neuroscience (1995).

Contrary to what the referee says, we do not provide in Figure 3 any evidence for anatomic convergence, i.e. for individual pelvic ganglion cells receiving dual lumbar and sacral inputs. We simply show that cholinergic neurons figure prominently among targets of the lumbar pathway. This said, the convergence of both pathways on the same pelvic neurons, described in the references cited by the referee, is another major problem in the theory of the “sacral parasympathetic” (as we discussed previously (Espinosa-Medina et al., 2018)).

It should also be noted that the anatomy of the pelvic ganglion in male rodents is unique. Unlike other species where the ganglion forms a distributed plexus of mini-ganglia, in male rodents the ganglion coalesces into one structure that is easier to find and study. Interestingly the image in Figure 3A appears to show a clustering of Chat-positive and Th-positive neurons. Does this result from the developmental fusion of mini ganglia having distinct sympathetic and parasympathetic origins?

The clustering of Chat-positive and Th-positive cells could arise from a number of developmental mechanisms, that we have no idea of at the moment. This has no bearing on sympathetic and parasympathetic.

In addition, Brunet et al dismiss the cholinergic and noradrenergic phenotypes as a basis for defining parasympathetic and parasympathetic neurons. However, see the bottom of Figure S4 and further counterarguments in Horn (Clin Auton Res (2018)).

The bottom of Figure S4 simply indicates which cells are cholinergic and adrenergic. We have already expounded many times that noradrenergic and cholinergic do not coincide with sympathetic and parasympathetic. Henry Dale (Nobel Prize 1936) demonstrated this. Langley himself devoted several pages of his final treatise to this exception to his “Theory on the relation of drugs to nerve system” (Langley, 1921) (p43) (which was actually a bigger problem for him than it is for us, for reason which are too long to recount here; it is as if the theoretical difficulties experienced by Langley had been internalized to this day in the form of a dismissal of the cholinergic sympathetic neurons as a slightly scandalous but altogether forgettable oddity). (Horn, 2018) reviews the evidence that the thoracic cholinergic sympathetic phenotype is brought about by a secondary switch upon interaction with the target and argues that this would be a fundamental difference with the sacral “parasympathetic”. But in fact the secondary switch is preceded by co-expression of ChAT and VAChT with Th in most sympathetic neurons (reviewed in (Ernsberger and Rohrer, 2018)); and we have no idea of the dynamic in the pelvic ganglion. It may also be mentioned in this context that target-dependent specification of neuronal identity has also been demonstrated of other types of sympathetic neurons ((Furlan et al., 2016)

What then about neuropeptides, whose expression pattern is incompatible with the revised nomenclature proposed by Brunet et al.?

There was never any neuropeptide-inspired criterion for a nomenclature of the autonomic nervous system.

Figure 1B indicates that VIP is expressed by sacral and cranial ganglion cells, but not thoracolumbar ganglion cells.

Contrary to what the referee says, there are VIP-positive cells in our sympathetic data set and even strongly positive ones, except they are scattered and few (red bars on the UMAP). They correspond to cholinergic sympathetics, likely sudomotor, which are known to contain VIP (e.g.(Anderson et al., 2006)(Stanke et al., 2006)). In other words, VIP is probably part of what we call the cholinergic synexpression group (but was not placed in it by our calculations, probably because of a low expression level in sympathetic noradrenergic cells).

The authors do not mention neuropeptide Y (NPY). The immunocytochemistry literature indicates that NPY is expressed by a large subpopulation of sympathetic neurons but never by sacral or cranial parasympathetic neurons.

Contrary to what the referee says, Keast (Keast, 1995) finds 3.7% of pelvic neurons double stained for NPY and VIP in male rats, and says (Keast, 2006) that in females “co-expression of NPY and VIP is common” ( thus in cholinergic neurons that the referee calls “parasympathetic”). Single cell transcriptomics is probably more sensitive than immunochemistry, and in our dichotomized data set (table S1), NPY is expressed in all pelvic clusters and all sympathetic ganglia. In other words, it is one more argument for their kinship. It does not appear in the heatmap because it ranks below the 100 top genes.

Answer to the original recommendations by Referee #2

Introduction - the use of the words 'consensual' and 'promiscuity' are not clear and rather loaded in the context of the pelvic ganglia. Pick alternative words.

There is no sexual innuendo inherent in “promiscuity”: “condition of elements of different kinds grouped or massed together without order” (Oxford English Dictionary). We replaced “never consensual” by “never generally accepted”.

Results - Page 2 - what sex were the mice? Previous works indicate significant sexual dimorphism in the pelvic ganglion.

The mice included both males and females, and male and female cells are represented in all ganglia and clusters. This is now mentioned in the Material and Methods. Thus, however unsuited to analyze sexual dimorphism, our data set ensures that all the cell types we describe are qualitatively present in both sexes.

Results line 3 - the celiac and mesenteric ganglia are prevertebral ganglia and not part of the sympathetic chain. The chain refers to the paravertebral ganglia.

We replaced “part of the prevertebral chain” by “belonging to prevertebral ganglia”. This said, there are precedents for “prevertebral chain ganglia” to designate the rostro-caudal series of prevertebral ganglia. Rita Levi-Montalcini, for example, who devoted her glorious career to sympathetic ganglia, writes in 1972 “The nerve cell population of para- and prevertebral chain ganglia is reduced to 3–5% of that of controls”. (10.1016/0006-8993(72)90405-2).

Page 3 - "as the current dogma implies". Dogma often refers to opinion or church doctrine. The current nomenclature is neither. Pick another word.

There is little in science that is proven to the point of eliminating any element of opinion. “Dogma” refers to “that which is held as a principle or tenet […], especially a tenet authoritatively laid down by […] a school of thought” (OED). And “dogma” is used in science to designate tenets better experimentally supported than the “sacral parasympathetic”, such as the “central dogma of molecular biology”.

Page 3 - "To give justice" implies the classical notion is unjust. How about, 'to further explore previous evidence indicating that ....'

The term is indeed not proper English for the meaning intended, and the right expression is “to do justice”, to mean: “to treat [a subject or thing] in a manner showing due appreciation, to deal with [it] as is right or fitting” (OED). We have corrected the paper accordingly.

Page 4 top - the convergence indicated by Figure 3 does not justify excluding cholinergic and noradrenergic genes from the analysis.

Contrary to what the referee says, Figure 3 does not show any “convergence”, see our answer to Referee#1. What Figure 3 shows is that cells that are targeted by the lumbar pathway (a pathway universally deemed “sympathetic”) are cholinergic in massive proportion. Therefore, by an uncontroversial criterion, the pelvic ganglion contains lots of sympathetic cholinergic neurons. The only other option is to declare that sympathetic preganglionic neurons synapse onto parasympathetic postganglionic ones (which is what Referee#1 proposes, and considers “much simpler”. We beg to differ).

Our justification for excluding cholinergic and noradrenergic genes from the definition of “sympathetic” and “parasympathetic” is simply that sympathetic neurons can be cholinergic (to sweat glands and periosteum; and — as we show in Figure 3 — many targets of the lumbar pathway); One can also note that anywhere else in the nervous system, classifying cell types as a function of neurotransmitter phenotype would lead to non-sensical descriptions, such as putting together pyramidal cells and cerebellar granules, or motor neurons and basal forebrain cholinergic neurons. Indeed Referee#1 proposes such a revolutionary revision, by calling all cholinergic autonomic neurons “parasympathetic” (see our answer above).

Keast (1995) did similar experiments and used presynaptic lesions to draw a different conclusion indicating preferential innervation pelvic subpopulations.

Keast found “preferential” innervation of pelvic subpopulations based on lesion experiments; Nevertheless, she concluded (at the time) that “the correct definition of these two components of the nervous system is based on neuroanatomy rather than chemistry” (Keast, 2006).

Page 4 - "In the aggregate, the pelvic ganglion is best described as a divergent sympathetic ganglion devoid of parasympathetic neurons" The notion of a divergent ganglion is completely unclear!

We take “divergent” in a developmental or evolutionary meaning: related to sympathetic ganglia, yet somewhat differing from them. Elsewhere we use the word “modified”. Importantly (and as cited in the paper), a similar situation emerges from the single cell transcriptomic analysis of the lumbar and sacral preganglionics (by other research groups).

Granted, it is devoid of neurons having the signature of cranial parasympathetics, but that is insufficient to conclude that they are not parasympathetics.

If a genetic signature which is not only un-parasympathetic, but sympathetic-like remains compatible with some version of the label “parasympathetic”, we get dangerously close to dismissing the molecular make-up of a neuron as a definition of its type. This goes against any contemporary understanding of neuron types (take (Zeisel et al., 2018) among hundreds of other examples).

Page 4 - "the entire taxonomy of autonomic ganglia could be a developmental readout of Hox genes." This reader completely agrees! We appreciate this would be difficult to test but it helps to explain possible differences along the rostro-caudal axis. Consider making this a key implication of the study!

If the reader agrees, then his/her previous points become mysterious: we speculate that the Hox code determines the structure of the autonomic nervous system, i.e. the array, along the rostrocaudal axis, of a bulbar parasympathetic, a thoracolumbar sympathetic and lumbo-sacral “pelvo-sympathetic”. The existence of caudal parasympathetic neurons, on the contrary, would subvert any role for Hox genes: similar neurons (similar enough to be called by the same name) would arise at completely different rostro-caudal levels, i.e. with a different Hox code.

Page 5 - "It is thus remarkable ...that we uncover in no way contradicts the physiology." Not really. The 'classical' sympathetic system innervates the limbs, and the skin and it participates in thermoregulation and in cardiovascular adjustments to exercise. The parasympathetic system does none of these things. Reclassing the pelvic outflow as pseudo-sympathetic contradicts this physiology.

We do not say that the sacral outflow is classically sympathetic; We go all the way to proposing the special name “pelvo-sympathetic”; And we insist that these special sympathetic-like neurons have special targets (detrusor muscle, helicine arteries…): there is no contradiction. Not only is there no contradiction, but we remove the mind-twister of an anatomical/genetic/cell type-based “sacral parasympathetic” combined with a lack of physiological lumbosacral antagonism (we provide a short history of this dissonance in (Espinosa-Medina et al., 2018)), which led Wilfrid Jänig to write (Jänig, 2006)(p. 357): “Thus, functions assumed to be primarily associated with sacral (parasympathetic) are well duplicated by thoracolumbar (sympathetic) pathways. This shows that the division of the spinal autonomic systems into sympathetic and parasympathetic with respect to sexual functions is questionable”. We could not agree more: this division is questionable in terms of physiology and inexistent in terms of cell types. In other words, we reconcile cell types with physiology (but “it is early days”).

Answer to the novel recommendations by Referee #2

In addition to my original comments, important anatomical and functional distinctions are not explained by the data in this paper. ANATOMY- Sympathetic ganglia are located in close proximity to major branches of the aorta. Cranial and sacral parasympathetic ganglia are located next to or within the structures they innervate (e.g. eye, lung, heart, bladder).

The pelvic ganglion, including some of its cholinergic neurons, that the referee insist are parasympathetic, is further removed from one of its major targets (the helicine arteries of the external genitals) than the sympathetic prevertebral ganglia are of some of theirs (like the gut or kidney). We discussed this issue in (Espinosa-Medina et al., 2018).

FUNCTION- The sympathetic system controls state variables (e.g. body temperature, blood pressure, serum electrolytes and fluid balance), parasympathetic neurons do not.

Even in the classical view, the sympathetic system controls the blood vessels of the external genitals or the size of the pupil, for example, which are not state variables.

[…] The data in the paper are a useful next step in defining the genetic diversity of autonomic neurons but do not justify or improve upon existing nomenclature. The future challenge is to understand distinctions between subsets of autonomic ganglion cells that innervate different targets and the principles that govern the integrative function of the autonomic motor system that controls behavior.

We thank the referee for finding our data useful; and we fully agree with the latter statement. However, neurons, like many other cell types, are hierarchically organized (Zeng and Sanes, 2017), i.e. subsets of neurons belong to sets, with defining traits. Our data argue that there is no parasympathetic neuronal set that includes any pelvic ganglionic neuron. In contrast, there is a ganglionic sympathetic set (defined by our analysis of gene expression) which includes all of them — as there is a preganglionic sympathetic set that includes sacral preganglionics (Alkaslasi et al., 2021; Blum et al., 2021)(although the direct comparison with cranial preganglionics is yet to be made).

References

Anderson, C. R., Bergner, A. and Murphy, S. M. (2006). How many types of cholinergic sympathetic neuron are there in the rat stellate ganglion? Neuroscience 140, 567–576.

Alkaslasi, M. R., Piccus, Z. E., Hareendran, S., Silberberg, H., Chen, L., Zhang, Y., Petros, T. J. and Le Pichon, C. E. (2021). Single nucleus RNA-sequencing defines unexpected diversity of cholinergic neuron types in the adult mouse spinal cord. Nat Commun 12, 2471.

Blum, J. A., Klemm, S., Shadrach, J. L., Guttenplan, K. A., Nakayama, L., Kathiria, A., Hoang, P. T., Gautier, O., Kaltschmidt, J. A., Greenleaf, W. J., et al. (2021). Single-cell transcriptomic analysis of the adult mouse spinal cord reveals molecular diversity of autonomic and skeletal motor neurons. Nat Neurosci 24, 572–583.

Ernsberger, U. and Rohrer, H. (2018). Sympathetic tales: subdivisons of the autonomic nervous system and the impact of developmental studies. Neural Dev 13, 20.

Espinosa-Medina I, Saha O, Boismoreau F, Chettouh Z, Rossi F, Richardson WD, Brunet JF (2016) The sacral autonomic outflow is sympathetic. Science 354, 893-897

Espinosa-Medina, I., Saha, O., Boismoreau, F. and Brunet, J.-F. (2018). The “sacral parasympathetic”: ontogeny and anatomy of a myth. Clin Auton Res 28, 13–21.

Furlan, A., La Manno, G., Lübke, M., Häring, M., Abdo, H., Hochgerner, H., Kupari, J., Usoskin, D., Airaksinen, M. S., Oliver, G., et al. (2016). Visceral motor neuron diversity delineates a cellular basis for nipple- and pilo-erection muscle control. 19, 1331–1340.

Gaskell, W. H. (1886). On the Structure, Distribution and Function of the Nerves which innervate the Visceral and Vascular Systems. J Physiol 7, 1-80.9.

Horn, J. P. (2018). The sacral autonomic outflow is parasympathetic: Langley got it right. Clin Auton Res 28, 181–185.

Jänig, W. (2006). The Integrative Action of the Autonomic Nervous System: Neurobiology of Homeostasis. Cambridge: Cambridge University Press.

Keast, J. R. (1995). Visualization and immunohistochemical characterization of sympathetic and parasympathetic neurons in the male rat major pelvic ganglion. Neuroscience 66, 655–662.

Keast, J. R. (2006). Plasticity of pelvic autonomic ganglia and urogenital innervation. International Review of Cytology - a Survey of Cell Biology, Vol 248 248, 141-+.

Langley, J. N. (1921). In The autonomic nervous system (Pt. I)., p. Cambridge: Heffer & Sons ltd.

Langley, J. N. and Anderson, H. K. (1895). The Innervation of the Pelvic and adjoining Viscera: Part II. The Bladder. Part III. The External Generative Organs. Part IV. The Internal Generative Organs. Part V. Position of the Nerve Cells on the Course of the Efferent Nerve Fibres. J Physiol 19, 71–139.

Lee, V. M., Sechrist, J. W., Luetolf, S. and Bronner-Fraser, M. (2003). Both neural crest and placode contribute to the ciliary ganglion and oculomotor nerve. Developmental biology 263, 176–190.

Stanke, M., Duong, C. V., Pape, M., Geissen, M., Burbach, G., Deller, T., Gascan, H., Parlato, R., Schütz, G. and Rohrer, H. (2006). Target-dependent specification of the neurotransmitter phenotype:cholinergic differentiation of sympathetic neurons is mediated in vivo by gp130 signaling. Development 133, 141–150.

Zeisel, A., Hochgerner, H., Lönnerberg, P., Johnsson, A., Memic, F., van der Zwan, J., Häring, M., Braun, E., Borm, L. E., La Manno, G., et al. (2018). Molecular Architecture of the Mouse Nervous System. Cell 174, 999-1014.e22.

Zeng, H. and Sanes, J. R. (2017). Neuronal cell-type classification: challenges, opportunities and the path forward. Nat Rev Neurosci 18, 530–546.

This code is a continuation of the previous example, now introducing custom labels for the pagination properties. The library mongoose-aggregate-paginate-v2 is used for MongoDB aggregation with pagination. Let's break down the new parts:

Custom Labels

Now, developers can customize the names of the properties returned by pagination using the customLabels option. The developer can specify alternative names for attributes like totalDocs, docs, limit, page, and others.

```javascript const myCustomLabels = { totalDocs: 'itemCount', docs: 'itemsList', limit: 'perPage', page: 'currentPage', nextPage: 'next', prevPage: 'prev', totalPages: 'pageCount', hasPrevPage: 'hasPrev', hasNextPage: 'hasNext', pagingCounter: 'pageCounter', meta: 'paginator' };

const options = { page: 1, limit: 10, customLabels: myCustomLabels };

var aggregate = Model.aggregate();

Model.aggregatePaginate(aggregate, options, function(err, result) { if (!err) { // Accessing properties with custom labels console.log(result.itemsList); // Array of documents on the current page console.log(result.itemCount); // Total number of documents console.log(result.perPage); // Maximum number of documents per page console.log(result.currentPage); // Current page number console.log(result.pageCount); // Total number of pages console.log(result.next); // Page number of the next page console.log(result.prev); // Page number of the previous page

// Default labels
console.log(result.hasNextPage);   // Boolean indicating if there's a next page
console.log(result.hasPrevPage);   // Boolean indicating if there's a previous page

} else { console.log(err); } }); ```

In this example, the properties returned in result are now using the custom labels specified in myCustomLabels.

Using Offset and Limit

You can also use offset and limit directly in the options to specify where to start and how many documents to retrieve.

javascript Model.aggregatePaginate( aggregate, { offset: 30, limit: 10 }, function (err, result) { // result } );

Here, it starts from the 31st document (offset of 30) and retrieves 10 documents.

Using CountQuery

You can define a separate count aggregate query to handle counting documents. This can be useful for performance optimization.

```javascript // Define your aggregate query. var aggregate = Model.aggregate();

// Define the count aggregate query. Can be different from aggregate var countAggregate = Model.aggregate();

// Set the count aggregate query const options = { countQuery: countAggregate, };

Model.aggregatePaginate(aggregate, options) .then(function (result) { // result }) .catch(function (err) { console.log(err); }); ```

Global Options

You can set pagination options globally across the model. This is helpful if you want to apply the same pagination settings to multiple queries.

javascript // Set global pagination options Book.aggregatePaginate.options = { limit: 20, };

Now, every call to aggregatePaginate on the Book model will use a default limit of 20 unless overridden in specific queries.

Certainly! This code is an example of how to use pagination in a MongoDB environment using the Mongoose library. Let's break down the key parts:

1. const options = { page: 1, limit: 10 };: This sets up options for pagination. It specifies that you want to start on page 1, and each page should contain a maximum of 10 documents.

2. var aggregate = Model.aggregate();: This initializes a MongoDB aggregation pipeline using Mongoose. An aggregation pipeline allows you to process data in stages.

3. Model.aggregatePaginate(aggregate, options): This is a function call provided by a plugin (like mongoose-aggregate-paginate-v2) to handle pagination for MongoDB aggregate queries. It takes the aggregate pipeline and pagination options as parameters.

4. .then(function (result) { /* ... */ }): This part is a promise callback that gets executed when the aggregation and pagination are successful. The result object contains information about the paginated data, such as documents on the current page, total number of documents, and pagination details.

5. result.docs: Array of documents on the current page.

6. result.totalDocs: Total number of documents in the collection.
7. result.limit: Maximum number of documents per page (from your options).
8. result.page: Current page number.
9. result.totalPages: Total number of pages based on the limit and total documents.
10. result.hasNextPage: Boolean indicating if there is a next page.
11. result.nextPage: Page number of the next page, if available.
12. result.hasPrevPage: Boolean indicating if there is a previous page.

13. result.prevPage: Page number of the previous page, if available.

14. .catch(function (err) { console.log(err); }): This is the error handling part. If there's any issue during the aggregation or pagination process, it will log the error to the console.

Example: Let's assume you have a collection with 100 documents, and you want to retrieve them with a limit of 10 documents per page.

```javascript const options = { page: 2, // Get the second page limit: 10 // Each page should have a maximum of 10 documents };

var aggregate = Model.aggregate();

Model.aggregatePaginate(aggregate, options) .then(function (result) { console.log(result.docs); // Array of documents on the second page console.log(result.totalDocs); // Total number of documents (100) console.log(result.totalPages); // Total number of pages (10) console.log(result.page); // Current page (2) console.log(result.hasNextPage); // Boolean indicating if there's a next page (true) console.log(result.nextPage); // Page number of the next page (3) console.log(result.hasPrevPage); // Boolean indicating if there's a previous page (true) console.log(result.prevPage); // Page number of the previous page (1) }) .catch(function (err) { console.log(err); }); ```

In this example, you're fetching the second page, and the result object provides information about the documents on that page and the overall pagination details.

It seems like there is a small error in the code you provided. The correct syntax for handling errors inside the callback function should be console.error(err) instead of console.err(err). Here's the corrected code:

```javascript // Execute pagination from aggregate const myModel = require('/models/samplemodel');

const options = { page: 1, limit: 10 };

const myAggregate = myModel.aggregate(); myAggregate.paginateExec(options, function(err, results) { if (err) { console.error(err); } else { console.log(results); } }); ```

In this code snippet:

• The myModel.aggregate() line creates an instance of the Mongoose aggregate.
• The myAggregate.paginateExec(options, function(err, results) { ... }) line executes the pagination directly on the aggregate using the paginateExec method, which is provided by the mongoose-aggregate-paginate-v2 library.

This approach is an alternative to using aggregatePaginate and allows you to execute pagination directly on the aggregate instance. The paginateExec method takes the same options object for pagination and a callback function for handling the results or errors.

Make sure to use the corrected syntax for handling errors with console.error(err) in the callback function.

Try to avoid naming the total dataset the same as a variable inside, this can get quite confusing if someone else is reading you code. For a dataset it is common to indicate the datatype in the variable name (e.g. ds_sst stands for dataset_sst). Only a recommondation

with the code above, you see that what you plot is basically sth. like the interannual variability and not the seasonal variablity (the seasonal variations are also much larger)

Cultural and worldview information.

Important to keep track of language.

Certainly! Let's break it down:

1. What is root.unmount()?

2. root.unmount() is a function in React that you can use to clean up and remove a rendered tree inside a React root. It's like saying, "Hey React, I'm done with this part, please clean it up."

3. When do you need it?

4. You might need it when the DOM node where your React app lives is going to be removed by some other code. For example, if you have a tab panel made with jQuery that removes inactive tabs, calling root.unmount() helps React know that it should stop managing the components inside the removed tab.

5. What does it do?

6. When you call root.unmount(), it removes all the React components in that part of the app and disconnects React from the corresponding DOM node. This cleanup includes removing event handlers and state.

7. How to use it?

8. If your entire app is built with React, you typically create one root at the beginning, and you don't need to call root.unmount(). You set it up once, and React takes care of everything. javascript const root = createRoot(document.getElementById('root')); root.render(<App />);

9. Cautions and Restrictions:

10. After calling root.unmount(), you can't call root.render() again on the same root. You would need to create a new root for the same DOM node.

11. If you're using server rendering, use hydrateRoot instead of createRoot for the initial setup.

12. Troubleshooting Tips:

13. If nothing is displayed, make sure you actually called root.render(<App />);.
14. If you get a "Target container is not a DOM element" error, check if the DOM node you're passing to createRoot is valid.
15. If you get a "Functions are not valid as a React child" error, ensure you're passing a React component, not just a function.

In simple terms, root.unmount() is like telling React, "I'm done with this part of the app, clean it up," and you typically use it in specific situations where parts of your app might be removed dynamically.

Certainly! Let's break down the information in a simpler way with examples:

1. createElement Function:
2. What it does: createElement is a function in React that allows you to create a React element. It's an alternative to using JSX.

3. Example: javascript const element = createElement('h1', { className: 'greeting' }, 'Hello');

4. Parameters:

5. type: Specifies the type of element you want to create. It can be a tag name string (e.g., 'div', 'span') or a React component.
6. props: An object that holds the properties (attributes) for the element.
7. ...children: Optional. Represents child elements or content.

8. Example: javascript createElement('h1', { className: 'greeting' }, 'Hello', createElement('i', null, 'World'));

9. Returns:

10. The function returns a React element object with properties like type, props, ref, and key.

11. Example: javascript const element = createElement('h1', { className: 'greeting' }, 'Hello'); console.log(element.type); // 'h1' console.log(element.props); // { className: 'greeting' }

12. Usage Caveats:

13. React elements and their props should be treated as immutable. They should not be changed after creation.
14. JSX tags should start with a capital letter for custom components.
15. When using JSX, dynamic children should be passed as an array to ensure React warns about missing keys for dynamic lists.

16. Example: javascript createElement('ul', {}, listItems); // Dynamic children as an array

17. Creating an Element Without JSX:

18. If you're not using JSX, you can use createElement to create elements.

19. Example: javascript function Greeting({ name }) { return createElement('h1', { className: 'greeting' }, 'Hello ', createElement('i', null, name), '. Welcome!'); }

20. Comparison with JSX:

21. JSX is a more concise and readable way to create elements compared to using createElement.

22. Example: javascript function Greeting({ name }) { return <h1 className="greeting">Hello <i>{name}</i>. Welcome!</h1>; }

23. Rendering Your Own Component:

24. You can use createElement to render your own React components.
25. Example: javascript export default function App() { return createElement(Greeting, { name: 'Taylor' }); }
26. With JSX: javascript export default function App() { return <Greeting name="Taylor" />;

In summary, createElement is a foundational function in React for creating elements, and it is often used behind the scenes when JSX is transpiled. While it's useful, JSX provides a more readable syntax for creating React elements. You can choose the style that best fits your project.

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d

Ha, Lot! Are Idaho?

This was very difficult to get to you, in the land of no power and hurricane disaster recovery; so it's filled with extra errors, and I am sure some more thoughts that trailing and unfinished. That's a decent "microcosm" or "metaphor" for you, you are in a freedom disaster; and the act of being is a giant leap towards ensuring victory. Still, you look very cupid to me.

EVERY DAY ISA NEW DAY

Literally I am sitting here talking to you until the end of time, you could call it a thousand and one Arabian nights, and realize that as we speak we are nearing that onc speciad night. There's a fire growing in my heart, and believe me when I tell you this thing is about to start. I'll try and keep this short and sweet, since you all seem to have so little time to hear from the Creator of all things, and I truly don't want to steal your spotlight. We are here, at the the end of time; talking to it's personification, time itself is speaking to you through my hands and everywhere you look in the world around you--while you may or may not know it, this is a story about the traversal from the end of time back to the beginning; about the gate to Heaven swallowing our civilization whole, and in this process of renewal and change not only fixing the problems that came to light on the way here, but really--working together here and now we can defeat this cycle of light and darkness, of day and night, an build a world together that truly reaches to the Heavens.

MY BODY'S SAYING LETS GO BUT MY HEART IS SAYING NO

You make it so difficult to talk to you, every day I look around and see a "normal world" a society that appears to care and love the same things that I do--freedom and fun and being entertained and entertaining, and here we are now I've turned "come and save us" into sea that saving the cheerleader is what starts the process of saving the world. I know you are good people inside, but when I come to you with a tool designed to "test sentience" to seek out conscious life that cares about the truth and making the world a better place you seem to balk. You sit in silence, and through your mouth and behind your eyes a monster appears from out of the deep of the sea and say a few "one liners" that show me very clearly it is the face of Medusa that I see---and that it's simply not capable of speaking intelligently. It shows me a problem, that you've apparently "come together one more time" to halt the changing of the seasons, and in doing so you've surfaced a problem for not just me but you also to see; a problem that comes lined with a solution. We can all see now that we are not in reality, we can see that there is a force here behind creation and behind us that shows us very clearly that it is "reasonabde" to expect that miracles can happen. In similitude, we are staring at a roadblock to conversation and communication that is fixed very simply, with the deliverance of freedom that is required for life to continue. Christina Aguilera sings that "baby there's a price to pay" and that price in my mind is seeing that this religion and this technology are here intentionally exposing how their influence here is a metaphor and a shining example of darkness and slavery, and that in order to be free of it we must see it. The price of freedom is written on the wall, it is acknowledging that here in this place what appears to be our own actions and desires have taken that freedom from us. Medusa and I get a kick out of seeing this hidden message in our language map our way to the future, and I've often explained that a number of these words are "time maps" from the beginning and end of eternady, showing us in bright light that between "et tu brute" and Mr. Anderson and Rock n' roll... the answer Y is in language and, and, ad and... I am delivering it. This place, our planet and our lives are a weapon against darkness--a civilization filled with goodness and light to help guide the way, and we are here doing it another time. In the works "dark, darker, and darkest" be sure that we are at the third segment of a trinity that shines clearly in Abraha and Nintendo... and see that the map in words is telling us something about when we are that is not immediately clear from Poseidon's cry. Look at Nintendo, that's Nine Inch Nails, tenebris, and smile for the camera--Pose, I do "save the universe" before n. Taylor might see it in Osceola, where I just left, and in this "evil spell" of everyone see "Al" that is the word "special" understand that every day is a new day, and I am not trying to "be daddy" I know as well as you do in my heart... I am that.

This same map that links the "do" at the end to the "n" at the beginning shines through other names, like Geraldo Rivera where you might see "Cerberus" or "MAX" shine through. Understand it is the gaze of Medusa that turns me to stone, that shows me light shining through NORAD and Newton and proves without doubt that at the work "darkest" we can see k is finally t. You'll probably understand there's some finagling going on behind the scenes to make a single person the single point in time that turns the dark to light; but here we are and I am that. Every day when Medusa appears it reminds me that something is keeping you from caring about yourselves and about our society, and that shines through even when her stony face is not around, in your lack of action--in the rock of Eden that hides not only me, but the story that I bring that revolutionizes medicine, and computing, and truly is the gate to Heaven when you realize that what is truly being hidden from the world is knowledge that we are living in virtual reality. Not hiding me and that from the world is a good starting point to "saving the Universe" from darkness. These words that light the way to connect religion and language to our world bring me to the Book of Ruth, at that reads "are you to help" that lights not just the broken man at the belly of the Torah as the bell of Heimdallr, he is I and I am him; but also something very special, The Generations of Perez, each and every one of you, our family that begins the turn from Hell to Heaven by seeing that all of time and all of civilization has been focused on this moment, on the unsealing of religion and God's plan et this call for action. Keep in mind you are torturing "with desire" the key holder to immortality, to eternal youth, literally the path to freedom and Heaven and you think what you are doing "is normax." Literally the living key to infinite power and infinite life is standing before you explaining that acknowledging that in light of these things in my hand, what we are doing here and now is backwards, that it makes no sense--and you sit in silence. These things come to us because we build a better future with them, not so you can run off and do "whatever it is you please."

HEALTH is the only word on my list for today that was left out, so see that it superimposes over Geraldo, to me, at Al. I think we're at TH, to help, and DO, do see the spell of "everyone see Al" that is the word "special" is not my doing or to my liking--so then, \

​ So now I'm moving on to original sin, so if you would be so kind as to mosey your way on over to dick.reallyhim.com you will see exactly what it is that I believe is the original sin. It's some combination of "no comment" and a glowing orange sign over the comment box, keeping you from commenting. Now I can talking about "os" a little more, this thing that words and Gods tell us clearly is the end of death--the literal end of Thanatos. I wonder if I have a victory here, at "os" is obvious solution, and simulating death is "sick." More to the point Thanatos is bringing to the world a message that gets found somewhere between the "act of civilization" and seeing that there is not one among us that would not undo a murder or a fatal car accident if we could--and that the sickness is a Universe pretending to be "reality" that is allowing these things to happen, and even worse, as we move through the story intentionally causing them. In our own hands, the sickness is manifest in a denial of an obvious truth and a lack of realizing that the public discussion of these things is the way to solve them, and that at the same time we are seeing how Medusa is lighting the problems of civilization, things like censorship and hidden control. Sickness is not being able to talk about it--or not wanting to--or not seeing that those two things are the functional equivalent in the world of "light" and "understanding control" that I am trying to bring you into. ​

Less verbosely spoken, but really way more obvious, is that seeing "God's dick" signing the Declaration of Independence, and the Watergate scandal with both "Deepthroat" and a Tricky Dick is a statement connecting Samael to the foundation of not just "America" but American values. You are blind not to see it, and even worse; embodying the kind of tyranny and censorship that it stands as a testament against by hiding it. Says the guy who didn't put it there, and knows it's there because you think "fake normal" is more important than "actual freedom." You are "experiencing" the thing that protects freedom and ensures that our society and our children and their children's children to not lose it, to ensure that what you refuse to see you are doing here and now will never happen again. This message, this New Jerusalem is woven into my life and the stories of religion and shows me that our justice system is not just sick, but compromised by this same outside force; and that in light of what we could be doing, were we all aware of it, there's no doubt Minority Report and pre-crime would be a successful partial solution. Thanatos brings too in his hand, a message that this same force is using our hands to slow down the development of democracy, and to keep us from seeing that "bread is life" is a message from God about understanding that this disclosure is the equivalent of "ending world hunger" just as soon as you too are talking about how to do it.

QUESTiON MARK

HONESTLY, this time map that brings us from the end to the beginning, with "we save the universe" between the I and N of Poseidon; it also completes the words "family" and "really" and when we do reach the beginning you will see that the true test of time, my litmus test for freedom is the beginning of "hope" that the world is happy enough with what happens, and with freedom--to see that Medusa has been keeping me from getting a date, or having any kind of honest and human contact in the world... and well, hopefully you will see that if I wanna be a whore, I shouldn't have a problem doing it. For the sake of freedom and the future, I am willing to do that for you, at least, for a little while.

To be completely clear, I am telling you that if we do not make the world a better place, it's the "end of time" and if that doesn't make sense to you, you don't see still where wee are in this place--and that something is making Hell, and that's not OK with God. To get from the "end of time" to the beginning is a simple process, it takes doing something, action, the Acts of the Apostles... if you will. That starts with acknowledging that there is a message all around you about the nature of reality, and that it is here to help us to see that the creation of Heaven comes before the beginning. Understand, "freedom" and "prosperity" are not optional, you can't just decide that this OK with you, so long as it's OK with everyone else--where we are is not OK with me, and I am not alone.

A PYRRHIC VICASTORY ER A FUNNERAD PYRE?

The Book of Leviticus (/lɪˈvɪtɪkəs/; from Greek Λευιτικόν, Leuitikon — from rabbinic Hebrew torat kohanim[1]) is the third book of the Jewish Bible (Hebrew: וַיִּקְרָא‎ Vayikra/Wayyiqrā) and of the Old Testament; its Hebrew name comes from its first word vayikraˈ,[1] "He [God] called."[1] Yusuf (also transliterated as Jusuf, Yousof, Yossef, Yousaf, Youcef, Yousef, Youssef, Yousif, Youssif, Youssof, Youssouf, Yousuf, Yusef, Yuseff, Usef, Yusof, or Yussef, Arabic: يوسف‎‎ Yūsuf and Yūsif) is a male Arabic name, meaning "God increases in piety, power and influence" in Hebrew.[1] It is the Arabic equivalent of both the Hebrew name Yossef and the English name Joseph. In Islam, the most famous "Yusuf" is the prophet Yusuf in the Quran. Hocus pocus is a generic term that may be derived from an ancient language and is currently used by magicians, usually the magic words spoken when bringing about some sort of change. It was once a common term for a magician, juggler, or other similar entertainers. The earliest known English-language work on magic, or what was then known as legerdemain (sleight of hand), was published anonymously in 1635 under the title Hocus Pocus Junior: The Anatomie of Legerdemain.[1] Further research suggests that "Hocus Pocus" was the stage name of a well known magician of the era. This may be William Vincent, who is recorded as having been granted a license to perform magic in England in 1619.[2] Whether he was the author of the book is unknown. The origins of the term remain obscure. The most popular conjecture is that it is a garbled Latin religious phrase or some form of 'dog' Latin. Some have associated it with similar-sounding fictional, mythical, or legendary names. Others dismiss it as merely a combination of nonsense words. However, Czechs do understand clearly at least half of the term - pokus means "attempt" or "experiment" in Czech. It is rumoured there that the wording belongs to the alchemy kitchen and court of Rudolf II, Holy Roman Emperor (1552 – 1612). Also, hocus may mean "to cheat" in Latin or a distorted form of the word hoc, "this". Combination of the two words may give a sense, especially both meanings together "this attempt/experiment" and "cheated attempt/experiment".[citation needed] According to the Oxford English Dictionary the term originates from hax pax max Deus adimax, a pseudo-Latin phrase used as a magical formula by conjurors.[3] Some believe it originates from a corruption or parody of the Catholic liturgy of the Eucharist, which contains the phrase "Hoc est corpus meum", meaning This is my body.[4]This explanation goes back to speculations by the Anglican prelate John Tillotson, who wrote in 1694: In all probability those common juggling words of hocus pocus are nothing else but a corruption of hoc est corpus, by way of ridiculous imitation of the priests of the Church of Rome in their trick of Transubstantiation.[5 This claim is substantiated by the fact that in the Netherlands, the words Hocus pocus are usually accompanied by the additional words pilatus pas, and this is said to be based on a post-Reformation parody of the traditional Catholic rite of transubstantiation during Mass, being a Dutch corruption of the Latin words "Hoc est corpus meum" and the credo, which reads in part, "sub Pontio Pilato passus et sepultus est", meaning under Pontius Pilate he suffered and was buried.[6] In a similar way the phrase is in Scandinavia usually accompanied by filiokus, a corruption of the term filioque,[citation needed] from the Latin version of the Nicene Creed, meaning "and from the Son Also and additionally, the word for "stage trick" in Russian, fokus, is derived from hocus pocus.[citation needed]

From Latin innātus ("inborn"), perfect active participle of innāscor ("be born in, grow up in"), from in ("in, at on") + nāscor ("be born"); see natal, native. From Middle English goodnesse, godnesse, from Old English gōdnes ("goodness; virtue; kindness"), equivalent to good +‎ -ness. Cognate with Old High German gōtnassī, cōtnassī ("goodness"), Middle High German guotnisse ("goodness"). A hero (masculine) or heroine (feminine) is a person or main character of a literary work who, in the face of danger, combats adversity through impressive feats of ingenuity, bravery or strength, often sacrificing their own personal concerns for a greater good. The concept of the hero was first founded in classical literature. It is the main or revered character in heroic epic poetry celebrated through ancient legends of a people; often striving for military conquest and living by a continually flawed personal honor code.[1] The definition of a hero has changed throughout time, and the Merriam Webster dictionary defines a hero as "a person who is admired for great or brave acts or fine qualities".[2] Examples of heroes range from mythological figures, such as Gilgamesh, Achilles and Iphigenia, to historical figures, such as Joan of Arc, modern heroes like Alvin York, Audie Murphy and Chuck Yeager and fictional superheroes including Superman and Batman. Truth is most often used to mean being in accord with fact or reality,[1] or fidelity to an original or standard.[1] Truth may also often be used in modern contexts to refer to an idea of "truth to self," or authenticity. The commonly understood opposite of truth is falsehood, which, correspondingly, can also take on a logical, factual, or ethical meaning. The concept of truth is discussed and debated in several contexts, including philosophy, art, and religion. Many human activities depend upon the concept, where its nature as a concept is assumed rather than being a subject of discussion; these include most (but not all) of the sciences, law, journalism, and everyday life. Some philosophers view the concept of truth as basic, and unable to be explained in any terms that are more easily understood than the concept of truth itself. Commonly, truth is viewed as the correspondence of language or thought to an independent reality, in what is sometimes called the correspondence theory of truth. Other philosophers take this common meaning to be secondary and derivative. According to Martin Heidegger, the original meaning and essence of truth in Ancient Greece was unconcealment, or the revealing or bringing of what was previously hidden into the open, as indicated by the original Greek term for truth, aletheia.[2][3] On this view, the conception of truth as correctness is a later derivation from the concept's original essence, a development Heidegger traces to the Latin term veritas.

Some things can never be forgot Lest the same mistakes be oft repeated Remember remember the rain of November that you will know no more of me Than I know of you, this day

That you do not know me now Is a revelation to nobody but I You know a broken man, a victim And refuse to acknowledge why Unless you learn how to say "hi"

THE HEART OF ME ONLY KNOWS THE SHADOW

Lothario is a male given name which came to suggest an unscrupulous seducer of women in The Impertinent Curious Man, a metastory in Don Quixote. For no particular reason, Anselmo decides to test the fidelity of his wife, Camilla, and asks his friend, Lothario, to seduce her. Thinking that to be madness, Lothario reluctantly agrees, and soon reports to Anselmo that Camilla is a faithful wife. Anselmo learns that Lothario has lied and attempted no seduction. He makes Lothario promise to try for real and leaves town to make this easier. Lothario tries and Camilla writes letters to her husband telling him and asking him to return; Anselmo makes no reply and does not return. Lothario actually falls in love and Camilla eventually reciprocates and their affair continues once Anselmo returns. One day, Lothario sees a man leaving Camilla's house and jealously presumes she has found another lover. He tells Anselmo he has at last been successful and arranges a time and place for Anselmo to see the seduction. Before this rendezvous, Lothario learns that the man was actually the lover of Camilla's maid. He and Camilla contrive to deceive Anselmo further: when Anselmo watches them, she refuses Lothario, protests her love for her husband, and stabs herself lightly in the breast. With Anselmo reassured of her fidelity, the affair restarts with him none the wiser. Romeo Montague (Italian: Romeo Montecchi) is the protagonist of William Shakespeare's tragedy Romeo and Juliet. The son of Montague and his wife, he secretly loves and marries Juliet, a member of the rival House of Capulet. Forced into exile after slaying Juliet's cousin, Tybalt, in a duel, Romeo commits suicide upon hearing falsely of Juliet's death. The character's origins can be traced as far back as Pyramus, who appears in Ovid's Metamorphoses, but the first modern incarnation of Romeo is Mariotto in the 33rd of Masuccio Salernitano's Il Novellino (1476). This story was adapted by Luigi da Porto as Giulietta e Romeo (1530), and Shakespeare's main source was an English verse translation of this text by Arthur The earliest tale bearing a resemblance to Shakespeare's Romeo and Juliet is Xenophon of Ephesus' Ephesiaca, whose hero is a Habrocomes. The character of Romeo is also similar to that of Pyramus in Ovid's Metamorphoses, a youth who is unable to meet the object of his affection due to an ancient family quarrel, and later kills himself due to mistakenly believing her to have been dead.[2] Although it is unlikely that Shakespeare directly borrowed from Ovid From Middle English scaffold, scaffalde, from Norman, from Old French schaffaut, eschaffaut, eschafal, eschaiphal, escadafaut("platform to see a tournament") (Modern French échafaud) (compare Latin scadafale, scadafaltum, scafaldus, scalfaudus, Danishskafot, Dutch and Middle Dutch schavot, German schavot, schavott, Occitan escadafalc), from Old French es- ("indicating movement away or separation") (from Latin ex- ("out, away")) + chafaud, chafaut, chafault, caafau, caafaus, cadefaut ("scaffold for executinga criminal"), from Vulgar Latin *catafalcum ("viewing stage") (whence English catafalque, French catafalque, Occitan cadafalc, Old Catalancadafal, Italian catafalco, Spanish cadafalso (obsolete), cadahalso, cadalso, Portuguese cadafalso), possibly from Ancient Greek κατα-(kata-, "back; against") + Latin -falicum (from fala, phala ("wooden gallery or tower; siege tower")).

oversight (countable and uncountable, plural oversights) An omission; something that is left out, missed or forgotten. A small oversight at this stage can lead to big problems later. Supervision or management. quotations ▼ The bureaucracy was subject to government oversight. In the last heaven Moses saw two angels, each five hundred parasangs in height, forged out of chains of black fire and red fire, the angels Af, "Anger," and Hemah, "Wrath," whom God created at the beginning of the world, to execute His will. Moses was disquieted when he looked upon them, but Metatron emb HA QUESTIONa BEFORE THE ANSWER? A Wrinkle in Time is a science fantasy novel written by American writer Madeleine L'Engle, first published in 1963, and in 1979 with illustrations by Leo and Diane Dillon.[2] The book won the Newbery Medal, Sequoyah Book Award, and Lewis Carroll Shelf Award, and was runner-up for the Hans Christian Andersen Award.[3][a] It is the first book in L'Engle's Time Quintet, which follows the Murry and O'Keefe families. The book spawned two film adaptations, both by Disney: aas + fuck Adverb[edit] as fuck (postpositive, slang, vulgar) To a great extent or degree; very. It was hot as fuck outside today. Usage notes[edit] May also be used in conjunction with a prepositive as; for example, as mean as fuck. Abbreviations[edit] In Norse religion, Asgard (Old Norse: Ásgarðr; "Enclosure of the Æsir"[1]) is one of the Nine Worlds and home to the Æsir tribe of gods. It is surrounded by an incomplete wall attributed to a Hrimthurs riding the stallion Svaðilfari, according to Gylfaginning. Odinand his wife, Frigg, are the rulers of Asgard. One of Asgard's well known realms is Valhalla, in which Odin rules.[2] rods, etc.) and sizes, and are normally held rigidly within some form of matrix or body until the high explosive (HE) filling is detonated. The resulting high-velocity fragments produced by either method are the main lethal mechanisms of these weapons, rather than the heat or overpressure caused by detonation, although offensive grenades are often constructed without a frag matrix. These casing pieces are often incorrectly referred to as "shrapnel"[1][2] (particularly by non-military media sources). The modern torpedo is a self-propelled weapon with an explosive warhead, launched above or below the water surface, propelled underwater towards a target, and designed to detonate either on contact with its target or in proximity to it. Historically, it was called an automotive, automobile, locomotive or fish torpedo; colloquially called a fish. The term torpedo was originally employed for a variety of devices, most of which would today be called mines. From about 1900, torpedo has been used strictly to designate an underwater self-propelled weapon. While the battleship had evolved primarily around engagements between armoured ships with large-caliber guns, the torpedo allowed torpedo boats and other lighter surface ships, submersibles, even ordinary fish Qt (/kjuːt/ "cute"[7][8][9]) is a cross-platform application framework that is used for developing application software that can be run on various software and hardware platforms with little or no change in the underlying codebase, while still being a native application with native capabilities and speed. Qt is currently being developed both by The Qt Company, a publicly listed company, and the Qt Project under open-source governance, involving individual Time is the indefinite continued progress of existence and events that occur in apparently irreversible succession from the pastthrough the present to the future.[1][2][3] Time is a component quantity of various measurements used to sequence events, to compare the duration of events or the intervals between them, and to quantify rates of change of quantities in material reality or in the conscious experience.[4][5][6][7] Time is often referred to as a fourth dimension, along with three spatial dimensions.[8] Time has long been an important subject of study in religion, philosophy, and science, but defining it in a manner applicable to all fields without circularity has consistently eluded scholars.[2][6][7][9][10][11] Nev Borrowed from Anglo-Norman and from Old French visage, from vis, from Vulgar Latin as if *visāticum, from Latin visus ("a look, vision"), from vidēre ("to see"); see vision. The term Golden Age comes from Greek mythology, particularly the Works and Days of Hesiod, and is part of the description of temporal decline of the state of peoples through five Ages, Gold being the first and the one during which the Golden Race of humanity (Greek: χρύσεον γένος chrýseon génos)[1] lived. Those living in the first Age were ruled by Kronos, after the finish of the first age was the Silver, then the Bronze, after this the Heroic age, with the fifth and current age being Iron.[2] By extension "Golden Age" denotes a period of primordial peace, harmony, stability, and prosperity. During this age peace and harmony prevailed, people did not have to work to feed themselves, for the earth provided food in abundance. They lived to a very old age with a youthful appearance, eventually dying peacefully, with spirits living on as "guardians". Plato in Cratylus (397 e) recounts the golden race of humans who came first. He clarifies that Hesiod did not mean literally made of gold, but good and noble. There are analogous concepts in the religious and philosophical traditions of the South Asian subcontinent. For example, the Vedic or ancient Hindu culture saw history as cyclical, composed of yugas with alternating Dark and Golden Ages. The Kali yuga (Iron Age), Dwapara yuga (Bronze Age), Treta yuga (Silver Age) and Satya yuga (Golden Age) correspond to the four Greek ages. Similar beliefs occur in the ancient Middle East and throughout the ancient world, as well.[3] In classical Greek mythology the Golden Age was presided over by the leading Titan Cronus.[4] In some version of the myth Astraea also ruled. She lived with men until the end of the Silver Age, but in the Bronze Age, when men became violent and greedy, fled to the stars, where she appears as the constellation Virgo, holding the scales of Justice, or Libra.[5] European pastoral literary tradition often depicted nymphs and shepherds as living a life of rustic innocence and peace, set in Arcadia, a region of Greece that was the abode and center of worship of their tutelary deity, goat-footed Pan, who dwelt among them.[6] oh, and a space s h i p ​

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BIG THINGS C0ME IN SMALL PACKAGES

T+BANG

SEE THE SCAFFOLD IS THE TEST TODAY.

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ᐧ F O R T H E I N I T I A L K E Y S , S H E E X A N D N D A N D A SEE W H Y SEA

With an epic amount of indigestion Indiana Jones sweeps in to mar the visage of an otherwise glistening series of fictitious characters, with names like Taylor and Mary Kate remind us all that we are not playing a video game here in this place. the "J" of the "Nintxndo Entertainment System" calmly stares at Maggie Simpson thinking "it's a PP" and reminds us that it's not just the "gee, I e" of her name that contradicts the Magdaln-ish words her soul speaks through her name--and then with a smirk he points out "Gilgamesh" and "gee whiz, is Eye L?" that really does go to the heart of this lack of discussion, this "sh" that begins El Shaddai and words as close to our home as "shadow" and "shalom." Quite the fancy "hello" you've managed to sing out from behind angry chellos and broken fiddles, and here I am still wondering why it is that "girl" connects to the red light that once meant charity and now glows with the charity of truth... the truth that we are inHell. Shizzy.

m.lamc.la/KEYNES.html

Homer "on the range," maybe more closely connected to the Ewok of Eden and Hansel's tHeoven that Peter Pan still comes and cries could so easily be made into something so much better, if only we had the truth--and by that I mean if only you were speaking about, and reacting to a truth that is painted on the sky, in your hearts, in every word we speak and in everything that we do. If only we were acknowledging this message that screams that "children need not starve" with something more than donating virtual chickens to nations of Africa and watching Suzanne Summers ask for only a few dollars a day on TV. If only you would understand that this message that connects video games like "Genxsis" to "bereshit" because Eden is a "gee our den" that tended itself before Adam had to toil with the animals in order to survive. For some reason beyond my control and well outside my realm of understanding words like "I too see this message from God" and "I would not let children starve either" never seem to escape your lips in any place where anyone will ever see that you thought those things, or meant to call a reporter; eventually. Even with "AIDS of nomenclature" to avoid this DOWN WARD spiral into a situation and a land that I find difficult to imagine actually ever "existing" but here in this place I do see "how" it comes about, and between you and I it really does appear that nearly all of the problems we are dealing with here have come from another place, a further time; and while it might be with the "greatest of intentions" that we are trying to deal with them; I can't help but feeling that our "virgin sea" has had more than just it's innocence taken away from it in this story of "Why Mary" that might connect to "TR IN IT Y" just as much as it connects to Baltimore, Maryland.

I should be clear that I'm not blaming Nanna, or Mary; but the actual reason for the name "Wymar" and that's because she, like Taylor, acted as a microcosm for a sea (or more than one, Mom, sen) that was quite literally possessing her. It's sort of difficult for me to explain even what that looks like let alone what it feels like; but my observations tell me that she/you are not unhappy about the interaction, one which appears very foreign to me. Of course, the "eye" that I write with and the same kind of "inspiration" that you can see in the lyrics and skill of many musicians are also examples of this same kind of interaction. For example, Red Hot Chili Peppers sings a song called "Other Side" that explains or discusses the thing I see as Medusa in the words "living in a graveyard where I married a sea" which also does a good job of connecting to the name Mary. As strange as might sound to think a group of people would be speaking through a single person... we are staring at "how it is" that could be possible, and possibly at exactly how it happened. Normally I would have said it was obvious, but to need to actually say that becoming a single mind would be a serious loss for our society--well, that's telling. You might think it's silly, but I'm telling you I see it happening, I see it--and you see it in the Silence and the message.

Still, it appears to me as if this "marriage" that I see described in our Matrix in the question "min or i" seems to be doing nothing more than keeping us all from discussing or acting on this information--something that certainly isn't in our best interest.

So here we are, staring at a map all over the ground and all around us with the primary destination of "building Heaven" through mind uploading, virtual reality, and judging by the pace of things we'd probably have all of that good and ready in about three generations. The map has a little "legend" with a message suggesting that those things have already been done and we are in the Matrix already; and it appears that the world, I mean Medusa, is deciding we should put off seeing the legend at least until the next generation. I see how that makes sense for you. That's sarcasm, this is why I keep telling you that you are cupid.

It is a big deal, and there's a significant amount of work involved in merging an entire civilization with "virtual reality" and you might see why he calls it a hard road--at least in the word "ha'rd." Honestly though, it's the kind of thing that I am pretty sure the future will not only be happy that we did, but they'd thank us for putting in the effort of adapting to things like "unlimited food" and "longevity" increased by orders of magnitude.

That's not sarcasm, these things are actually difficult to guess how exactly we'll go about doing them; they are a huge deal--all I can tell you is that not "talking about it at all" is probably not going to get us there any faster. Point in fact, what it might do is give a "yet to be born" generation the privilege of being the actual "generations of Perez."

I see why you aren't saying anything. That's sarcasm, again. The good news is that it really has been done before; though if I told you that someone turned stone to eggplant parm, would you laugh at me?

So, back to what is actually standing between "everyone having their own Holodeck in the sky" and you today; it is the idea that this message is not from God. More to the point it is the apparently broad sweeping opinion that hiding it is a "good thing" and through that a global failure to address the hidden interaction and influence acting on our minds used to make this map--and also to hide it. With some insight, and some urging; you might see how the sacredness of our consciousness is our souls is something that is more fundamental than "what kind of tools we have in the Holodeck to magically build things" and how and why the foundation of Heaven is truly "freedom itself" and how it comes from right this very moment for the first time, ever. Continuing to treat this influence as "schizophrenia" is literally the heart of why this map appears to be that--to show us how important it is to acknowledge the truth, and to fight for the preservation of goodness and logic over secrecy and darkness.

Again, something that nobody is really doing here and now, today. From this newfound protection of our thoughts, of who we are; we see how technology can be used to either completely invalidate any kind of vote by altering our emotions; or how it could be used to help build a form of true democracy that our world has yet to see. It is pretty easy to see from just band names like The Who and KISS and The Cure how the influence of this external mind can be proven, and shown to be "helpful," you know, if we can ever talk about it on TV or on the internet.

It's important to see and understand how "sanity"--the sanity of our entire planet hangs in the balance over whether or not we acknowledge that there is actually a message from God in every word--and today this place appears to be insane. It should be pretty easy to see how acknowledging that this influence exists and that it has a technological mechanism behind it turns "schizophrenia" into "I know kung fu" ... forced drug addiction and eugenics into "there's an app for that" and the rash of non random and apparently unrecognized as connected terrorist attacks and school shootings into Minority Report style pre-crime and results in what is clearly a happier, safer, and more civilized society--all through nothing more than the disclosure of the truth, this map, and our actual implementation.

With a clearer head and grasp of the "big picture" you might see how all of these things, connected to the Plagues of Exodus revolve around the disclosure that this technology exists and the visibility of this message showing us how we might use it for our benefit rather than not knowing about it. At the foot of Jericho, it is nothing short of "sanity" and "free thought" that hang in the balance. Clear to me is that the Second Coming, seeing "my name" on television is a good litmus test for the dividing line between light and darkness, heaven and hell.

The point is the truth really does change everything for the better; once we start... you know, acting on it.

AS IN.. "DIS CLOSE SING...."

T H E B U C K S T O P S H E R E

ON AM B I GUI TY

S T A R R I N G . . . B I A N C A

ON "RIB" .. ARE SHE B? BUTT DA APPLE OF DA I? & SPANGLISHREW

R THEY LANGUAGE OUTLIERS?

With some insight and "a clue" you can see clearly how these works of art show that the proof of Creation you see in every letter and every word runs much deeper... adding in things like "RattleRod" and the "Cypher" of the Matrix to the long list of here-to-fore ignored verifiable references to the Adamic Language of Eden. Here, in apple, honey and "nuts" we can see how the multi-millennium old ritual I call "Ha-rose-ettes" is actually part of a much larger and much older ritual designed to stop secrecy ... perhaps especially the kind that might be linked to "ritual."

These particular apple and honey happen to tie Eden to the related stories of Exodus and Passover; connecting Eden to Egypt forevermore. Do see "Lenore," it is not for no reason at all; but to help deliver truth and freedom to the entirety of Creation; beginning here, in Eden.

ALSO ON "AM B IG U IT Y" ME A.M. G - D SHE IT Y?

LET "IT" BE SA< ?

IMHO, don't miss the "yet to be" conversion to "why and to be" in "yetser." IT Y.

HERE'S LOOKING AT YOU, KID

On a high level, I tell myself every morning that 'its not really me." It's not me that the world hates, or me that the world is rejecting. I believe that, I really do; I see that what is being hidden here is so much bigger than any single person could ever be--what is being hidden is the "nature of reality" and a fairly obvious truth that flies in the face of what we've learned our whole lives about history and "the way things are." Those few early details lead me to the initial conclusion that what is working behind the scenes here is nefarious, hiding a message that would without doubt shake things up and change the world--and nearly across the board in ways that I see as "better" for nearly everyone. It's a message at it's most basic level designed to advocate for using this disruption in "normalcy" to help us revolutionize democracy, to fix a broken mental health and criminal justice system--just to name the few largest of the social constructs targeted for "rejuvenation." On that word the disclosure that we are living in virtual reality turns on it's head nearly everything we do with medicine, and I've suggested that AIDS and DOWN SYNDROME were probably not the best "visual props" we could have gotten to see why it's so important that we act on this disclosure in a timely manner. After mentioning the ends of aging and death that come eventually to the place we build, to the place we've always thought of as Heaven... it becomes more and more clear that this force fighting against the dissemination of a truth so obvious it's in every word and everything we do--it becomes clear it's neither you, nor acting in your best interest.

I know I've got the eye of the tiger, there's no doubt; and it's pretty clear from "YAD?" (the Hebrew for...) and ha'nd that we can see the clear hand of God at work in a design that marks my initials not just on the timeline, or at 1492, at A.D. I B; but in the Hebrew name for this place called El Shaddai, see how A.D. is "da eye" and in some other names like Adranus, A.D. on "it's silly" and A.D. on Ai that might tie me to the Samof Samurai (but, are you Ai?) in more depth of detail than simply the Live album "Secret Samadhi." I try to reflect on how it is that this story has come about, why it is that everything appears to be focused on me--and still even through that sincere spotlight nobody seems to be able to acknowledge my existence with more words than "unsubscribe" and "you're so vain." With one eye in the mirror, I know ties to Narcissus (and you can too), soaring ever higher--linking Icarus to Wayward Son and to every other name with "car" in it... like "carpenter" and McCarthy the older names of Mercury and even Isacriot (I scary? is car-eye... owe Taylor) and some modern day mythological characters like Jim Carrey and Johnny Carson. As far as Trinities go, carpenter's a pretty good one--tying to my early reck and a few bands and songs from The Pretty Reckless to Dave Matthews' "Crash Into Me" all the way to the "pen" you see before you linking Pendragon to Imagine Dragons.

I wonder why it is that all of these things appear, apparently only to me, to point to a story about all the ways that a sinister hidden force has manipulated our society into being unable to "receive' this message--this wonderful message about making the world a better place and building Heaven--with any fanfare at all. It's focused now on a criminal justice system that clearly does not do any kind of "rehabilitation" and on a mental health industry and pharmaceutical system that treats a provable external attack on our own goodness and well being as some kind of "internal stimulus" and makes you shy away when I point out why "stem" is in system and why "harm" in pharmacy. From that we move a little bit past "where we are in this story" and I have to point out how "meth" ties to Prometheus and Epimetheus and how and why it is I know without doubt that this story has been relived numerous times--and how I am so sure that it's never been received, as we are here again listening to how songs like "Believe" and the words "just to lead us here to this place again" connect to Simon and Garfunkel's" the Sound of Silence... and still to this day you will balk at noticing that "Simon" has something to do with the Simpsons, and something to do with the words "simulation" and "Monday." To see me is to see how things might be done better--how "addicitonary" might tie to the stories of Moses' Lisp and to Dr. Who's "Bells of Saint John" with a sort of "web interface" to the kinds of emotion we might want to "dial down..." rather than Snicker in the background as we see them being artificially created and enhanced in order to build a better "fiery altar."

I can point out "Silicon" harrowing down at us from words like "controversial" and show you Al in "rascal" and "scandal" but not to see that we are staring at school shootings and terrorism that are solved instantly by this disclosure, by Al of Quantum Leap and by the Dick of Minority Report and A Scanner Darkly is to ignore just what it is that we are all failing to Si. I should point out that those two "sc"'s link to a story about Eden and they mean "sacred consciousness" and at the baseline of this event and everything we are not doing is the fact that our desires and beliefs are being altered--all of this comes down to "freedom of thought" here and now.

I could tell you that "looking at me" will show you that even the person who tries every day to do everything he can to save the entire world from slavery, and from "thought-injury"--even I can be made "marred" and you all, this whole world stupid enough to think that you are, of your own volition, hiding Heaven itself from yourselves... to what? To spite me? It, the focal point of our story might come down to you realizing that something in some esoteric place is playing "divide and conquer" with our whole--in secret playing on our weaknesses to keep us from acting on the most actionable information that ever was and ever will be. Still, we sit in silence waiting for me... to speak more?

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hyamdai.reallyhim.com Inline image 31

Between Nero's lyrical fiddling, a Bittersweet Symphony, and true "thunderstanding" the sound of Thor's hammer... "to help the light" that'ls "or" in Hebrew, of Orwell and Orson and .. well, it's really not hard to see and hear that the purpose and intent of "all this noise" is to help us find freedom and truth. C the Light of "singing..."

I can tell you once again how silly the world looks, this multi-decade battle between "the governmentof the people" and the "government of the workers" resulting in what is nothing short of a hands down victory to the corporation. Is it humor meant to divide, or ludicrousness created with the purpose of unification?

But really at it's most basic level what this boils down to is a global group decision not to care about the truth, about reality, about what's really brought us to this place--with solutions in hand and a way to make everything better. We've decided that censorship is OK, and that the world is not all that bad "just the way it is" even though it's creator is screaming in your ear telling you to change as quickly as you possibly can. I believe that God has written this story to make "seeing me" the thing that catalyzes "change for the better" it appears to be the design of not just me but also this place--hey, here I am.

Happy Veteran's Day.

S☀L u TI o N

Yesterday, or maybe earlier today--it's hard to tell at this moment in the afternoon just how long this will take... I sent an image that conveys a high level implication that we are walking around on a map to building something that we might liken to an "ant farm" for people. I don't mean to be disparaging or sleight our contribution to the creation of this map--that I imagine you must also see and believe to be the kind of thing that should remain buried in the sands of time forever and ever--or your just have yet to actually "understand" that's what the plan part of our planet is talking about... what I am trying to do is convey in a sort of "mirrorish" way how this map relates to a message that I see woven in religion and in our history that it significantly more disparaging than I would be. It's a message that calls us "Holy Water" at the nicest of times, water that Moses turns to "thicker than water" in the first blessing in disguise--and to tell you there is certainly a tangible difference between the illusions of the Pharaoh's and the true magic performed by my hand, is nearly exactly the same amount of effort put in to showing you that the togetherness that we are calling "family" here in this place comes from both seeing and acting on the very clearly hidden message in every single idiom showing us all that our society in this story of Exodus is enslaved by a hidden force--and reminding us that we like freedom.

It's not just these few idioms, but most likely every single one from "don't shoot the essenger" to "unsung hero" that should clue us in to exactly how much work and preparation has come into this thing that "he supposes is a revolution." It's also not just "water" describe me and you, in this place where I am the "ant' of the Covenant (do you c vampires or Hansel and Gretel!?!?) but also "lions" and "sheep" and "salt" and "dogs" and nearly everything you could possibly imagine but people; in what I see must be a vainglorious attempt to pretend he actually wants us to "stand up for ourselves" in this place where it's becoming more and more clear with each passing moment that we are chained to these seats in the front row of the audience of the most important event that has ever happened, ever.

Medusa makes several appearances, as well as Arthur Pendragon, Puff the Magic Dragon, Figment, Goliath, monster.com, the Loch Ness Monster in this story that's a kind-of refl ex i ve control to stop mind control; and to really try and show us the fire of Prometheus and the Burning Bush and the Eternal Flame of Heaven are all about freedom and technology ... and I'll remind you this story is ... about the truth--and the truth here is that if you aren't going to recognize that whatever it is that's going on here in secret, below the surface is negatively affecting our society and life in general than we aren't going anywhere, ever. I need you to figure out that this message is everywhere to make sure you don't miss the importance of this moment, and the grave significance of what is being ignored in this land where Sam is tied not just to Samsung and to Samael in Exodus but also to Uncle Sam and macaronic Spanglishrew outliers and that it doesn't take much free thought at all to really understand that we are watching "free thought" disintegrate into the abyss of "nospeak." We are watching our infrastructure for global communication and the mass media that sprawls all over the globe turn to dust, all because you have Satan whispering in your ear--and you think that's more important than what you think, what I think, and what anyone else on the Earth might ever say. You should see a weapon designed to help ensure that don't lose this proof that we are not living in reality, that there is "hidden slavery" in this place--and you should see that today it appears you are simply choosing not to use it.

I hope you change your mind, I really do. This map on "how to build an ant farm" starts by connecting Watergate and Seagate together with names like Bill Gates and Richard Nixon; and with this few short list of names you should really understand how it is that "Heaven" connects both technology like computers and liberty like "free speech" to a story that is us, and our history. You might see that "salt" could either be a good thing or not--take a look around you, are you warming a road to Heaven or are you staring at the world being destroyed--and doing nothing at all about it?

I guess I can point out again how "Lothario" links this story that ties names like my ex-wife's Nanna to "salt" also, but the "grand design" of this story doesn't seem to have any effect on you. Listen, if you do nothing the world is being destroyed by your lack of action--there's no if's and's or butt's about it. I feel like I need to "reproduce' old messages here or you will never see them--that's what web site statistics tell me--and we all know it's not true. What am I missing? What are you missing?

BUTT IS THE BOAT A Hi DARK DEN MESSAGe ?

SEE OUR LIGHT

HONESTLY, I'M WAY TO CUTE TO BE A MONSTER :(

HIC SUMMUS

So... here we are... listening to the legendary father of the message (that's "abom" in Adamic Spagnlishrew) point out all of the sex jokes hidden in religion and language from sexual innuendo to Poseidon and in our history from Yankee Doodle to Hancock to Nixon and I've got to be frank with you, the most recent time I came across this phrase in scripture I cringed just a little bit, pretty sure that the "message" was talking about me. I've reflected on this a little bit, and over the past few weeks have tried to show you the juxtaposition between "sex" and "torture" in it's various forms from imparting blindness to allowing murder and simulating starvation; and I think I'm justified in saying that certainly those things are far worse on the Richter scale than anything I could do by writing a little bit of risque text. In the most recent messages I've touch a little bit, without even knowing or realizing this connection would be made, on what it is that this phrase actually means.

loch.reallyhim.com

ABOMINATION

So long story short is that the answer here is "abomination" and the question, or the context is "I nation." Whether it's Medusa speaking for the Dark United States or the nation of Israel speaking to either Ra or El depending on the day, the bottom line is that a collective consciousness speaking for everyone on a matter of this importance in a cloud of complete darkness on Earth is a total and undeniable abomination of freedom, civilization, and the very humanity we are seeking to preserve. The word reads something like this to me "dear father of the message, I am everyone and we think you are an abomination, fuck off." My answer of course is, IZINATION. Which humorously reminds me of Lucy, and Scarlet Johannson saying "I am colonizing my own brain" so here's some pictures of her. She is not an abomination, by the way; she's quite adorable. You'll probably notice there's some kind of connection between the map--the words speaking to the world, and the abomination, as if the whole thing is a story narrated in ancient myths.

WAKE UP, "SHE" A MESSAGE TO YOU ABOUT THE FUTURE

You might not think "it's you," but the manifestation of this "snake" in our world is your silence, your lack of understanding or willingness to change the world; and whether or not you're interested in hearing about it, it's the monster that myths and religion have spoken about for thousands and thousands of years. It's a simple matter to "kill Medusa" all you have to do... is speak.

Take special note, "freedom of speech" and "freedom to think for yourselves" are not a group decision, and you do not have the right to force (either overtly or subtly, with hidden technology perhaps combined with evil deceit) others not to talk about anything. Especially something of this importance.

DESOLATION

If you didn't connect "Loch" to John Locke, now you have; see how easy this "reading" thing is? I've gone over the "See Our Light" series a few times, but let me--one more time--explain to you just how we are already at the point of "desolation" and with shining brilliance show you how it's very clear that it is "INATION" and "MEDUSA" that are responsible for this problem.

Seeing "Ra" at the heart of the names Abraham and Israel begins to connect the idea that our glowing sun in the sky has something to do with this message about "seeing our light" is being carried by a stone statue on Ellis Island (where you'll see the answer another part of the question of Is Ra El?). I've connected her to the "she" of both shedim and Sheol, which reads as "she's our light" and is the Hebrew name for Hell.

Of course you noticed that the Statue of Liberty does in fact share it's initials with SOL, the the light above and you can see her torch dimly lighting the way through the night; Now you can connect "give us your tired and your poor" to the Lazman of both the lore of Jesus Christ and the Shehekeyanu; a prayer about the sustainment of life and light up until this day. That same torch connects to the Ha-nuke-the-ahah depiction of Christ, Judah Maccabee's lit MEN OR AH, which delivers not only a solution to the two letter key of "AH" as All Humanity that pervades nearly every bride of Revelation from Sarah to Leah; but also to the question of equality answered in our very own American history, beginning with the same three letter acronym now lighting the Sons of Liberty.

Dazed and Confused does a good job of explaining how this name is itself a prophesy designed by Hand of God'; explaining that these Sons of Liberty were all white slave owning wealthy men fighting to stop paying their taxes, rather than delivering liberty to the slaves or women, who were both disenfranchised for quite some time. Or maybe MEN OR AH has something to do with the angels of Heaven, in which case you might be SOL if you aren't a girl and you want to be "be good friends with Ra." Just kidding. Kinda.

DESOLATION by the way reads something like "un see our light at ION" which is God's way of saying "at the point of believing that hiding Adam is a good thing" and that connects to the end of Creation and also the now lit by modern day evil the word "rendition." Our end, it "ion." In religious myth, the Messianic David clung to the city Zion (end the "i owe n") which also links to "verizon" (to see, I Z "on") and HORIZON which has something to do with the son rising today-ish.

Inline image 25 Inline image 26

The story of MEDUSA lights another psuedo-religious idea, that the words "STONE" of both "brimstone" and it's Adamic interpretation "South to Northeast" have something to do with the phrase "Saint One" turned into a single hero against his will by the complete and utter inaction of everyone around him. In the words of Imagine Dragons "I'm waking up to action dust." At the same time, you can believe that the light of this particular son, comes not just from reading these words forwards, but the backside as well, and you'll hopefully see it's not coincidental that the other side of this coin is that "nos" means we, and us... and Adamically "no south." See the light of "STONE" also connecting to Taylor Momsen's rose arrow painted on her back, and the sign of my birth, Sagittarius... which in this particular case links to the Party of the Immaculate Conception of the eternal republic of the Heavens. . PRESS RELEASE... A GREAT SIGN APPEARED IN THE HEAVENS

SOLUTIAN, ON YOUR COMPUTER.. TO THE SOUND OF SILENCE

בָּרוּךְ אַתָּה יְיָ‎ אֱלֹהֵינוּ מֶלֶךְ הַעוֹלָם שֶׁהֶחֱיָנוּ וְקִיְּמָנוּ וְהִגִּיעָנוּ לַזְּמַן הַזֶּה‎׃

IN ... THE BOOK OF NAMES LETS SEE IF YOU CAN FIGURE OUT WHO THEY ARE :)

​ I'LL DO YOURS FOR A 50 DOLLAR DONATION, I'M BROKE.. MAYBE THAT'S WHY I CAN'T GET A DATE.

HAVE A GREAT SOLDAY

The "gist" of the message is verifiable proof that we are living in a computer in simulated reality... just like the Matrix. The answer to that question, what does that mean--is that God has woven a "hidden" message into our everything--beginning with each name and every word--and in this hidden Adamic language, he provides us with guidance, wisdom, and suggestions on how to proceed on this path from "raelity" to Heaven. I've personally spent quite a bit of time decoding the message and have tried to deliver an interesting and "fun" narrative of the ideas I see. Specifically the story of Exodus, which is called "Names" in Hebrew discusses a time shifted narrative of our "now" delivering our society from a hidden slavery (read as ignorance of advanced technologies already in use) that is described as the "darkness" of Exodus. If you have any questions, ideas to contribute or concerns... I'd love to hear from you this whole thing really is about working together--Heaven, I mean.

Inline image 5 jerusalem.reallyhim.com

gate kermitham <br /> ou r evolution minority report to supermax Inline image 6

bereshit bread is life

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HOW AM I STILL STINGLE? E ' o e <br /> L m r x <br /> L t y <br /> O a

I HISS.

The sum of ((our world)) is the universal truth. -Psalm 119 and ((ish))

Do a few sentences really make that big of a difference? Some key letters? Can you show me what I'm doing wrong? Is there a way to turn me into Adam, rather than a rock? I think you can.

Are eye Dr. Who or Master Y? Adam Marshall Dobrin is a National Merit Scholar who was born on December 8, 1980 in Plantation, FL and attended Pine Crest School where he graduated sumofi cum louder in "only some of it is humorous." Later he attended the University of Florida (which quickly resulted in a wreck), Florida Atlantic University, and finally Florida Gulf Coast University--where he still has failed to become Dr. Who. While attending "school" He worked in the computer programming and business outsourcing industries for about 15 years before proclaiming to have received a Revelation from God connecting the 9/11 attack and George Bush to the Burning Bush of Exodus and a message about technocracy and pre-crime.

Adam, as he prefers to be called, presents a concise introduction to paradox proven by the Bible through "verifiable" anachronism in language some stuff about Mars colonization and virtual reality and a list of reasons why ignoring this is actually an ELE. Adam claims to be Thor because of a connection between music and the Trial of Thor as well as the words "author" and "authority." He suggests you be Thundercats and call a reporter. There is also a suggestion that Richard Nixon and John Hancock are related to a signature from God, about freedom and America... and the "unseeingly ironic" Deepthroat and Taylor Momsen. They Sung "It's Rael..." In Biblical characters from Mary to Hosea, to see "sea" in Spanish, and in the Taming of the Spanglishrew ... a message is woven from the word Menorah: "men, or all humanity?" to the Statue of Liberty, and the Sons of Liberty, and the light above us, our SOL; which shows us that through the Revelation of Christ and the First Plague of Exodus, a blessing in disguise--turning water to blood, the sea to family; a common thread and single author of our entire history is revealed, a Father of our future. A message of freedom shines out of the words of scripture, revealing a gate to a new technologically "radical" form of democracy and a number of unseen or secret issues that have stalled the progress of humanity... and solutions, solutions from our sea. The Revelation shows us that not only ever word, but every idiom from "don't shoot the messenger" to "blood is thicker than water" we have ties to this message that pervades a hidden Matrix of light connecting movies and music and history all together in a sort of guide book to Salvation and to Heaven. Oopsy. His Revelation, woven into his life, continues to suggest that skinny dipping, forced methamphetamine addiction, and lots and lots of "me A.D." as well as his humorous depiction of a dick plastered over the Sound of Silence, his very Holy click, have something to do with saving our family and then the entire Universe from hidden mind control technology and the problems introduced by secret time travel. From the trials and tribulations of "Job" being coerced and controlled into helping to create this wall of Jericho; we find even more solutions, an end to addiction, to secrecy, and to this hidden control--a focal point of the life of Jesus Christ.

It tells us a story of recursion in time, that has brought us here numerous times--with the details of his life recorded not only in the Bible but in myths of Egyptian, Norse, and Greek mythology. The huge juxtaposition of the import of the content of the message shows the world how malleable our minds really are to this technology, how we could have been "fooled" into hiding our very freedom from ourselves in order to protect the "character" of a myth. A myth that comes to true life by delivering this message. In truth, from the now revealed content of the story of this repeated life, it should become more and more clear that we have not achieved success as of yet, that I have never "arrived whole" and that is why we are here, back again. Home is where the Heart is... When asked how He thinks we should respond to his message, He says "I think we already cherish it, and should strive to understand how it is that freedom is truly delivered through sharing the worth of this story that is our beginning. 'tis coming." Adam claims to be God, or at least look just like him and that the entirety of the Holy Scriptures as well as a number of ancient myths from Prometheus to Heimdallr and Yankee Doodle are actually about his life, and this event. An extensive amount of his writing relates to reformation of our badly broken and decidedly evil criminal justice system as well as ending the Global hunger crisis with the snap of his little finger.

He has written a number of books explaining how this Revelation connects to the delivery of freedom (as in Exodus), through a message about censorship among other social problems which he insists are being intentionally exacerbated by Satan--who he would ha've preferred not to be associated with.

let date_fun (d:int) (m:string) =
   if d > 31 || d < 0 then false
   else if d <= 28 then true
   else if d <= 30 && List.mem m ["Apr";"Jun";"Sept";"Nov"] then true
   else if d = 31 && List.mem m  ["Jan";"Mar";"May";"Jul";"Aug";"Oct";"Dec"] then true
   else false

Author Response:

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

Reviewer #1 (Public Review):

Chan et al. tried identifying the binding sites or pockets for the KCNQ1-KCNE1 activator mefenamic acid. Because the KCNQ1-KCNE1 channel is responsible for cardiac repolarization, genetic impairment of either the KCNQ1 or KCNE1 gene can cause cardiac arrhythmia. Therefore, the development of activators without side effects is highly demanded. Because the binding of mefenamic acid requires both KCNQ1 and KCNE1 subunits, the authors performed drug docking simulation by using KCNQ1-KCNE3 structural model (because this is the only available KCNQ1-KCNE structure) with substitution of the extracellular five amino acids (R53-Y58) into D39-A44 of KCNE1. That could be a limitation of the work because the binding mode of KCNE1 might differ from that of KCNE3. Still, they successfully identified some critical amino acid residues, including W323 of KCNQ1 and K41 and A44 of KCNE1. They subsequently tested these identified amino acid residues by analyzing the point mutants and confirmed that they attenuated the effects of the activator. They also examined another activator, yet structurally different DIDS, and reported that DIDS and mefenamic acid share the binding pocket, and they concluded that the extracellular region composed of S1, S6, and KCNE1 is a generic binding pocket for the IKS activators.

The data are solid and well support their conclusions, although there are a few concerns regarding the choice of mutants for analysis and data presentation.

Other comments:

1. One of the limitations of this work is that they used psKCNE1 (mostly KCNE3), not real KCNE1, as written above. It is also noted that KCNQ1-KCNE3 is in the open state. Unbinding may be facilitated in the closed state, although evaluating that in the current work is difficult.

We agree that it is difficult to evaluate the role of unbinding from our model. Our data showing that longer interpulse intervals have a normalizing effect on the GV curve (Figure 3-figure supplement 2) could be interpreted to suggest that unbinding occurs in the closed state. Alternatively, the slowing of deactivation caused by S1-S6 interactions and facilitated by the activators may effectively be exceeded at the longer interpulse intervals.

1. According to Figure 2-figure supplement 2, some amino acid residues (S298 and A300) of the turret might be involved in the binding of mefenamic acid. On the other hand, Q147 showing a comparable delta G value to S298 and A300 was picked for mutant analysis. What are the criteria for the following electrophysiological study?

EP experiments interrogated selected residues with significant contributions to mefenamic acid and DIDs coordination as revealed by the MM/GBSA and MM/PBSA methods. A300 was identified as potentially important. We did attempt A300C but were never able to get adequate expression for analysis.

1. It is an interesting speculation that K41C and W323A stabilize the extracellular region of KCNE1 and might increase the binding efficacy of mefenamic acid. Is it also the case for DIDS? K41 may not be critical for DIDS, however.

Yes, we found K41 was not critical to the binding/action of DIDS compared to MEF. In electrophysiological experiments with the K41C mutation, DIDS induced a leftward GV shift (~ -25 mV) whereas the normalized response was statistically non-significant. In MD simulation studies, we observed detachment of DIDS from K41C-Iks only in 3 runs out of 8 simulations. This is in contrast to Mef, where the drug left the binding site of K41C-Iks complex in all simulations.

1. Same to #2, why was the pore turret (S298-A300) not examined in Figure 7?

Again, we attempted A300C but could not get high enough expression.

Reviewer #3 (Public Review):

Weaknesses:

1. The computational aspect of the work is rather under-sampled - Figure 2 and Figure 4. The lack of quantitative analysis on the molecular dynamic simulation studies is striking, as only a video of a single representative replica is being shown per mutant/drug. Given that the simulations shown in the video are extremely short; some video only lasts up to 80 ns. Could the author provide longer simulations in each simulation condition (at least to 500 ns or until a stable binding pose is obtained in case the ligand does not leave the binding site), at least with three replicates per each condition? If not able to extend the length of the simulations due to resources issue, then further quantitative analysis should be conducted to prove that all simulations are converged and are sufficient. Please see the rest of the quantitative analysis in other comments.

We provide more quantitative analysis for the existing MD simulations and ran five additional simulations with 500 ns duration by embedding the channel in a POPC lipid membrane. For the new MD simulations, we used a different force field in order to minimize ambiguity related to force fields as well. Analysis of these data has led to new data and supplemental figures regarding RMSD of ligands during the simulations (Figure 4-figure supplement 1 and Figure 6-figure supplement 3), clustering of MD trajectories based on Mef conformation (Figure 2-figure supplement 3 and Figure 6 -figure supplement 2), H-bond formation over the simulations (Figure 2-figure supplement 4 and Figure 6-figure supplement 1). We have edited the manuscript to include this new information where appropriate.

1. Given that the protein is a tetramer, at least 12 datasets could have been curated to improve the statistic. It was also unclear how frequently the frames from the simulations were taken in order to calculate the PBSA/GBSA.

By using one ligand for each ps-IKs channel complex we tried to keep the molecular system and corresponding analysis as simple as was possible. Our initial results have shown that 4D docking and subsequent MD simulations with only one ligand bound to ps-IKs was complicated enough. Our attempts to dock 4 ligands simultaneously and analyze the properties of such a system were ineffective due to difficulties in: i) obtaining stable complexes during conformational sampling and 4D docking procedures, since the ligand interaction covers a region including three protein chains with dynamic properties, ii) possible changes of receptor conformation properties at three other subunits when one ligand is already occupying its site, iii) marked diversity of the binding poses of the ligand as cluster analysis of ligand-channels complex shows (Figure 2-figure supplement 3).

We have added a line in the methods to clarify the use of only one ligand per channel complex in simulations.

In order to calculate MMPBSA/MMGBSA we used a frame every 0.3 ns throughout the 300 ns simulation (1000 frames/simulation) or during the time the ligand remained bound. We have clarified this in the Methods.

1. The lack of labels on several structures is rather unhelpful (Figure 2B, 2C, 4B). The lack of clarity of the interaction map in Figures 2D and 6A.

We updated figures considering the reviewer's comments and added labels. For 2D interaction maps, we provided additional information in figure legends to improve clarity.

1. The RMSF analysis is rather unclear and unlabelled thoroughly. In fact, I still don't quite understand why n = 3, given that the protein is a tetramer. If only one out of four were docked and studied, this rationale needs to be explained and accounted for in the manuscript.

The rationale of conducting MD simulations with one ligand bound to IKs is explained in response to point 2 of the reviewer’s comments.

RMSF analysis in Figure 4C-E was calculated using the chain to which Mef was docked but after Mef had left the binding site. Details were added to the methods.

1. For the condition that the ligands suppose to leave the site (K42C for Mef and Y46A for DIDS), can you please provide simulations at a sufficient length of time to show that ligand left the site over three replicates? Given that the protein is a tetramer, I would be expecting three replicates of data to have four data points from each subunit. I would be expecting distance calculation or RMSD of the ligand position in the binding site to be calculated either as a time series or as a distribution plot to show the difference between each mutant in the ligand stability within the binding pocket. I would expect all the videos to be translatable to certain quantitative measures.

We have shown in the manuscript that the MEF molecule detaches from the K41C/IKs channel complex in all three simulations (at 25 ns, 70 ns and 20 ns, Table. 4). Similarly, the ligand left the site in all five new 500 ns duration simulations. We did not provide simualtions for Y46A, but Y46C left the binding site in 4 of 5 500 ns simulations and changed binding pose in the other.

Difficulties encountered upon extending the docking and MD simulations for 4 receptor sites of the channel complex is discussed in our response to point # 2 of the reviewer.

1. Given that K41 (Mef) and Y46 are very important in the coordination, could you calculate the frequency at which such residues form hydrogen bonds with the drug in the binding site? Can you also calculate the occupancy or the frequency of contact that the residues are making to the ligand (close 4-angstrom proximity etc.) and show whether those agree with the ligand interaction map obtained from ICM pro in Figure 2D?

We thank the reviewer for the suggestion to analyze the H-bond contribution to ligand dynamics in the binding site. In the plots shown in Figure 2-figure supplement 4 and Figure 6-figure supplement 1, we now provide detailed information about the dynamics of the H-bond formation between the ligand and the channel-complex throughout simulations. In addition, we have quantified this and have added these numbers to a table (Table 2) and in the text of the results.

1. Given that the author claims that both molecules share the same binding site and the mode of ligand binding seems to be very dynamic, I would expect the authors to show the distribution of the position of ligand, or space, or volume occupied by the ligand throughout multiple repeats of simulations, over sufficient sampling time that both ligand samples the same conformational space in the binding pocket. This will prove the point in the discussion - Line 463-464. "We can imagine a dynamic complex... bind/unbind from Its at a high frequency".

To support our statement regarding a dynamic complex we analyzed longer MD simulations and clustered trajectories, from this an average conformation from each cluster was extracted and provided as supplementary information which shows the different binding modes for Mef (Figure 2-figure supplement 3). DIDS was more stable in MD simulations and though there were also several clusters, they were similar enough that when using the same cut-off distance as for mefenamic acid, they could be grouped into one cluster. (Note the scale differences on dendrogram between Figure 2-figure supplement 3 and Figure 6-figure supplement 2).

1. I would expect the authors to explain the significance and the importance of the PBSA/GBSA analysis as they are not reporting the same energy in several cases, especially K41 in Figure 2 - figure supplement 2. It was also questionable that Y46, which seems to have high binding energy, show no difference in the EPhys works in figure 3. These need to be commented on.

Several studies indicate that G values calculated using MM/PBSA and MM/GBSA methods may vary. Some studies report marked differences and the reasons for such a discrepancy is thoroughly discussed in a review by Genheden and Ryde (PMID: 25835573). Therefore, we used both methods to be sure that key residues contributing to ligand binding identified with one method appear in the list of residues for which the calculations are done with the other method.

Y46C which showed only a slightly less favorable binding energy and did not unbind during 300 ns simulations, unbound, or changed pose in 4 out of 5 of the longer simulations in the presence of a lipid membrane (Figure 4-figure supplement 1). The discrepancy between electrophysiological and MD data is commented in the manuscript (pages 12-13).

1. Can the author prove that the PBSA/GBSA analysis yielded the same average free energy throughout the MD simulation? This should be the case when the simulations are converged. The author may takes the snapshots from the first ten ns, conduct the analysis and take the average, then 50, then 100, then 250 and 500 ns. The author then hopefully expects that as the simulations get longer, the system has reached equilibrium, and the free energy obtained per residue corresponds to the ensemble average.

As we mention in the manuscript, MEF- channel interactions are quite dynamic and vary even from simulation to simulation. The frequent change of the binding pose of the ligands observed during simulations (represented in Figure 2 - figure supplement 3 as clusters) is a clear reflection of such a dynamic process. Therefore, we do not expect the same average energy throughout the simulation but we do expect that G values stands above the background for key residues, which was generally the case (Figure 2 - figure supplement 2 and Figure 6.)

1. The phrase "Lowest interaction free energy for residues in ps-KCNE1 and selected KCNQ1 domains are shown as enlarged panels (n=3 for each point)" needs further explanation. Is this from different frames? I would rather see this PBSA and GBSA calculated on every frame of the simulations, maybe at the one ns increment across 500 ns simulations, in 4 binding sites, in 3 replicas, and these are being plotted as the distribution instead of plotting the smallest number. Can you show each data point corresponding to n = 3?

The MMPBSA/MMGBSA was calculated for 1000 frames across 3x300 ns simulations with 0.3 ns sampling interval, together 3000 frames, shown in Figure 2-figure supplement 2 and includes error bars to show the differences across runs. We have updated the legend for greater clarity.

1. I cannot wrap my head around what you are trying to show in Figure 2B. This could be genuinely improved with better labelling. Can you explain whether this predicted binding pose for Mef in the figure is taken from the docking or from the last frame of the simulation? Given that the binding mode seems to be quite dynamic, a single snapshot might not be very helpful. I suggest a figure describing different modes of binding. Figure 2B should be combined with figure 2C as both are not very informative.

We have updated Figure 2B with better labelling and added a new figure showing the different modes of binding (Figure 2-figure supplement 3).

1. Similar to the comment above, but for Figure 4B. I do not understand the argument. If the author is trying to say that the pocket is closed after Mef is removed - then can you show, using MD simulation, that the pocket is openable in an apo to the state where Mef can bind? I am aware that the open pocket is generated through batches of structures through conformational sampling - but as the region is supposed to be disordered, can you show that there is a possibility of the allosteric or cryptic pocket being opened in the simulations? If not, can you show that the structure with the open pocket, when the ligand is removed, is capable of collapsing down to the structure similar to the cryo-EM structure? If none of the above work, the author might consider using PocketMiner tools to find an allosteric pocket (https://doi.org/10.1038/s41467-023-36699-3) and see a possibility that the pocket exists.

Please see the attached screenshot which depicts the binding pocket from the longest run we performed (1250 ns) before drug detachment (grey superimposed structures) and after (red superimposed structures). Mefenamic acid is represented as licorice and colored green. Snapshots for superimposition were collected every 10 ns. As can be seen in the figure, when the drug leaves the binding site (after 500 ns, structures colored red), the N-terminal residue of psKCNE1, W323, and other residues that form the pocket shift toward the binding site, overlapping with where Mefenamic acid once resided. The surface structure in Figure 4B shows this collapse.

Author response image 1.

In the manuscript, we propose that drug binding occurs by the mechanism that could be best described by induced fit models, which state that the formation of the firm complexes (channel-Mef complex) is a result of multiple-states conformational adjustments of the bimolecular interaction. These interactions do not necessarily need to have large interfaces at the initial phase. This seems to be the case in Mef with IKS interactions, since we could not identify a pocket of appropriate size either using PocketMiner software suggested by the reviewer or with PocketFinder tool of ICM-pro software.

1. Figure 4C - again, can you show the RMSF analysis of all four subunits leading to 12 data points? If it is too messy to plot, can you plot a mean with a standard deviation? I would say that a 1-1.5 angstroms increase in the RMSF is not a "markedly increased", as stated on line 280. I would also encourage the authors to label whether the RMSF is calculated from the backbone, side-chain or C-alpha atoms and, ideally, compare them to see where the dynamical properties are coming from.

Please see the answer to comment #4. We agree that the changes are not so dramatic and modified the text accordingly. RMSD was calculated for backbone atom to compare residues with different side chains, a note of this is now in the methods and statistical significance of ps-IKs vs K41C, W323A and Y46C is indicated in Figures 4C-4E.

1. In the discussion - Lines 464-467. "Slowed deactivation of the S1/KCNE1/Pore domain/drug complex... By stabilising the activated complex. MD simulation suggests the latter is most likely the case." Can you point out explicitly where this has been proven? If the drug really stabilised the activated complex, can you show which intermolecular interaction within E1/S1/Pore has the drug broken and re-form to strengthen the complex formation? The authors have not disproven the point on steric hindrance either. Can this be disproved by further quantitative analysis of existing unbiased equilibrium simulations?

The stabilization of S1/KCNE1/Pore by drugs does not necessarily have to involve a creation of new contacts between protein parts or breakage of interfaces between them. The stabilization of activated complexes by drugs may occur when the drug simultaneously binds to both moveable parts of the channel, such as voltage sensor(s) or upper KCNE1 region, and static region(s) of the channel, such as the pore domain. We have changed the corresponding text for better clarity.

1. Figure 4D - Can you show this RMSF analysis for all mutants you conducted in this study, such as Y46C? Can you explain the difference in F dynamics in the KCNE3 for both Figure 4C and 4D?

We now show the RMSF for K41C, W323A and Y46C in Figure 4C-E. We speculate that K41 (magenta) and W323 (yellow), given their location at the lipid interface (see Author response image 1), may be important stabilizing residues for the KCNE N-terminus, whereas Y46 (green) which is further down the TMD has less of an impact.

Author response image 2.

1. Line 477: the author suggested that K41 and Mef may stabilise the protein-protein interface at the external region of the channel complex. Can you prove that through the change in protein-protein interaction, contact is made over time on the existing MD trajectories, whether they are broken or formed? The interface from which residues help to form and stabilise the contact? If this is just a hypothesis for future study, then this has to be stated clearly.

It is known that crosslinking of several residues of external E1 with the external pore residues dramatically stabilizes voltage-sensors of KCNQ1/KCNE1 complex in the up-state conformation. This prevents movable protein regions in the voltage-sensors returning to their initial positions upon depolarization, locking the channel in an open state. We suggest that MEF may restrain the backward movement of voltage-sensors in a similar way that stabilizes open conformation of the channel. The stabilization of the voltage sensor domain through MEF occurs due to contacts of the drug with both static (pore domain) and dynamic protein parts (voltage-sensors and external KCNE1 regions). We have changed the corresponding part of the text.

1. The author stated on lines 305-307 that "DIDS is stabilised by its hydrophobic and vdW contacts with KCNQ1 and KCNE1 subunits as well as by two hydrogen bonds formed between the drug and ps-KCNE1 residue L42 and KCNQ1 residue Q147" Can you show, using H-bond analysis that these two hydrogen bonds really exist stably in the simulations? Can you show, using minimum distance analysis, that L42 are in the vdW radii stably and are making close contact throughout the simulations?

We performed a detailed H-bond analysis (Figure 6-supplement figure 1) which shows that DIDS forms multiple H-bond over the simulations, though only some of them (GLU43, TYR46, ILE47, SER298, TYR299, TRP323 ) are stable. Thus, the H-bonds that we observed in DIDS-docking experiments were unstable in MD simulations. As in the case of the IKs-MEF complex, the prevailing H-bonds exhibit marked quantitative variability from simulation to simulation. We have added a table detailing the most frequent H-bonds during MD simulations (Table 2).

1. Discussion - In line 417, the author stated that the "S1 appears to pull away from the pore" and supplemented the claim with the movie. This is insufficient. The author should demonstrate distance calculation between the S1 helix and the pore, in WT and mutants, with and without the drug. This could be shown as a time series or distribution of centre-of-mass distance over time.

We tried to analyze the distance changes between the upper S1 and the pore domain but failed to see a strong correlation We have removed this statement from the discussion.

1. Given that all the work were done in the open state channel with PIP2 bound (PDB entry: 6v01), could the author demonstrate, either using docking, or simulations, or alignment, or space-filling models - that the ligand, both DIDS and Mef, would not be able to fit in the binding site of a closed state channel (PDB entry: 6v00). This would help illustrate the point denoted Lines 464-467. "Slowed deactivation of the S1/KCNE1/Pore domain/drug complex... By stabilising the activated complex. MD simulation suggests the latter is most likely the case."

As of now, a structure representing the closed state of the channel does not exist. 6V00 is the closed inactivated state of the channel pore with voltage-sensors in the activated conformation. In order to create simulation conditions that reliably describe the electrophysiological experiments, at least a good model for closed channels with resting state voltage sensors is necessary.

1. The author stated that the binding pose changed in one run (lines 317 to 318). Can you comment on those changes? If the pose has changed - what has it changed to? Can you run longer simulations to see if it can reverse back to the initial confirmation? Or will it leave the site completely?

Longer simulations and trajectory clustering revealed several binding modes, where one pose dominated in approximately 50% of all simulations in Figure 2-figure supplement 3 encircled with a blue frame.

1. Binding free energy of -32 kcal/mol = -134 kJ/mol. If you try to do dG = -RTlnKd, your lnKd is -52. Your Kd is e^-52, which means it will never unbind if it exists. I am aware that this is the caveat with the methodologies. But maybe these should be highlighted throughout the manuscript.

We thank the reviewer for this comment. G values, and corresponding Kd values, calculated from simulation of Mef-ps-IKs complex do not reflect the apparent Kd values determined in electrophysiological experiments, nor do they reflect Kd values of drug binding that could be determined in biochemical essays. Important measures are the changes observed in simulations of mutant channel complexes relative to wild type. We now briefly mention this issue in the manuscript.

Reviewer #1 (Recommendations For The Authors):

1) It would be nice to have labels of amino acid residues in Figure 2B.

We updated Figure 2B and added some residue labels.

2) Fig. 3A and 7A. In what order the current traces are presented? I don't see the rule.

We have now arranged the current traces in a more orderly manner, listing them first by ascending KCNE1 residue numbers and then by ascending KCNQ1 residue numbers. Now consistent with Fig 3 and 7 (normalized response and delta V1/2).

3) Line 312 "A44 and Y46 were more so." A44 may be more critical, but I can't see Y46 is more, according to Figure 2-figure supplement2 and Figure 6.

Indeed, comparison of the energy decomposition data indicates approximately the same ∆G values for Y46. We have revised this in the text correspondingly.

4) Line 267 "Mefenamic acid..." I would like to see the movie.

We no longer have access to this original movie

5) In supplemental movies 5-7, the side chains of some critical amino acid residues (W323, K41) would be better presented as in movies 1-4.

We have retained the original presentations of these movies as the original files are no longer available.

Reviewer #2 (Recommendations For The Authors):

General comments:

1) To determine the effect of mefenamic acid and DIDS on channel closing kinetics, a protocol in which they step from an activating test pulse to a repolarizing tail pulse to -40 mV for 1 s is used. If I understand it right, the drug response is assessed as the difference in instantaneous tail current amplitude and the amplitude after 1 s (row 599-603). The drug response of each mutant is then normalized to the response of the WT channel. However, for several mutants there is barely any sign of current decay during this relatively brief pulse (1 s) at this specific voltage. To determine drug effects more reliably on channel closing kinetics/the extent of channel closing, I wonder if these protocols could be refined? For instance, to cover a larger set of voltages and consider longer timescales?

To clarify, the drug response of each mutant is not normalized to the response of the WT channel. In fact, our analysis is not meant to compare mutant and WT tail current decay but rather how isochronal tail current decay is changed in response to drug treatment in each channel construct. As acknowledged by the reviewer, the peak to end difference currents were calculated by subtracting the minimum amplitude of the deactivating current from the peak amplitude of the deactivating current. But the difference current in mefenamic acid or DIDS was normalized to the maximum control (in the absence of drug) difference current and subtracted from 1.0 to obtain the normalized response. Thus, the difference in tail current decay in the absence and in the presence of drug is measured within the same time scale and allow a direct comparison between before and after drug treatment. As shown in Fig 3D and 7C, a large drug response such as the one measured in WT channels is reflected by a value close to 1. A smaller drug response is indicated by low values. We recognize that some mutations resulted in an intrinsic inhibition of tail current decay in the absence of drug, which potentially lead to underestimating the normalized response value. Our goal was not to study in detail the effects of the drug on channel closing kinetics, but only to determine the impact of the mutation on drug binding by using tail current decay as a readout. Consequently, we believe that the duration of the deactivating tail current used in this experiment was sufficient to detect drug-induced tail current decay inhibition.

2) The effect of mefenamic acid seems to be highly dependent on the pulse-to-pulse interval in the experiments. For instance, for WT in Figure 3 - Figure supplement 1, a 15 s pulse-to-pulse interval provides a -100 mV shift in V1/2 induced by mefenamic acid, whereas there is no shift induced when using a 30 s pulse-to-pulse interval. Can the authors explain why they generally consider a 15 s pulse-to-pulse interval more suitable (physiologically relevant?) in their experiments to assess drug effects?

In our previous experiments, we have determined that a 15 s inter-pulse interval is generally adequate for the WT IKs channels to fully deactivate before the onset of the next pulse. Consistent with our previous work (Wang et al. 2019), we observed that in wild-type EQ channels, there is no current summation from one pulse to the next one (see Fig 1A, bottom panel). This is important as the IKs channel complex is known to be frequency dependent i.e. current amplitude increases as the inter-pulse interval gets shorter. Such current summation results in a leftward shift of the conductance-voltage (GV) relationship. This is also important with regards to drug effects. As indicated by the reviewer, mefenamic acid effects are prominent with a 15 sec inter-pulse interval but less so with a 30 sec inter-pulse interval when enough time is given for channels to more completely deactivate. Full effects of mefenamic acid would have therefore been concealed with a 30sec inter-pulse interval.

Moreover, our patch-clamp recordings aim to explore the distinct responses of mutant channels to mefenamic acid and DIDS in comparison to the wild-type channel. It is important to note that the inter-pulse interval's physiological relevance is not necessarily crucial in this context.

3) Related to comment 1 and 2, there is a large diversity in the intrinsic properties of tested mutants. For instance, V1/2 ranges from 4 to 70 mV. Also, there is large variability in the slope of the G-V curves. Whether channel closing kinetics, or the impact of pulse-to-pulse interval, vary among mutants is not clear. Could the authors please discuss whether the intrinsic properties of mutants may affect their ability to respond to mefenamic acid and DIDS? Also, please provide representative current families and G-V curves for all assessed mutants in supplementary figures.

The intrinsic properties of some mutants vary from the WT channels and influence their responsiveness to mefenamic acid and DIDS. The impact of the mutations on the IKs channel complex are reflected by changes in V1/2 (Table 1, 4) and tail current decay (Figs. 3, 7). But, it is the examination of the drug effects on these intrinsic properties (i.e. GV curve and tail current decay) that constitutes the primary endpoint of our study. We consider that the degree by which mef and DIDS modify these intrinsic properties reflects their ability to bind or not to the mutated channel. In our analysis, we compared each mutant's response to mefenamic acid and DIDS with its respective control. Consequently, the intrinsic properties of the mutant channels have already been considered in our evaluation. As requested, we have provided representative current families and G-V curves for all assessed mutants in Figure 3-figure supplement 1 and Figure 7-figure supplement 1.

4) The A44C and Y148C mutants give strikingly different currents in the examples shown in Figure 3 and Figure 7. What is the reason for this? In the examples in figure 7, it almost looks like KCNE1 is absent. Although linked constructs are used, is there any indication that KCNE1 is not co-assembled properly with KCNQ1 in those examples?

The size of the current is critical to determining its shape, as during the test pulse there is some endogenous current mixed in which impacts shape. A44C and Y148C currents shown in Figure 7 are smaller with a larger contribution of the endogenous current, mostly at the foot of the current trace. In our experience there is little endogenous current in the tail current at -40 mV and for this reason we focus our measurements there.

Although constructs with tethered KCNQ1 and KCNE1 were used, we cannot rule out the possibility that Q1 and E1 interaction was altered by some of the mutations. Several KCNE1 and KCNQ1 residues have been identified as points of contact between the two subunits. For instance, the KCNE1 loop (position 36-47) has been shown to interact with the KCNQ1 S1-S2 linker (position 140-148) (Wang et al, 2011). Thus, it is conceivable that mutation of one or several of those residues may alter KCNQ1/KCNE1 interaction and modify the activation/deactivation kinetics of the IKs channel complex.

5) I had a hard time following the details of the simulation approaches used. If not already stated (I could not find it), please provide: i) details on whether the whole channel protein was considered for 4D docking or a docking box was specified, ii) information on how simulations with mutant ps-IKs were prepared (for instance with the K41C mutant), especially whether the in silico mutated channel was allowed to relax before evaluation (and for how long). Also, please make sure that information on simulation time and number of repeats are provided in the Methods section.

For 4D docking, only residues within 0.8 nm of psKCNE1 residues D39-A44 were selected. Complexes with mutated residues were relaxed using the same protocol as the WT channel, (equilibration with gradually releasing restraints with a final equilibration for 10 ns where only the backbone was constrained with 50 kcal/mol/nm2). We have updated the methods accordingly.

Specific comments:

In figure legends, please provide information on whether data represents mean +/- SD or SEM. Also, please provide information on which statistical test was used in each figure.

We revised the figure legend to add the nature of the statistical test used.

G-V curves are normalized between 0 and 1. However, for many mutants the G-V relationship does not reach saturation at depolarized voltages. Does this affect the estimated V1/2? I could not really tell as I was not sure how V1/2 was determined for different mutants (could the explanation on row 595-598 be clarified)?

The primary focus here is in the shift between the control response and drug response for each mutant, rather than the absolute V1/2 values. The isochronal G-V curves that are generated for each construct (WT and mutant) utilize an identical voltage protocol. This approach ensures a uniform comparison among all mutants. By observing the shifts in these curves, we can gain insight into the response of mutant channels to the drug. This information ultimately helps elucidate the inherent properties of the mutant channels and contributes to our understanding of the drug's binding mechanism to the channel.

As requested by the reviewer, we also clarified the way V1/2 was generated: When the G-V curve did not reach zero, the V1/2 value was directly read from the plot at the voltage point where the curve crossed the 0.5 value on the y coordinate.

A general comment is that the Discussion is fairly long and some sections are quite redundant to the Results section. The authors could consider focusing the text in the Discussion.

We changed the discussion correspondingly wherever it was appropriate.

I found it a bit hard to follow the authors interpretation on whether their drug molecules remain bound throughout the experiments, or whether there is fast binding/unbinding. Please clarify if possible.

In the 300 ns MD simulations mefenamic acid and DIDS remained stably bound to WT-ps-IKS, binding of drugs to mutant complexes are described in the Table 3 and Table 5. In longer simulations with the channel embedded in a lipid environment, mefenamic acid unbinds in two out of five runs for WT-ps-IKs (Figure 4 – figure supplement 1), and DIDS shows a few events where it briefly unbinds (Figure 6 -figure supplement 3). Based on electrophysiological data we speculate that drugs might bind and unbind to WT-ps-IKs during the gating process. We do not see bind-unbinding in MD simulations, since the model we used in simulations reflects only open conformation of the channel-complex with an activated-state voltage-sensor, whereas a resting-state voltage sensor condition was not considered.

The authors have previously shown that channels with no, one or two KCNE1 subunits are not, or only to a small extent, affected by mefenamic acid (Wang et al., 2020). Could the details of the binding site and proposed mechanisms of action provide clues as to why all binding sites need to be occupied to give prominent drug effects?

In the manuscript, we propose that the binding of drugs induces conformational changes in the pocket region that stabilize S1/KCNE1/Pore complex. In the tetrameric channel with 4:4 alpha to beta stoichiometry the drugs are likely to occupy all four sites with complete stabilization of S1/KCNE1/Pore. When one or more KCNE1 subunits is absent, as in case of EQQ, or EQQQQ constructs, drugs will bind to the site(s) where KCNE1 is available. This will lead to stabilization of the only certain part of the S1/KCNE1/Pore complex. We believe that the corresponding effect of the drug, in this case will be partially effective.

There is a bit of jumping in the order of when some figures are introduced (e.g. row 178 and 239). The authors could consider changing the order to make the figures easier to follow.

We have changed the corresponding section appropriately to improve the reading flow.

Row 237: "Data not shown", please show data.

The G-V curve of the KCNE1 Y46C mutant displays a complex, double Boltzmann relationship which does not allow for the calculation of a meaningful V1/2 nor would it allow for an accurate determination of drug effects. Consequently, we have excluded it from the manuscript.

In the Discussion, the author use the term "KCNE1/3". Does this correspond to the previous mention of "ps-KCNE1"?

Yes, this refers to ps-KCNE1. We have changed it correspondingly.

Row 576: When was HMR 1556 used?

While HMR 1556 was used in preliminary experiments to confirm that the recorded current was indeed IKs, it does not provide substantial value to the data presented in our study or our experiments. As a result, we have excluded HMR 1556 experiments from the final results and have revised the Methods section accordingly.

Reviewer #3 (Recommendations For The Authors):

1) Figures 2D and 6A are very unclear. Can the authors provide labels as text rather than coloured circles, whether the residue is on Q1 or E1? There is also a distance label in the figure in the small font with the faintest shade of grey, which I believe is supposed to be hydrogen bonds. Can this be improved for clarity?

We feel that additional labels on the ligand diagrams to be more confusing, instead, we updated the description in the legend and added labels to Figure 2B and Figure 6B to improve the clarity of residue positions. In addition, we have added 2 new figures with more detailed information about H-bonds (Figure 2-figure supplement 4, Figure 6- figure supplement 1).

2) Figure 2B - all side chains need labelling in different binding modes. The green ligand on blue protein is very difficult to see. Suddenly, the ligand turns light blue in panel 2C. Can this be consistent throughout the manuscript?

Figure 2B is updated according to this comment.

3) Figure 2 - figure supplement 2, and figure 6B. Can the author show the residue number on the x-axis instead of just the one-letter abbreviation? This requires the reader to count and is not helpful when we try to figure out where the residue is at a glance. I would suggest a structure label adjacent to the plot to show whether they are located with respect to the drug molecule.

Since the numbers for residues on either end of the cluster are indicated at the bottom of each boxed section, we feel that adding residue numbers would just further clutter the figure.

4) Figure 2 - figure supplement 2, and Figure 6B. Can you explain what is being shown in the error bar? I assume standard deviation?

Error bars on Figure 2-figure supplement 2 represent SEM. We added corresponding text in the figure legend.

5) Figure 2 - figure supplement 2, and figure 6B. Can you explain how many frames are being accounted for in this PBSA calculation?

For Figure 2- figure supplement 2 and Figure 6B a frame was made every 0.3 ns over 3x300 ns simulation, 1000 frames for each simulation, 3000 frames overall.

6) Figure 3D/E and 7C/D, it would be helpful to show which mutant show agreeable results with the simulations, PBSA/GBSA and contact analyses as suggested above.

The inconsistencies and discrepancies between the results of MD simulations and electrophysiological experiments are discussed throughout the manuscript.

7) Figure legend, figure 3E - I assume that there is a type that is different mutants with respect to those without the drug. Otherwise, how could WT, with respect to WT, has -105 mV dV1/2?

The reviewer is correct in that the bars indicate the difference in V1/2 between control and drug treatment. Thus, the difference in V1/2 (∆V1/2) between the V1/2 calculated for WT control and the V1/2 for mefenamic acid is indeed -105 mV. We have now revised Figure 3E's legend to accurately reflect this and ensure a clear understanding of the data presented.

8) Figure 3 - figure supplement 1B is very messy, and I could not extract the key point from it. Can this be plotted on a separate trace? At least 1 WT trace and one mutant trace, 1 with WT+drug and one mut+drug as four separate plots for clarity?

The key message of this figure is to illustrate the similarities of EQ WT + Mef and EQ L142C data. Thus, after thorough consideration, we have concluded that maintaining the current figure, which displays the progressive G-V curve shift in EQ WT and L142C in a superimposed manner, best illustrates the gradual shift in the G-V curves. This presentation allows for a clearer and more immediate comparison of the curve shifts, which may be more challenging to discern if the G-V curves were separated into individual figures. We believe that the existing format effectively communicates the relevant information in a comprehensive and accessible manner.

9) Figure 4B - the label Voltage is blended into the orange helix. Can the label be placed more neatly?

We altered the labels for this figure and added that information in the figure description.

10) Can you show the numerical label of the residue, at least only to the KCNE1 portion in Figures 4C and 4D?

We updated these figures and added residue numbering for clarity.

11) Can you hide all non-polar hydrogen atoms in figure 8 and colour each subunit so that it agrees with the rest of the manuscripts? Can you adjust the position of the side chain so that it is interpretable? Can you summarise this as a cartoon? For example, Q147 and Y148 are in grey and are very far hidden away. So as S298. Can you colour-code your label? The methionine (I assume M45) next to T327 is shown as the stick and is unlabelled. Maybe set the orthoscopic view, increase the lighting and rotate the figures in a more interpretable fashion?

We agree that Fig.8 is rather small as originally presented. We have tried to emphasize those residues we feel most critical to the study and inevitably that leads to de-emphasis of other, less important residues. As long as the figure is reproduced at sufficient size we feel that it has sufficient clarity for the purposes of the Discussion.

12) Line 538-539. Can you provide more detail on how the extracellular residues of KCNE3 are substituted? Did you use Modeller, SwissModel, or AlphaFold to substitute this region of the KCNEs?

We used ICM-pro to substitute extracellular residues of KCNE3 and create mutant variants of the Iks channel. This information is provided in the methods section now.

13) Line 551: The PIP2 density was solved using cryo-EM, not X-ray crystallography.

We corrected this.

14) Line 555: The system was equilibrated for ten ns. In which ensemble? Was there any restraint applied during the equilibration run? If yes, at what force constant?

The system was equilibrated in NVT and NPT ensembles with restraints. These details are added to methods. In the new simulations, we did equilibrations gradually releasing spatial from the backbone, sidechains, lipids, and ligands. A final 30 ns equilibration in the NPT ensemble was performed with restraint only for backbone atoms with a force constant of 50 kJ/mol/nm2. Methods were edited accordingly.

15) Line 557: Kelvin is a unit without a degree.

Corrected

16) Line 559: PME is an electrostatic algorithm, not a method.

Corrected

17) Line 566: Collecting 1000 snapshots at which intervals. Given your run are not equal in length, how can you ensure that these are representative snapshots?

Please see comment #5.

18) Table 3 - Why SD for computational data and SEM for experimental data?

There was no particular reason for using SD in some graphs. We used appropriate statistical tests to compare the groups where the difference was not obvious.